WorldWideScience

Sample records for 120-kd surface protein

  1. PSC: protein surface classification.

    Science.gov (United States)

    Tseng, Yan Yuan; Li, Wen-Hsiung

    2012-07-01

    We recently proposed to classify proteins by their functional surfaces. Using the structural attributes of functional surfaces, we inferred the pairwise relationships of proteins and constructed an expandable database of protein surface classification (PSC). As the functional surface(s) of a protein is the local region where the protein performs its function, our classification may reflect the functional relationships among proteins. Currently, PSC contains a library of 1974 surface types that include 25,857 functional surfaces identified from 24,170 bound structures. The search tool in PSC empowers users to explore related surfaces that share similar local structures and core functions. Each functional surface is characterized by structural attributes, which are geometric, physicochemical or evolutionary features. The attributes have been normalized as descriptors and integrated to produce a profile for each functional surface in PSC. In addition, binding ligands are recorded for comparisons among homologs. PSC allows users to exploit related binding surfaces to reveal the changes in functionally important residues on homologs that have led to functional divergence during evolution. The substitutions at the key residues of a spatial pattern may determine the functional evolution of a protein. In PSC (http://pocket.uchicago.edu/psc/), a pool of changes in residues on similar functional surfaces is provided.

  2. Hydrophobic patches on protein surfaces

    NARCIS (Netherlands)

    Lijnzaad, P.

    2007-01-01

    Hydrophobicity is a prime determinant of the structure and function of proteins. It is the driving force behind the folding of soluble proteins, and when exposed on the surface, it is frequently involved in recognition and binding of ligands and other proteins. The energetic cost of exposing hydroph

  3. Competitive Protein Adsorption - Multilayer Adsorption and Surface Induced Protein Aggregation

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin

    2009-01-01

    In this study, competitive adsorption of albumin and IgG (immunoglobulin G) from human serum solutions and protein mixtures onto polymer surfaces is studied by means of radioactive labeling. By using two different radiolabels (125I and 131I), albumin and IgG adsorption to polymer surfaces...... is monitored simultaneously and the influence from the presence of other human serum proteins on albumin and IgG adsorption, as well as their mutual influence during adsorption processes, is investigated. Exploring protein adsorption by combining analysis of competitive adsorption from complex solutions...... of high concentration with investigation of single protein adsorption and interdependent adsorption between two specific proteins enables us to map protein adsorption sequences during competitive protein adsorption. Our study shows that proteins can adsorb in a multilayer fashion onto the polymer surfaces...

  4. Neurocognitive derivation of protein surface property from protein aggregate parameters

    OpenAIRE

    Mishra, Hrishikesh; Lahiri, Tapobrata

    2011-01-01

    Current work targeted to predicate parametric relationship between aggregate and individual property of a protein. In this approach, we considered individual property of a protein as its Surface Roughness Index (SRI) which was shown to have potential to classify SCOP protein families. The bulk property was however considered as Intensity Level based Multi-fractal Dimension (ILMFD) of ordinary microscopic images of heat denatured protein aggregates which was known to have potential to serve as...

  5. Surface waves of Min-proteins

    Science.gov (United States)

    Fischer-Friedrich, Elisabeth; Nguyen van yen, Romain; Kruse, Karsten

    2007-03-01

    In the bacterium Escherichia coli, the Min-proteins show pronounced pole-to-pole oscillations. They are functional for suppressing cell division at the cell ends, leaving the center as the only possible site for division. Analyzing different models of Min-protein dynamics in a bacterial geometry, we find waves on the cytoplasmic membrane. Interestingly, the surface wave solutions of different models belong to different symmetry classes. We suggest that experiments on Min-protein surface waves in vitro are helpful in distinguishing between different classes of models of Min-protein dynamics.

  6. Neurocognitive derivation of protein surface property from protein aggregate parameters

    Science.gov (United States)

    Mishra, Hrishikesh; Lahiri, Tapobrata

    2011-01-01

    Current work targeted to predicate parametric relationship between aggregate and individual property of a protein. In this approach, we considered individual property of a protein as its Surface Roughness Index (SRI) which was shown to have potential to classify SCOP protein families. The bulk property was however considered as Intensity Level based Multi-fractal Dimension (ILMFD) of ordinary microscopic images of heat denatured protein aggregates which was known to have potential to serve as protein marker. The protocol used multiple ILMFD inputs obtained for a protein to produce a set of mapped outputs as possible SRI candidates. The outputs were further clustered and largest cluster centre after normalization was found to be a close approximation of expected SRI that was calculated from known PDB structure. The outcome showed that faster derivation of individual protein’s surface property might be possible using its bulk form, heat denatured aggregates. PMID:21572883

  7. Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein

    Energy Technology Data Exchange (ETDEWEB)

    Dooley, J.S.G.; Trust, T.J.

    1988-02-01

    The surface protein composition of members of a serogroup of Aeromonas hydrophila was examined. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed a 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase /sup 125/I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size required for A. hydrophila S-layer anchoring. The native S layer was permeable to /sup 125/I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behavior characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein.

  8. Protein-mediated surface structuring in biomembranes

    Directory of Open Access Journals (Sweden)

    Maggio B.

    2005-01-01

    Full Text Available The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein, integral (Folch-Lees proteolipid protein and amphitropic (c-Fos and c-Jun proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase, in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

  9. Organising Atoms, Clusters and Proteins on Surfaces

    Science.gov (United States)

    Palmer, Richard E.

    2008-10-01

    This talk will discuss new developments in the creation of nanoscale surface features and their applications in biomedicine. Electron-surface interactions and plasma methods play a crucial role in both the production and analysis of these ``atomic architectures.'' At the extreme limit, electron injection from the tip of a scanning tunnelling microscope (STM) enables bond-selective manipulation of individual polyatomic molecules [1]. On a more practical level, the controlled deposition of size-selected clusters [2], generated by magnetron sputtering and gas condensation followed by mass selection, represents a surprisingly efficient route to the fabrication of surface features of size 1-10 nm, the size scale of biological molecules such as proteins. STM and AFM measurements show the clusters can act as binding sites for individual protein molecules. For example, the pinning of size-selected AuN clusters (N = 1--2000) to the (hydrophobic) graphite surface presents bindings site for sulphur atoms and thus for the cysteine residues in protein molecules. Systematic studies of different proteins [3] provide ``ground rules'' for residue-specific protein immobilisation by clusters and have led to the development of a novel biochip for protein screening by a spin-off company. The 3D atomic structure of the clusters is highly relevant to such applications. We show that measurement of the scattered electron beam intensity - specifically, the high angle annular dark field (HAADF) signal - in the scanning transmission electron microscope (STEM) allows us (a) to count the number of atoms in a cluster on the surface and (b) to determine a 3D atom-density map of the cluster when an aberration-corrected STEM is used [4]. 1. P.A. Sloan and R.E. Palmer, Nature 434 367 (2005). 2. S. Pratontep, P. Preece, C. Xirouchaki, R.E. Palmer, C.F. Sanz-Navarro, S.D. Kenny and R. Smith, Phys. Rev. Lett. 90 055503 (2003). 3. R.E. Palmer, S. Pratontep and H.-G. Boyen, Nature Materials 2 443 (2003

  10. Cell surface engineering with edible protein nanoshells.

    Science.gov (United States)

    Drachuk, Irina; Shchepelina, Olga; Harbaugh, Svetlana; Kelley-Loughnane, Nancy; Stone, Morley; Tsukruk, Vladimir V

    2013-09-23

    Natural protein (silk fibroin) nanoshells are assembled on the surface of Saccharomyces cerevisiae yeast cells without compromising their viability. The nanoshells facilitate initial protection of the cells and allow them to function in encapsulated state for some time period, afterwards being completely biodegraded and consumed by the cells. In contrast to a traditional methanol treatment, the gentle ionic treatment suggested here stabilizes the shell silk fibroin structure but does not compromise the viability of the cells, as indicated by the fast response of the encapsulated cells, with an immediate activation by the inducer molecules. Extremely high viability rates (up to 97%) and preserved activity of encapsulated cells are facilitated by cytocompatibility of the natural proteins and the formation of highly porous shells in contrast to traditional polyelectrolyte-based materials. Moreover, in a high contrast to traditional synthetic shells, the silk proteins are biodegradable and can be consumed by cells at a later stage of growth, thus releasing the cells from their temporary protective capsules. These on-demand encapsulated cells can be considered a valuable platform for biocompatible and biodegradable cell encapsulation, controlled cell protection in a synthetic environment, transfer to a device environment, and cell implantation followed by biodegradation and consumption of protective protein shells.

  11. Identification and characterization of the surface proteins of Clostridium difficile

    Energy Technology Data Exchange (ETDEWEB)

    Dailey, D.C.

    1988-01-01

    Several clostridial proteins were detected on the clostridial cell surface by sensitive radioiodination techniques. Two major proteins and six minor proteins comprised the radioiodinated proteins on the clostridial cell surface. Cellular fractionation of surface radiolabeled C. difficile determined that the radioiodinated proteins were found in the cell wall fraction of C. difficile and surprisingly were also present in the clostridial membrane. Furthermore, an interesting phenomenon of disulfide-crosslinking of the cell surface proteins of C. difficile was observed. Disulfide-linked protein complexes were found in both the membrane and cell wall fractions. In addition, the cell surface proteins of C. difficile were found to be released into the culture medium. In attempts to further characterize the clostridial proteins recombinant DNA techniques were employed. In addition, the role of the clostridial cell surface proteins in the interactions of C. difficile with human PMNs was also investigated.

  12. Protein adsorption on materials surfaces with nano-topography

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Protein adsorption behavior on the surfaces of biomedical materials is highly related to the biocompatibility of the materials. In the past, numerous research reports were mainly focused on the effect of chemical components of a material's surface on protein adsorption. The effect of surface topography on protein adsorption, the topic of this review, has recently receuvedkeen interest. The influence of surface nano-topographic factors, including roughness, curvature and geometry, on protein adsorption as well as the protein adsorption behavior, such as the amount of protein adsorbed, the activity and morphology of adsorbed protein, is introduced.

  13. The Electrophoretic Mobility of Proteins near Surfaces

    Science.gov (United States)

    Ramasamy, Perumal; Singh, Avtar; Rafailovich, Miriam; Sokolov, Jonathan

    2004-03-01

    We have attempted to apply the methods developed for surface DNA electrophoresis (1) for proteomics. Droplets of FITC stained Abumin, Poly- L-Lysine, or Casein purchased from Sigma were deposited on glass cover slips. The droplets were then place in contact with a TBE buffer solution contained in a cell molded from PDMS. Pt electrodes were inserted into the cell and a voltage was a applied. The motion of the protein was then imaged with a Leica Confocal microscope as a function of buffer concentration, distance from the surface, and applied voltage. The mobilities were then compared with those of uncharged one micron florescent Polystyrene beads. References: 1)Henzel WJ, Watanabe C, Stults JT., !0 Protein Identification: The Origins of Peptide Mass Fingerprinting. !1 J. American Society for Mass Spectrometry. 14 (September 2003): 931-942 2)Mathesius U, Imin N, Natera SH, Rolfe BG., !0 Proteomics as a functional genomics tool. !1 Methods of Molecular Biology 236: 395-414. *Work supported in part by the NSF-MRSEC program

  14. Protein surface patterning using nanoscale PEG hydrogels.

    Science.gov (United States)

    Hong, Ye; Krsko, Peter; Libera, Matthew

    2004-12-01

    We have used focused electron-beam cross-linking to create nanosized hydrogels and thus present a new method with which to bring the attractive biocompatibility associated with macroscopic hydrogels into the submicron length-scale regime. Using amine-terminated poly(ethylene glycol) thin films on silicon substrates, we generate nanohydrogels with lateral dimensions of order 200 nm which can swell by a factor of at least five, depending on the radiative dose. With the focused electron beam, high-density arrays of such nanohydrogels can be flexibly patterned onto silicon surfaces. Significantly, the amine groups remain functional after e-beam exposure, and we show that they can be used to covalently bind proteins and other molecules. We use bovine serum albumin to amplify the number of amine groups, and we further demonstrate that different proteins can be covalently bound to different hydrogel pads on the same substrate to create multifunctional surfaces useful in emerging bio/proteomic and sensor technologies.

  15. Trichomonas vaginalis surface proteins: a view from the genome

    DEFF Research Database (Denmark)

    Hirt, R. P.; Noel, C. J.; Sicheritz-Pontén, Thomas

    2007-01-01

    Surface proteins of mucosal microbial pathogens play multiple and essential roles in initiating and sustaining the colonization of the heavily defended mucosa. The protist Trichomonas vaginalis is one of the most common human sexually transmitted pathogens that colonize the urogenital mucosa....... However, little is known about its surface proteins. The recently completed draft genome sequence of T. vaginalis provides an invaluable resource to guide molecular and cellular characterization of surface proteins and to investigate their role in pathogenicity. Here, we review the existing data on T....... vaginalis surface proteins and summarize some of the main findings from the recent in silico characterization of its candidate surface proteins....

  16. Calreticulin: Roles in Cell-Surface Protein Expression

    Directory of Open Access Journals (Sweden)

    Yue Jiang

    2014-09-01

    Full Text Available In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins.

  17. POLY(N-VINYLPYRROLIDONE)-MODIFIED SURFACES REPEL PLASMA PROTEIN ADSORPTION

    Institute of Scientific and Technical Information of China (English)

    Xiao-li Liu; Zhao-qiang Wu; Dan Li; Hong Chen

    2012-01-01

    The present work aimed to study the interaction between plasma proteins and PVP-modified surfaces under more complex protein conditions.In the competitive adsorption of fibrinogen (Fg) and human serum albumin (HSA),the modified surfaces showed preferential adsorption of HSA.In 100% plasma,the amount of Fg adsorbed onto PVP-modified surfaces was as low as 10 ng/cm2,suggesting the excellent protein resistance properties of the modified surfaces.In addition,immunoblots of proteins eluted from the modified surfaces after plasma contact confirmed that PVP-modified surfaces can repel most plasma proteins,especially proteins that play important roles in the process of blood coagulation.

  18. Identification of surface proteins in Enterococcus faecalis V583

    Directory of Open Access Journals (Sweden)

    Eijsink Vincent GH

    2011-03-01

    Full Text Available Abstract Background Surface proteins are a key to a deeper understanding of the behaviour of Gram-positive bacteria interacting with the human gastro-intestinal tract. Such proteins contribute to cell wall synthesis and maintenance and are important for interactions between the bacterial cell and the human host. Since they are exposed and may play roles in pathogenicity, surface proteins are interesting targets for drug design. Results Using methods based on proteolytic "shaving" of bacterial cells and subsequent mass spectrometry-based protein identification, we have identified surface-located proteins in Enterococcus faecalis V583. In total 69 unique proteins were identified, few of which have been identified and characterized previously. 33 of these proteins are predicted to be cytoplasmic, whereas the other 36 are predicted to have surface locations (31 or to be secreted (5. Lipid-anchored proteins were the most dominant among the identified surface proteins. The seemingly most abundant surface proteins included a membrane protein with a potentially shedded extracellular sulfatase domain that could act on the sulfate groups in mucin and a lipid-anchored fumarate reductase that could contribute to generation of reactive oxygen species. Conclusions The present proteome analysis gives an experimental impression of the protein landscape on the cell surface of the pathogenic bacterium E. faecalis. The 36 identified secreted (5 and surface (31 proteins included several proteins involved in cell wall synthesis, pheromone-regulated processes, and transport of solutes, as well as proteins with unknown function. These proteins stand out as interesting targets for further investigation of the interaction between E. faecalis and its environment.

  19. RPE cell surface proteins in normal and dystrophic rats

    Energy Technology Data Exchange (ETDEWEB)

    Clark, V.M.; Hall, M.O.

    1986-02-01

    Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE.

  20. Adsorption of HP Lattice Proteins on Patterned Surfaces

    Science.gov (United States)

    Wilson, Matthew; Shi, Guangjie; Landau, David P.; Li, Ying Wai; Wuest, Thomas

    2014-03-01

    The HP lattice model[2] is a course-grained, yet useful tool for modeling protein sequences where amino acids are treated as either hydrophobic (H) or polar (P) monomers. With the use of Wang-Landau sampling and an efficient set of Monte-Carlo moves[3], HP lattice proteins adsorbed on patterned surfaces are studied. Each substrate is modeled as a periodically bounded pattern of lattice sites that interact with either H or P monomers in the lattice protein, where the energy contributions of the surface are determined by assigned coupling strengths. By analyzing energy degeneracies, along with the thermodynamic and structural quantities of the protein, both the protein folding and surface adsorption can be observed. The adsorption behavior of the lattice proteins on patterned surfaces will be compared to those interacting with uniform surfaces. Research supported by NSF.

  1. Structures of multidomain proteins adsorbed on hydrophobic interaction chromatography surfaces.

    Science.gov (United States)

    Gospodarek, Adrian M; Sun, Weitong; O'Connell, John P; Fernandez, Erik J

    2014-12-05

    In hydrophobic interaction chromatography (HIC), interactions between buried hydrophobic residues and HIC surfaces can cause conformational changes that interfere with separations and cause yield losses. This paper extends our previous investigations of protein unfolding in HIC chromatography by identifying protein structures on HIC surfaces under denaturing conditions and relating them to solution behavior. The thermal unfolding of three model multidomain proteins on three HIC surfaces of differing hydrophobicities was investigated with hydrogen exchange mass spectrometry (HXMS). The data were analyzed to obtain unfolding rates and Gibbs free energies for unfolding of adsorbed proteins. The melting temperatures of the proteins were lowered, but by different amounts, on the different surfaces. In addition, the structures of the proteins on the chromatographic surfaces were similar to the partially unfolded structures produced in the absence of a surface by temperature as well as by chemical denaturants. Finally, it was found that patterns of residue exposure to solvent on different surfaces at different temperatures can be largely superimposed. These findings suggest that protein unfolding on various HIC surfaces might be quantitatively related to protein unfolding in solution and that details of surface unfolding behavior might be generalized.

  2. Surface passivation for single-molecule protein studies.

    Science.gov (United States)

    Chandradoss, Stanley D; Haagsma, Anna C; Lee, Young Kwang; Hwang, Jae-Ho; Nam, Jwa-Min; Joo, Chirlmin

    2014-04-24

    Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

  3. Computational design of protein interactions: designing proteins that neutralize influenza by inhibiting its hemagglutinin surface protein

    Science.gov (United States)

    Fleishman, Sarel

    2012-02-01

    Molecular recognition underlies all life processes. Design of interactions not seen in nature is a test of our understanding of molecular recognition and could unlock the vast potential of subtle control over molecular interaction networks, allowing the design of novel diagnostics and therapeutics for basic and applied research. We developed the first general method for designing protein interactions. The method starts by computing a region of high affinity interactions between dismembered amino acid residues and the target surface and then identifying proteins that can harbor these residues. Designs are tested experimentally for binding the target surface and successful ones are affinity matured using yeast cell surface display. Applied to the conserved stem region of influenza hemagglutinin we designed two unrelated proteins that, following affinity maturation, bound hemagglutinin at subnanomolar dissociation constants. Co-crystal structures of hemagglutinin bound to the two designed binders were within 1Angstrom RMSd of their models, validating the accuracy of the design strategy. One of the designed proteins inhibits the conformational changes that underlie hemagglutinin's cell-invasion functions and blocks virus infectivity in cell culture, suggesting that such proteins may in future serve as diagnostics and antivirals against a wide range of pathogenic influenza strains. We have used this method to obtain experimentally validated binders of several other target proteins, demonstrating the generality of the approach. We discuss the combination of modeling and high-throughput characterization of design variants which has been key to the success of this approach, as well as how we have used the data obtained in this project to enhance our understanding of molecular recognition. References: Science 332:816 JMB, in press Protein Sci 20:753

  4. Deciphering fine molecular details of proteins' structure and function with a Protein Surface Topography (PST) method.

    Science.gov (United States)

    Koromyslova, Anna D; Chugunov, Anton O; Efremov, Roman G

    2014-04-28

    Molecular surfaces are the key players in biomolecular recognition and interactions. Nowadays, it is trivial to visualize a molecular surface and surface-distributed properties in three-dimensional space. However, such a representation trends to be biased and ambiguous in case of thorough analysis. We present a new method to create 2D spherical projection maps of entire protein surfaces and manipulate with them--protein surface topography (PST). It permits visualization and thoughtful analysis of surface properties. PST helps to easily portray conformational transitions, analyze proteins' properties and their dynamic behavior, improve docking performance, and reveal common patterns and dissimilarities in molecular surfaces of related bioactive peptides. This paper describes basic usage of PST with an example of small G-proteins conformational transitions, mapping of caspase-1 intersubunit interface, and intrinsic "complementarity" in the conotoxin-acetylcholine binding protein complex. We suggest that PST is a beneficial approach for structure-function studies of bioactive peptides and small proteins.

  5. Hydrophobic patches on the surfaces of protein structures

    NARCIS (Netherlands)

    Lijnzaad, P.; Berendsen, H.J.C.; Argos, P.

    1996-01-01

    A survey of hydrophobic patches on the surface of 112 soluble, monomeric proteins is presented, The largest patch on each individual protein averages around 400 Angstrom(2) but can range from 200 to 1,200 Angstrom(2). These areas are not correlated to the sizes of the proteins and only weakly to the

  6. VASCo: computation and visualization of annotated protein surface contacts

    Directory of Open Access Journals (Sweden)

    Thallinger Gerhard G

    2009-01-01

    Full Text Available Abstract Background Structural data from crystallographic analyses contain a vast amount of information on protein-protein contacts. Knowledge on protein-protein interactions is essential for understanding many processes in living cells. The methods to investigate these interactions range from genetics to biophysics, crystallography, bioinformatics and computer modeling. Also crystal contact information can be useful to understand biologically relevant protein oligomerisation as they rely in principle on the same physico-chemical interaction forces. Visualization of crystal and biological contact data including different surface properties can help to analyse protein-protein interactions. Results VASCo is a program package for the calculation of protein surface properties and the visualization of annotated surfaces. Special emphasis is laid on protein-protein interactions, which are calculated based on surface point distances. The same approach is used to compare surfaces of two aligned molecules. Molecular properties such as electrostatic potential or hydrophobicity are mapped onto these surface points. Molecular surfaces and the corresponding properties are calculated using well established programs integrated into the package, as well as using custom developed programs. The modular package can easily be extended to include new properties for annotation. The output of the program is most conveniently displayed in PyMOL using a custom-made plug-in. Conclusion VASCo supplements other available protein contact visualisation tools and provides additional information on biological interactions as well as on crystal contacts. The tool provides a unique feature to compare surfaces of two aligned molecules based on point distances and thereby facilitates the visualization and analysis of surface differences.

  7. Competitive protein adsorption to polymer surface from human serum

    DEFF Research Database (Denmark)

    Holmberg, Maria; Jensen, Karin Bagger Stibius; Larsen, Niels Bent;

    2008-01-01

    Surface modification by "soft" plasma polymerisation to obtain a hydrophilic and non-fouling polymer surface has been validated using radioactive labelling. Adsorption to unmodified and modified polymer surfaces, from both single protein and human serum solutions, has been investigated. By using ...

  8. Decorating microbes : surface display of proteins on Escherichia coli

    NARCIS (Netherlands)

    van Bloois, Edwin; Winter, Remko T.; Kolmar, Harald; Fraaije, Marco W.

    2011-01-01

    Bacterial surface display entails the presentation of recombinant proteins or peptides on the surface of bacterial cells. Escherichia coil is the most frequently used bacterial host for surface display and, as such, a variety of E. coil display systems have been described that primarily promote the

  9. SURF'S UP! – Protein classification by surface comparisons

    Indian Academy of Sciences (India)

    Joanna M Sasin; Adam Godzik; Janusz M Bujnicki

    2007-01-01

    Large-scale genome sequencing and structural genomics projects generate numerous sequences and structures for ‘hypothetical’ proteins without functional characterizations. Detection of homology to experimentally characterized proteins can provide functional clues, but the accuracy of homology-based predictions is limited by the paucity of tools for quantitative comparison of diverging residues responsible for the functional divergence. SURF’S UP! is a web server for analysis of functional relationships in protein families, as inferred from protein surface maps comparison according to the algorithm. It assigns a numerical score to the similarity between patterns of physicochemical features (charge, hydrophobicity) on compared protein surfaces. It allows recognizing clusters of proteins that have similar surfaces, hence presumably similar functions. The server takes as an input a set of protein coordinates and returns files with ``spherical coordinates” of proteins in a PDB format and their graphical presentation, a matrix with values of mutual similarities between the surfaces, and the unrooted tree that represents the clustering of similar surfaces, calculated by the neighbor-joining method. SURF’S UP! facilitates the comparative analysis of physicochemical features of the surface, which are the key determinants of the protein function. By concentrating on coarse surface features, SURF’S UP! can work with models obtained from comparative modelling. Although it is designed to analyse the conservation among homologs, it can also be used to compare surfaces of non-homologous proteins with different three-dimensional folds, as long as a functionally meaningful structural superposition is supplied by the user. Another valuable characteristic of our method is the lack of initial assumptions about the functional features to be compared. SURF’S UP! is freely available for academic researchers at http://asia.genesilico.pl/surfs_up/.

  10. ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

    Science.gov (United States)

    Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

    2010-10-01

    The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion.

  11. Hydration dynamics near a model protein surface

    Energy Technology Data Exchange (ETDEWEB)

    Russo, Daniela; Hura, Greg; Head-Gordon, Teresa

    2003-09-01

    The evolution of water dynamics from dilute to very high concentration solutions of a prototypical hydrophobic amino acid with its polar backbone, N-acetyl-leucine-methylamide (NALMA), is studied by quasi-elastic neutron scattering and molecular dynamics simulation for both the completely deuterated and completely hydrogenated leucine monomer. We observe several unexpected features in the dynamics of these biological solutions under ambient conditions. The NALMA dynamics shows evidence of de Gennes narrowing, an indication of coherent long timescale structural relaxation dynamics. The translational water dynamics are analyzed in a first approximation with a jump diffusion model. At the highest solute concentrations, the hydration water dynamics is significantly suppressed and characterized by a long residential time and a slow diffusion coefficient. The analysis of the more dilute concentration solutions takes into account the results of the 2.0M solution as a model of the first hydration shell. Subtracting the first hydration layer based on the 2.0M spectra, the translational diffusion dynamics is still suppressed, although the rotational relaxation time and residential time are converged to bulk-water values. Molecular dynamics analysis shows spatially heterogeneous dynamics at high concentration that becomes homogeneous at more dilute concentrations. We discuss the hydration dynamics results of this model protein system in the context of glassy systems, protein function, and protein-protein interfaces.

  12. Enhanced microcontact printing of proteins on nanoporous silica surface.

    Science.gov (United States)

    Blinka, Ellen; Loeffler, Kathryn; Hu, Ye; Gopal, Ashwini; Hoshino, Kazunori; Lin, Kevin; Liu, Xuewu; Ferrari, Mauro; Zhang, John X J

    2010-10-15

    We demonstrate porous silica surface modification, combined with microcontact printing, as an effective method for enhanced protein patterning and adsorption on arbitrary surfaces. Compared to conventional chemical treatments, this approach offers scalability and long-term device stability without requiring complex chemical activation. Two chemical surface treatments using functionalization with the commonly used 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were compared with the nanoporous silica surface on the basis of protein adsorption. The deposited thickness and uniformity of porous silica films were evaluated for fluorescein isothiocyanate (FITC)-labeled rabbit immunoglobulin G (R-IgG) protein printed onto the substrates via patterned polydimethlysiloxane (PDMS) stamps. A more complete transfer of proteins was observed on porous silica substrates compared to chemically functionalized substrates. A comparison of different pore sizes (4-6 nm) and porous silica thicknesses (96-200 nm) indicates that porous silica with 4 nm diameter, 57% porosity and a thickness of 96 nm provided a suitable environment for complete transfer of R-IgG proteins. Both fluorescence microscopy and atomic force microscopy (AFM) were used for protein layer characterizations. A porous silica layer is biocompatible, providing a favorable transfer medium with minimal damage to the proteins. A patterned immunoassay microchip was developed to demonstrate the retained protein function after printing on nanoporous surfaces, which enables printable and robust immunoassay detection for point-of-care applications.

  13. Enhanced microcontact printing of proteins on nanoporous silica surface

    Science.gov (United States)

    Blinka, Ellen; Loeffler, Kathryn; Hu, Ye; Gopal, Ashwini; Hoshino, Kazunori; Lin, Kevin; Liu, Xuewu; Ferrari, Mauro; Zhang, John X. J.

    2010-10-01

    We demonstrate porous silica surface modification, combined with microcontact printing, as an effective method for enhanced protein patterning and adsorption on arbitrary surfaces. Compared to conventional chemical treatments, this approach offers scalability and long-term device stability without requiring complex chemical activation. Two chemical surface treatments using functionalization with the commonly used 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were compared with the nanoporous silica surface on the basis of protein adsorption. The deposited thickness and uniformity of porous silica films were evaluated for fluorescein isothiocyanate (FITC)-labeled rabbit immunoglobulin G (R-IgG) protein printed onto the substrates via patterned polydimethlysiloxane (PDMS) stamps. A more complete transfer of proteins was observed on porous silica substrates compared to chemically functionalized substrates. A comparison of different pore sizes (4-6 nm) and porous silica thicknesses (96-200 nm) indicates that porous silica with 4 nm diameter, 57% porosity and a thickness of 96 nm provided a suitable environment for complete transfer of R-IgG proteins. Both fluorescence microscopy and atomic force microscopy (AFM) were used for protein layer characterizations. A porous silica layer is biocompatible, providing a favorable transfer medium with minimal damage to the proteins. A patterned immunoassay microchip was developed to demonstrate the retained protein function after printing on nanoporous surfaces, which enables printable and robust immunoassay detection for point-of-care applications.

  14. A coarse grain model for protein-surface interactions

    Science.gov (United States)

    Wei, Shuai; Knotts, Thomas A.

    2013-09-01

    The interaction of proteins with surfaces is important in numerous applications in many fields—such as biotechnology, proteomics, sensors, and medicine—but fundamental understanding of how protein stability and structure are affected by surfaces remains incomplete. Over the last several years, molecular simulation using coarse grain models has yielded significant insights, but the formalisms used to represent the surface interactions have been rudimentary. We present a new model for protein surface interactions that incorporates the chemical specificity of both the surface and the residues comprising the protein in the context of a one-bead-per-residue, coarse grain approach that maintains computational efficiency. The model is parameterized against experimental adsorption energies for multiple model peptides on different types of surfaces. The validity of the model is established by its ability to quantitatively and qualitatively predict the free energy of adsorption and structural changes for multiple biologically-relevant proteins on different surfaces. The validation, done with proteins not used in parameterization, shows that the model produces remarkable agreement between simulation and experiment.

  15. Arginine Inhibits Adsorption of Proteins on Polystyrene Surface

    Science.gov (United States)

    Shikiya, Yui; Tomita, Shunsuke; Arakawa, Tsutomu; Shiraki, Kentaro

    2013-01-01

    Nonspecific adsorption of protein on solid surfaces causes a reduction of concentration as well as enzyme inactivation during purification and storage. However, there are no versatile inhibitors of the adsorption between proteins and solid surfaces at low concentrations. Therefore, we examined additives for the prevention of protein adsorption on polystyrene particles (PS particles) as a commonly-used material for vessels such as disposable test tubes and microtubes. A protein solution was mixed with PS particles, and then adsorption of protein was monitored by the concentration and activity of protein in the supernatant after centrifugation. Five different proteins bound to PS particles through electrostatic, hydrophobic, and aromatic interactions, causing a decrease in protein concentration and loss of enzyme activity in the supernatant. Among the additives, including arginine hydrochloride (Arg), lysine hydrochloride, guanidine hydrochloride, NaCl, glycine, and glucose, Arg was most effective in preventing the binding of proteins to PS particles as well as activity loss. Moreover, even after the mixing of protein and PS particles, the addition of Arg caused desorption of the bound protein from PS particles. This study demonstrated a new function of Arg, which expands the potential for application of Arg to proteins. PMID:23967100

  16. Protein-surface interaction maps for ion-exchange chromatography.

    Science.gov (United States)

    Freed, Alexander S; Cramer, Steven M

    2011-04-05

    In this paper, protein-surface interaction maps were generated by performing coarse-grained protein-surface calculations. This approach allowed for the rapid determination of the protein-surface interaction energies at a range of orientations and distances. Interaction maps of lysozyme indicated that there was a contiguous series of orientations corresponding to several adjacent preferred binding regions on the protein surface. Examination of these orientations provided insight into the residues involved in surface interactions, which qualitatively agreed with the retention data for single-site mutants. Interaction maps of lysozyme single-site mutants were also generated and provided significant insight into why these variants exhibited significant differences in their chromatographic behavior. This approach was also employed to study the binding behavior of CspB and related mutants. The results indicated that, in addition to describing general trends in the data, these maps provided significant insight into retention data of the single-site mutants. In particular, subtle retention trends observed with the K12 and K13 mutants were well-described using this interaction map approach. Finally, the number of interaction points with energies stronger than -2 kcal/mol was shown to be able to semi-quantitatively predict the behavior of most of the mutants. This rapid approach for calculating protein-surface interaction maps is expected to facilitate future method development for separating closely related protein variants in ion-exchange systems.

  17. Protein-induced surface structuring in myelin membrane monolayers.

    Science.gov (United States)

    Rosetti, Carla M; Maggio, Bruno

    2007-12-15

    Monolayers prepared from myelin conserve all the compositional complexity of the natural membrane when spread at the air-water interface. They show a complex pressure-dependent surface pattern that, on compression, changes from the coexistence of two liquid phases to a viscous fractal phase embedded in a liquid phase. We dissected the role of major myelin protein components, myelin basic protein (MBP), and Folch-Lees proteolipid protein (PLP) as crucial factors determining the structural dynamics of the interface. By analyzing mixtures of a single protein with the myelin lipids we found that MBP and PLP have different surface pressure-dependent behaviors. MBP stabilizes the segregation of two liquid phases at low pressures and becomes excluded from the film under compression, remaining adjacent to the interface. PLP, on the contrary, organizes a fractal-like pattern at all surface pressures when included in a monolayer of the protein-free myelin lipids but it remains mixed in the MBP-induced liquid phase. The resultant surface topography and dynamics is regulated by combined near to equilibrium and out-of-equilibrium effects. PLP appears to act as a surface skeleton for the whole components whereas MBP couples the structuring to surface pressure-dependent extrusion and adsorption processes.

  18. A mass spectrometric-derived cell surface protein atlas.

    Directory of Open Access Journals (Sweden)

    Damaris Bausch-Fluck

    Full Text Available Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa. The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.

  19. Bap: a family of surface proteins involved in biofilm formation.

    Science.gov (United States)

    Lasa, Iñigo; Penadés, José R

    2006-03-01

    A group of surface proteins sharing several structural and functional features is emerging as an important element in the biofilm formation process of diverse bacterial species. The first member of this group of proteins was identified in a Staphylococcus aureus mastitis isolate and was named Bap (biofilm-associated protein). As common structural features, Bap-related proteins: (i) are present on the bacterial surface; (ii) show a high molecular weight; (iii) contain a core domain of tandem repeats; (iv) confer upon bacteria the capacity to form a biofilm; (v) play a relevant role in bacterial infectious processes; and (vi) can occasionally be contained in mobile elements. This review summarizes recent studies that have identified and assigned roles to Bap-related proteins in biofilm biology and virulence.

  20. Protein function annotation by local binding site surface similarity.

    Science.gov (United States)

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  1. Protein Adsorption to Surface Chemistry and Crystal Structure Modification of Titanium Surfaces

    Directory of Open Access Journals (Sweden)

    Ryo Jimbo

    2010-07-01

    Full Text Available Objectives: To observe the early adsorption of extracellular matrix and blood plasma proteins to magnesium-incorporated titanium oxide surfaces, which has shown superior bone response in animal models.Material and Methods: Commercially pure titanium discs were blasted with titanium dioxide (TiO2 particles (control, and for the test group, TiO2 blasted discs were further processed with a micro-arc oxidation method (test. Surface morphology was investigated by scanning electron microscopy, surface topography by optic interferometry, characterization by X-ray photoelectron spectroscopy (XPS, and by X-ray diffraction (XRD analysis. The adsorption of 3 different proteins (fibronectin, albumin, and collagen type I was investigated by an immunoblotting technique.Results: The test surface showed a porous structure, whereas the control surface showed a typical TiO2 blasted structure. XPS data revealed magnesium-incorporation to the anodic oxide film of the surface. There was no difference in surface roughness between the control and test surfaces. For the protein adsorption test, the amount of albumin was significantly higher on the control surface whereas the amount of fibronectin was significantly higher on the test surface. Although there was no significant difference, the test surface had a tendency to adsorb more collagen type I.Conclusions: The magnesium-incorporated anodized surface showed significantly higher fibronectin adsorption and lower albumin adsorption than the blasted surface. These results may be one of the reasons for the excellent bone response previously observed in animal studies.

  2. Surface plasmon resonance imaging for parallelized detection of protein biomarkers

    Science.gov (United States)

    Piliarik, Marek; Párová, Lucie; Vaisocherová, Hana; Homola, Jiří

    2009-05-01

    We report a novel high-throughput surface plasmon resonance (SPR) biosensor for rapid and parallelized detection of protein biomarkers. The biosensor is based on a high-performance SPR imaging sensor with polarization contrast and internal referencing which yields a considerably higher sensitivity and resolution than conventional SPR imaging systems (refractive index resolution 2 × 10-7 RIU). We combined the SPR imaging biosensor with microspotting to create an array of antibodies. DNA-directed protein immobilization was utilized for the spatially resolved attachment of antibodies. Using Human Chorionic Gonadotropin (hCG) as model protein biomarker, we demonstrated the potential for simultaneous detection of proteins in up to 100 channels.

  3. Protein adsorption to graphene surfaces controlled by chemical modification of the substrate surfaces.

    Science.gov (United States)

    Kamiya, Yasutaka; Yamazaki, Kenji; Ogino, Toshio

    2014-10-01

    We have investigated effects of the support substrate surfaces on properties of the attached graphene flakes by observing protein adsorption to the graphene surfaces on SiO2/Si substrates that are modified with self-assembled monolayers to control their hydrophilicity. Using atomic force microscopy operated in aqueous environment, we found that high-density clusters of agglomerated avidin molecules form on the graphene flakes in the areas supported by a hydrophobic substrate surface, whereas very low density of large avidin clusters form at the edge of graphene flakes in the area supported by a hydrophilic surface. These results demonstrate that hydrophilicity of the support surface affects hydrophilicity of the graphene surface also in aqueous environment and that surface modification of the support substrate is a useful technique to control protein adsorption phenomena on graphene surfaces for realization of high sensitive graphene biosensors.

  4. Surface (S)-layer proteins of Deinococcus radiodurans and their utility as vehicles for surface localization of functional proteins.

    Science.gov (United States)

    Misra, Chitra Seetharam; Basu, Bhakti; Apte, Shree Kumar

    2015-12-01

    The radiation resistant bacterium, Deinococcus radiodurans contains two major surface (S)-layer proteins, Hpi and SlpA. The Hpi protein was shown to (a) undergo specific in vivo cleavage, and (b) closely associate with the SlpA protein. Using a non-specific acid phosphatase from Salmonella enterica serovar Typhi, PhoN as a reporter, the Surface Layer Homology (SLH) domain of SlpA was shown to bind deinococcal peptidoglycan-containing cell wall sacculi. The association of SlpA with Hpi on one side and peptidoglycan on the other, localizes this protein in the 'interstitial' layer of the deinoccocal cell wall. Gene chimeras of hpi-phoN and slh-phoN were constructed to test efficacy of S-layer proteins, as vehicles for cell surface localization in D. radiodurans. The Hpi-PhoN protein localized exclusively in the membrane fraction, and displayed cell-based phosphatase activity in vivo. The SLH-PhoN, which localized to both cytosolic and membrane fractions, displayed in vitro activity but no cell-based in vivo activity. Hpi, therefore, emerged as an efficient surface localizing protein and can be exploited for suitable applications of this superbug.

  5. Identification of Uropathogenic Escherichia coli Surface Proteins by Shotgun Proteomics

    Science.gov (United States)

    Walters, Matthew S.; Mobley, Harry L.T.

    2009-01-01

    Uropathogenic Escherichia coli (UPEC) cause the majority of uncomplicated urinary tract infections in humans. In the process of identifying candidate antigens for a vaccine, two methods for the identification of the UPEC surface proteome during growth in human urine were investigated. The first approach utilized a protease to ‘shave’ surface-exposed peptides from the bacterial cell surface and identify them by mass spectrometry. Although this approach has been successfully applied to a Gram-positive pathogen, the adaptation to Gram-negative UPEC resulted in cytoplasmic protein contamination. In a more direct approach, whole-cell bacteria were labeled with a biotin tag to indicate surface-exposed peptides and two-dimensional liquid chromatography-tandem mass spectrometry (2-DLC-MS/MS) was used to identify proteins isolated from the outer membrane. This method discovered 25 predicted outer membrane proteins expressed by UPEC while growing in human urine. Nine of the 25 predicted outer membrane proteins were part of iron transport systems or putative iron-regulated virulence proteins, indicating the importance of iron acquisition during growth in urine. One of the iron transport proteins identified, Hma, appears to be a promising vaccine candidate is being further investigated. The method described here presents a system to rapidly identify the outer membrane proteome of bacteria, which may prove valuable in vaccine development. PMID:19426766

  6. Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

    Science.gov (United States)

    Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

    2015-10-01

    Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

  7. Interaction of Serum Proteins with Surface of Hemodialysis Fiber Membranes

    Science.gov (United States)

    Afrin, Rehana; Shirako, Yuji; Kishimoto, Kikuo; Ikai, Atsushi

    2012-08-01

    The poly(vinyl pyrrolidone)-covered hydrophilic surface of hollow-fiber membranes (fiber membrane, hereafter) for hemodialysis was mechanically probed using modified tips on an atomic force microscope (AFM) with covalent crosslinkers and several types of serum protein. The retraction part of many of the force extension (F-E) curves obtained with AFM tips coated with serum albumin had a long and smooth extension up to 200-300 nm indicating forced elongation of poly(vinyl pyrrolidone) chains. When fibrinogen-coated tips were used, long extension F-E curves up to 500 nm with multiple peaks were obtained in addition to smooth curves most likely reflecting the unfolding of fibrinogen molecules. The results indicated that individual polymer chains had a significant affinity toward serum proteins. The adhesion frequency of tips coated with serum proteins was lower on the poly(vinyl pyrrolidone) surface than on the uncoated hydrophobic polysulfone surface.

  8. Surface charge effects in protein adsorption on nanodiamonds.

    Science.gov (United States)

    Aramesh, M; Shimoni, O; Ostrikov, K; Prawer, S; Cervenka, J

    2015-03-19

    Understanding the interaction of proteins with charged diamond nanoparticles is of fundamental importance for diverse biomedical applications. Here we present a thorough study of protein binding, adsorption kinetics and structure on strongly positively (hydrogen-terminated) and negatively (oxygen-terminated) charged nanodiamond particles using a quartz crystal microbalance by dissipation and infrared spectroscopy. By using two model proteins (bovine serum albumin and lysozyme) of different properties (charge, molecular weight and rigidity), the main driving mechanism responsible for the protein binding to the charged nanoparticles was identified. Electrostatic interactions were found to dominate the protein adsorption dynamics, attachment and conformation. We developed a simple electrostatic model that can qualitatively explain the observed adsorption behaviour based on charge-induced pH modifications near the charged nanoparticle surfaces. Under neutral conditions, the local pH around the positively and negatively charged nanodiamonds becomes very high (11-12) and low (1-3) respectively, which has a profound impact on the protein charge, hydration and affinity to the nanodiamonds. Small proteins (lysozyme) were found to form multilayers with significant conformational changes to screen the surface charge, while larger proteins (albumin) formed monolayers with minor conformational changes. The findings of this study provide a step forward toward understanding and eventually predicting nanoparticle interactions with biofluids.

  9. A global optimization algorithm for protein surface alignment

    Directory of Open Access Journals (Sweden)

    Guerra Concettina

    2010-09-01

    Full Text Available Abstract Background A relevant problem in drug design is the comparison and recognition of protein binding sites. Binding sites recognition is generally based on geometry often combined with physico-chemical properties of the site since the conformation, size and chemical composition of the protein surface are all relevant for the interaction with a specific ligand. Several matching strategies have been designed for the recognition of protein-ligand binding sites and of protein-protein interfaces but the problem cannot be considered solved. Results In this paper we propose a new method for local structural alignment of protein surfaces based on continuous global optimization techniques. Given the three-dimensional structures of two proteins, the method finds the isometric transformation (rotation plus translation that best superimposes active regions of two structures. We draw our inspiration from the well-known Iterative Closest Point (ICP method for three-dimensional (3D shapes registration. Our main contribution is in the adoption of a controlled random search as a more efficient global optimization approach along with a new dissimilarity measure. The reported computational experience and comparison show viability of the proposed approach. Conclusions Our method performs well to detect similarity in binding sites when this in fact exists. In the future we plan to do a more comprehensive evaluation of the method by considering large datasets of non-redundant proteins and applying a clustering technique to the results of all comparisons to classify binding sites.

  10. Protein sequences bound to mineral surfaces persist into deep time

    Science.gov (United States)

    Demarchi, Beatrice; Hall, Shaun; Roncal-Herrero, Teresa; Freeman, Colin L; Woolley, Jos; Crisp, Molly K; Wilson, Julie; Fotakis, Anna; Fischer, Roman; Kessler, Benedikt M; Rakownikow Jersie-Christensen, Rosa; Olsen, Jesper V; Haile, James; Thomas, Jessica; Marean, Curtis W; Parkington, John; Presslee, Samantha; Lee-Thorp, Julia; Ditchfield, Peter; Hamilton, Jacqueline F; Ward, Martyn W; Wang, Chunting Michelle; Shaw, Marvin D; Harrison, Terry; Domínguez-Rodrigo, Manuel; MacPhee, Ross DE; Kwekason, Amandus; Ecker, Michaela; Kolska Horwitz, Liora; Chazan, Michael; Kröger, Roland; Thomas-Oates, Jane; Harding, John H; Cappellini, Enrico; Penkman, Kirsty; Collins, Matthew J

    2016-01-01

    Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated sequence (equivalent to ~16 Ma at a constant 10°C). DOI: http://dx.doi.org/10.7554/eLife.17092.001 PMID:27668515

  11. Comparison of surface and hydrogel-based protein microchips.

    Science.gov (United States)

    Zubtsov, D A; Savvateeva, E N; Rubina, A Yu; Pan'kov, S V; Konovalova, E V; Moiseeva, O V; Chechetkin, V R; Zasedatelev, A S

    2007-09-15

    Protein microchips are designed for high-throughput evaluation of the concentrations and activities of various proteins. The rapid advance in microchip technology and a wide variety of existing techniques pose the problem of unified approach to the assessment and comparison of different platforms. Here we compare the characteristics of protein microchips developed for quantitative immunoassay with those of antibodies immobilized on glass surfaces and in hemispherical gel pads. Spotting concentrations of antibodies used for manufacturing of microchips of both types and concentrations of antigen in analyte solution were identical. We compared the efficiency of antibody immobilization, the intensity of fluorescence signals for both direct and sandwich-type immunoassays, and the reaction-diffusion kinetics of the formation of antibody-antigen complexes for surface and gel-based microchips. Our results demonstrate higher capacity and sensitivity for the hydrogel-based protein microchips, while fluorescence saturation kinetics for the two types of microarrays was comparable.

  12. Preventing protein adsorption from a range of surfaces using an aqueous fish protein extract

    DEFF Research Database (Denmark)

    Pillai, Saju; Arpanaei, Ayyoob; Meyer, Rikke L.;

    2009-01-01

    We utilize an aqueous extract of fish proteins (FPs) as a coating for minimizing the adsorption of fibrinogen (Fg) and human serum albumin (HSA). The surfaces include stainless steel (SS), gold (Au), silicon dioxide (SiO2), and poly(styrene) (PS). The adsorption processes (kinetics and adsorbed...... Fg- or HSA-coated surfaces are exposed to the FPs, a significant increase in adsorbed mass occurs because the FPs are highly surface-active displacing Fg. Additionally, fluorescence microscopy confirms that very little Fg adsorbs to the FP-coated surfaces. We propose that FP coatings prevent protein...

  13. Protein sequences bound to mineral surfaces persist into deep time

    DEFF Research Database (Denmark)

    Demarchi, Beatrice; Hall, Shaun; Roncal-Herrero, Teresa;

    2016-01-01

    of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell......, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated...... sequence (equivalent to ~16 Ma at a constant 10°C)....

  14. Unique surface adsorption behaviors of serum proteins on chemically uniform and alternating surfaces

    Science.gov (United States)

    Song, Sheng

    With increasing interests of studying proteins adsorption on the surfaces with nanoscale features in biomedical field, it is crucial to have fundamental understandings on how the proteins are adsorbed on such a surface and what factors contribute to the driving forces of adsorption. Besides, exploring more available nanoscale templates would greatly offer more possibilities one could design surface bio-detection methods with favorable protein-surface interactions. Thus, to fulfill the purpose, the work in this dissertation has been made into three major sections. First, to probe the intermediate states which possibly exist between stable and unstable phases described in mean-field theory diagram, a solvent vapor annealing method is chosen to slowly induce the copolymer polystyrene-block-polyvinylpyridine (PS-b-PVP)'s both blocks undergoing micro-phase separations from initial spherical nanodomains into terminal cylindrical nanodomains. During this process, real time atomic force microscopy (AFM) has been conducted to capture other six intermediate states with different morphologies on the polymeric film surfaces. Secondly, upon recognizing each intermediate state, the solution of immunoglobulin gamma (IgG) proteins has been deposited on the surface and been rinsed off with buffer solution before the protein-bounded surface is imaged by AFM. It has been found IgG showing a strong adsorption preference on PS over P4VP block. Among all the six intermediate states, the proteins are almost exclusively adsorbed on PS nanodomains regardless the concentration and deposition time. Thirdly, a trinodular shape protein fibrinogen (Fg) is selected for investigating how geometry and surface charge of proteins would interplay with cylindrical nanodomains on a surface developed from Polystyrene -block-Poly-(methyl methacrylate) PS-b-PMMA. Also, Fg adsorptions on chemically homogeneous surfaces are included here to have a better contrast of showing how much difference it can make

  15. Conformal nanopatterning of extracellular matrix proteins onto topographically complex surfaces.

    Science.gov (United States)

    Sun, Yan; Jallerat, Quentin; Szymanski, John M; Feinberg, Adam W

    2015-02-01

    Our Patterning on Topography (PoT) printing technique enables fibronectin, laminin and other proteins to be applied to biomaterial surfaces in complex geometries that are inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface. Here, we used this method to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment.

  16. Protein-surface interactions on stimuli-responsive polymeric biomaterials.

    Science.gov (United States)

    Cross, Michael C; Toomey, Ryan G; Gallant, Nathan D

    2016-03-04

    Responsive surfaces: a review of the dependence of protein adsorption on the reversible volume phase transition in stimuli-responsive polymers. Specifically addressed are a widely studied subset: thermoresponsive polymers. Findings are also generalizable to other materials which undergo a similarly reversible volume phase transition. As of 2015, over 100,000 articles have been published on stimuli-responsive polymers and many more on protein-biomaterial interactions. Significantly, fewer than 100 of these have focused specifically on protein interactions with stimuli-responsive polymers. These report a clear trend of increased protein adsorption in the collapsed state compared to the swollen state. This control over protein interactions makes stimuli-responsive polymers highly useful in biomedical applications such as wound repair scaffolds, on-demand drug delivery, and antifouling surfaces. Outstanding questions are whether the protein adsorption is reversible with the volume phase transition and whether there is a time-dependence. A clear understanding of protein interactions with stimuli-responsive polymers will advance theoretical models, experimental results, and biomedical applications.

  17. Surface charge effects in protein adsorption on nanodiamonds

    Science.gov (United States)

    Aramesh, M.; Shimoni, O.; Ostrikov, K.; Prawer, S.; Cervenka, J.

    2015-03-01

    Understanding the interaction of proteins with charged diamond nanoparticles is of fundamental importance for diverse biomedical applications. Here we present a thorough study of protein binding, adsorption kinetics and structure on strongly positively (hydrogen-terminated) and negatively (oxygen-terminated) charged nanodiamond particles using a quartz crystal microbalance by dissipation and infrared spectroscopy. By using two model proteins (bovine serum albumin and lysozyme) of different properties (charge, molecular weight and rigidity), the main driving mechanism responsible for the protein binding to the charged nanoparticles was identified. Electrostatic interactions were found to dominate the protein adsorption dynamics, attachment and conformation. We developed a simple electrostatic model that can qualitatively explain the observed adsorption behaviour based on charge-induced pH modifications near the charged nanoparticle surfaces. Under neutral conditions, the local pH around the positively and negatively charged nanodiamonds becomes very high (11-12) and low (1-3) respectively, which has a profound impact on the protein charge, hydration and affinity to the nanodiamonds. Small proteins (lysozyme) were found to form multilayers with significant conformational changes to screen the surface charge, while larger proteins (albumin) formed monolayers with minor conformational changes. The findings of this study provide a step forward toward understanding and eventually predicting nanoparticle interactions with biofluids.Understanding the interaction of proteins with charged diamond nanoparticles is of fundamental importance for diverse biomedical applications. Here we present a thorough study of protein binding, adsorption kinetics and structure on strongly positively (hydrogen-terminated) and negatively (oxygen-terminated) charged nanodiamond particles using a quartz crystal microbalance by dissipation and infrared spectroscopy. By using two model proteins

  18. Quantifying protein-protein interactions in the ubiquitin pathway by surface plasmon resonance

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Gordon, Colin

    2005-01-01

    The commercial availability of instruments, such as Biacore, that are capable of monitoring surface plasmon resonance (SPR) has greatly simplified the quantification of protein-protein interactions. Already, this technique has been used for some studies of the ubiquitin-proteasome system. Here we...

  19. Surface energetics and protein-protein interactions: analysis and mechanistic implications

    Science.gov (United States)

    Peri, Claudio; Morra, Giulia; Colombo, Giorgio

    2016-04-01

    Understanding protein-protein interactions (PPI) at the molecular level is a fundamental task in the design of new drugs, the prediction of protein function and the clarification of the mechanisms of (dis)regulation of biochemical pathways. In this study, we use a novel computational approach to investigate the energetics of aminoacid networks located on the surface of proteins, isolated and in complex with their respective partners. Interestingly, the analysis of individual proteins identifies patches of surface residues that, when mapped on the structure of their respective complexes, reveal regions of residue-pair couplings that extend across the binding interfaces, forming continuous motifs. An enhanced effect is visible across the proteins of the dataset forming larger quaternary assemblies. The method indicates the presence of energetic signatures in the isolated proteins that are retained in the bound form, which we hypothesize to determine binding orientation upon complex formation. We propose our method, BLUEPRINT, as a complement to different approaches ranging from the ab-initio characterization of PPIs, to protein-protein docking algorithms, for the physico-chemical and functional investigation of protein-protein interactions.

  20. Sputter deposited bioceramic coatings: surface characterisation and initial protein adsorption studies using surface-MALDI-MS

    DEFF Research Database (Denmark)

    Boyd, A. R.; Burke, G. A.; Duffy, H.

    2011-01-01

    Protein adsorption onto calcium phosphate (Ca–P) bioceramics utilised in hard tissue implant applications has been highlighted as one of the key events that influences the subsequent biological response, in vivo. This work reports on the use of surface-matrix assisted laser desorption ionisation...... to a combination of growth factors and lipoproteins present in serum. From the data obtained here it is evident that surface-MALDI-MS has significant utility as a tool for studying the dynamic nature of protein adsorption onto the surfaces of bioceramic coatings, which most likely plays a significant role...

  1. Factor H-related proteins determine complement-activating surfaces.

    Science.gov (United States)

    Józsi, Mihály; Tortajada, Agustin; Uzonyi, Barbara; Goicoechea de Jorge, Elena; Rodríguez de Córdoba, Santiago

    2015-06-01

    Complement factor H-related proteins (FHRs) are strongly associated with different diseases involving complement dysregulation, which suggests a major role for these proteins regulating complement activation. Because FHRs are evolutionarily and structurally related to complement inhibitor factor H (FH), the initial assumption was that the FHRs are also negative complement regulators. Whereas weak complement inhibiting activities were originally reported for these molecules, recent developments indicate that FHRs may enhance complement activation, with important implications for the role of these proteins in health and disease. We review these findings here, and propose that FHRs represent a complex set of surface recognition molecules that, by competing with FH, provide improved discrimination of self and non-self surfaces and play a central role in determining appropriate activation of the complement pathway.

  2. Surface-protein interactions on different stainless steel grades: effects of protein adsorption, surface changes and metal release.

    Science.gov (United States)

    Hedberg, Y; Wang, X; Hedberg, J; Lundin, M; Blomberg, E; Wallinder, I Odnevall

    2013-04-01

    Implantation using stainless steels (SS) is an example where an understanding of protein-induced metal release from SS is important when assessing potential toxicological risks. Here, the protein-induced metal release was investigated for austenitic (AISI 304, 310, and 316L), ferritic (AISI 430), and duplex (AISI 2205) grades in a phosphate buffered saline (PBS, pH 7.4) solution containing either bovine serum albumin (BSA) or lysozyme (LSZ). The results show that both BSA and LSZ induce a significant enrichment of chromium in the surface oxide of all stainless steel grades. Both proteins induced an enhanced extent of released iron, chromium, nickel and manganese, very significant in the case of BSA (up to 40-fold increase), whereas both proteins reduced the corrosion resistance of SS, with the reverse situation for iron metal (reduced corrosion rates and reduced metal release in the presence of proteins). A full monolayer coverage is necessary to induce the effects observed.

  3. Proteomic inventory of "anchorless" proteins on the colon adenocarcinoma cell surface.

    NARCIS (Netherlands)

    Tjalsma, H.; Pluk, W.J.G.; Heuvel, L.P.W.J. van den; Peters, W.H.M.; Roelofs, R.H.W.M.; Swinkels, D.W.

    2006-01-01

    Surface proteins play important pathophysiological roles in health and disease, and accumulating proteomics-based studies suggest that several "non-membrane" proteins are sorted to the cell surface by unconventional mechanisms. Importantly, these proteins may comprise attractive therapeutic targets

  4. Surface display of proteins by Gram-negative bacterial autotransporters

    Directory of Open Access Journals (Sweden)

    Mourez Michael

    2006-06-01

    Full Text Available Abstract Expressing proteins of interest as fusions to proteins of the bacterial envelope is a powerful technique with many biotechnological and medical applications. Autotransporters have recently emerged as a good tool for bacterial surface display. These proteins are composed of an N-terminal signal peptide, followed by a passenger domain and a translocator domain that mediates the outer membrane translocation of the passenger. The natural passenger domain of autotransporters can be replaced by heterologous proteins that become displayed at the bacterial surface by the translocator domain. The simplicity and versatility of this system has made it very attractive and it has been used to display functional enzymes, vaccine antigens as well as polypeptides libraries. The recent advances in the study of the translocation mechanism of autotransporters have raised several controversial issues with implications for their use as display systems. These issues include the requirement for the displayed polypeptides to remain in a translocation-competent state in the periplasm, the requirement for specific signal sequences and "autochaperone" domains, and the influence of the genetic background of the expression host strain. It is therefore important to better understand the mechanism of translocation of autotransporters in order to employ them to their full potential. This review will focus on the recent advances in the study of the translocation mechanism of autotransporters and describe practical considerations regarding their use for bacterial surface display.

  5. Sputter deposited bioceramic coatings: surface characterisation and initial protein adsorption studies using surface-MALDI-MS.

    Science.gov (United States)

    Boyd, A R; Burke, G A; Duffy, H; Holmberg, M; O' Kane, C; Meenan, B J; Kingshott, P

    2011-01-01

    Protein adsorption onto calcium phosphate (Ca-P) bioceramics utilised in hard tissue implant applications has been highlighted as one of the key events that influences the subsequent biological response, in vivo. This work reports on the use of surface-matrix assisted laser desorption ionisation mass spectrometry (Surface-MALDI-MS) as a technique for the direct detection of foetal bovine serum (FBS) proteins adsorbed to hybrid calcium phosphate/titanium dioxide surfaces produced by a novel radio frequency (RF) magnetron sputtering method incorporating in situ annealing between 500°C and 700°C during deposition. XRD and XPS analysis indicated that the coatings produced at 700°C were hybrid in nature, with the presence of Ca-P and titanium dioxide clearly observed in the outer surface layer. In addition to this, the Ca/P ratio was seen to increase with increasing annealing temperature, with values of between 2.0 and 2.26 obtained for the 700°C samples. After exposure to FBS solution, surface-MALDI-MS indicated that there were significant differences in the protein patterns as shown by unique peaks detected at masses below 23.1 kDa for the different surfaces. These adsorbates were assigned to a combination of growth factors and lipoproteins present in serum. From the data obtained here it is evident that surface-MALDI-MS has significant utility as a tool for studying the dynamic nature of protein adsorption onto the surfaces of bioceramic coatings, which most likely plays a significant role in subsequent bioactivity of the materials.

  6. Protein antifouling and fouling-release in perfluoropolyether surfaces

    Science.gov (United States)

    Molena, Elena; Credi, Caterina; De Marco, Carmela; Levi, Marinella; Turri, Stefano; Simeone, Giovanni

    2014-08-01

    Perfluoropolyether polymers have been described as high performance fouling-release materials for marine coatings. Moreover, they have a good potential to be exploited in the biomedical field too. In this article several perfuoropolyether photopolymers were characterized in terms of surface and mechanical properties outlining the relationship between these properties and the polymer molecular structure. In particular the anti-fouling and fouling-release performances, evaluated using Bovine Serum Albumin as testing protein, was correlated to other material properties, like a parameter considering both surface tension components γ and elastic modulus E. A good correlation between the anti-fouling/fouling-release of perfluoropolyethers and (E*γpolar)1/2 can actually be established. Our results show that perfluoropolyether photopolymers are good protein anti-fouling/fouling-release materials.

  7. Structural changes in proteins resulting from homomolecular exchange at solid surfaces

    NARCIS (Netherlands)

    Giacomelli, C.E.; Norde, W.

    2003-01-01

    The overall protein adsorption process comprises various steps or stages: transport of the protein from the bulk solution into the interfacial region, attachment of the protein at the sorbent surface and relaxation of the protein on the surface, detachment from the surface, and transport back into t

  8. Characterization of the Eimeria maxima sporozoite surface protein IMP1.

    Science.gov (United States)

    Jenkins, M C; Fetterer, R; Miska, K; Tuo, W; Kwok, O; Dubey, J P

    2015-07-30

    The purpose of this study was to characterize Eimeria maxima immune-mapped protein 1 (IMP1) that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12h of sporulation with a considerable downregulation thereafter. Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence. The E. maxima (APU-1) IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors. Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence. Antisera specific for rEmaxIMP1 identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting. Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of E. maxima sporozoites, with negligible staining of merozoite stages. Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 h post-infection, and negligible staining thereafter. The expression of IMP1 during early stages of in vivo development and its location on the sporozoite surface may explain in part the immunoprotective effect of this protein against E. maxima infection.

  9. Ultrafast Hydration Dynamics Probed by Tryptophan at Protein Surface and Protein-DNA Interface

    Science.gov (United States)

    Qin, Yangzhong

    As we all live in a special water planet Earth, the significance of water to life has been universally recognized. The reason why water is so important to life has intrigued many researchers. This dissertation will focus on the ultrafast dynamics of protein surface water and protein-DNA interfacial water which have direct importance to the protein structure and function. Using tryptophan as an intrinsic fluorescence probe, combined with site-directed mutagenesis and ultrafast fluorescence up-conversion spectroscopy, we can achieve single residue spatial resolution and femtosecond temporal resolution. We can also precisely determine the local hydration water dynamics by monitoring the Stokes shift of tryptophan one at a time. Previously, the protein surface hydration has been extensively studied by our group. In this thesis, we will provide more details on the methods we are using to extract the hydration dynamics, and also validate our methods from both experimental and theoretical perspectives. To further interrogate the interfacial water hydration dynamics relative to the protein surface hydration, we studied two DNA polymerases: DNA Polymerase IV (Dpo4) and DNA Polymerase Beta (Pol beta). Both proteins show typical surface hydration pattern with three distinct time components including: (i) the ultrafast sub-picosecond component reflects the bulk type water motion; (ii) a few picoseconds component shows the inner water relaxation mainly corresponding to the local libration and reorientation; (iii) the tens to hundred picoseconds component represents the water-protein coupled motion involving the whole water network reorganization. Dpo4, a loosely DNA binding protein, exhibits very flexible interfacial water which resembles its surface water yet with a significantly reduced ultrafast component. Such dynamic interfacial water not only maintains interfacial flexibility, but also contributes to the low fidelity of the protein. In contrast to the Dpo4, pol beta

  10. Biological properties of Lactobacillus surface proteins 

    Directory of Open Access Journals (Sweden)

    Barbara Buda

    2013-04-01

    Full Text Available Lactobacillus, a genus of Gram-positive bacteria, includes many strains of probiotic microflora. Probiotics, by definition, are living microorganisms that exert beneficial effects on the host organism. The morphology and physiology of the Lactobacillus bacterial genus are described. The structure of the cell wall of Gram-positive bacteria is discussed. The surface S-layer of Lactobacillus composed of proteins (SLP with low molecular mass is presented. Cell surface proteins participating in the regulation of growth and survival of the intestinal epithelium cells are characterized. The influence of stress factors such as increased temperature, pH, and enzymes of gastric and pancreatic juice on SLP expression is described. The ability of binding of heavy metal ions by S-layer proteins is discussed. The characteristics of these structures, including the ability to adhere to epithelial cells, and the inhibition of invasion of pathogenic microflora of type Shigella, Salmonella, Escherichia coli and Clostridium and their toxins, are presented. 

  11. [Surface proteins of bacteria of the genus Bifidobacterium].

    Science.gov (United States)

    Dylus, Ewa; Buda, Barbara; Górska-Frączek, Sabina; Brzozowska, Ewa; Gamian, Andrzej

    2013-05-13

    Beneficial effects due to the presence of probiotic bacteria of the genus Bifidobacterium in the human intestinal tract are still an interesting object of study. So far activities have been confirmed of bifidobacteria in stimulation of the host immune system, stimulation of tumor cell apoptosis, improvement of bowel motility, alleviation of symptoms of lactose intolerance, cholesterol lowering capacity, prevention and treatment of diarrhea and irritable bowel syndrome, alleviation of allergy or atopic dermatitis, maintenance of homeostasis of the intestine, and stimulation of the development of normal intestinal microflora in infants. A multitude of therapeutic properties encourages researchers to investigate the possibility of using the potential of Bifidobacterium in the prevention and treatment of other conditions such as rheumatoid arthritis and depression. Although it is known that the beneficial effects are due to intestinal mucosal colonization by these bacteria, the cell components responsible for the colonization are still not determined. In addition to the beneficial effects of probiotic administration, there were also negative effects including sepsis. Therefore research has been directed to identify specific components of Bifidobacterium responsible for probiotic effects. Currently researchers are focused on identifying, isolating and evaluating the properties of surface proteins that are probably involved in the adhesion of bacterial cells to the intestinal epithelium, improving colonization. This paper is an overview of current knowledge on Bifidobacterium surface proteins. The ways of transport and anchoring proteins in Gram-positive bacterial cells, the assembly of cell wall, and a description of the genus Bifidobacterium are presented.

  12. Iron-regulated surface determinant (Isd) proteins of Staphylococcus lugdunensis.

    Science.gov (United States)

    Zapotoczna, Marta; Heilbronner, Simon; Speziale, Pietro; Foster, Timothy J

    2012-12-01

    Staphylococcus lugdunensis is the only coagulase-negative Staphylococcus species with a locus encoding iron-regulated surface determinant (Isd) proteins. In Staphylococcus aureus, the Isd proteins capture heme from hemoglobin and transfer it across the wall to a membrane-bound transporter, which delivers it into the cytoplasm, where heme oxygenases release iron. The Isd proteins of S. lugdunensis are expressed under iron-restricted conditions. We propose that S. lugdunensis IsdB and IsdC proteins perform the same functions as those of S. aureus. S. lugdunensis IsdB is the only hemoglobin receptor within the isd locus. It specifically binds human hemoglobin with a dissociation constant (K(d)) of 23 nM and transfers heme on IsdC. IsdB expression promotes bacterial growth in an iron-limited medium containing human hemoglobin but not mouse hemoglobin. This correlates with weak binding of IsdB to mouse hemoglobin in vitro. Unlike IsdB and IsdC, the proteins IsdJ and IsdK are not sorted to the cell wall in S. lugdunensis. In contrast, IsdJ expressed in S. aureus and Lactococcus lactis is anchored to peptidoglycan, suggesting that S. lugdunensis sortases may differ in signal recognition or could be defective. IsdJ and IsdK are present in the culture supernatant, suggesting that they could acquire heme from the external milieu. The IsdA protein of S. aureus protects bacteria from bactericidal lipids due to its hydrophilic C-terminal domain. IsdJ has a similar region and protected S. aureus and L. lactis as efficiently as IsdA but, possibly due to its location, was less effective in its natural host.

  13. Molecular cartography of proteins: surface relief analysis of the calf eye lens protein gamma-crystallin.

    Science.gov (United States)

    Chirgadze, Y u; Kurochkina, N; Nikonov, S

    1989-11-01

    Methods of calculating the protein molecular surface and different map representations are described. The maps are obtained by projection of the space-filling molecular model on the surface of the ellipsoid of inertia. A new approach to surface analysis is proposed which is based on the use of three general maps: an identification map with all residues outlined, a surface relief map and a coloured map with a specific colour for each of the surface atoms. Superposition of these maps greatly simplifies molecular surface analysis. The usefulness of such an approach has been demonstrated by the study of the relief of the calf eye lens protein gamma-crystallin II. Protrusions of the relief have been shown to be occupied generally by charged residues, but in some cases by the hydrophobic ones. It is interesting to note that in crystal medium the protruding residues are involved, in the majority of cases, in intermolecular contacts. The protruding regions have been found to be pseudosymmetrical to each other in accordance with the two-fold rotation axis of the molecule. However, the colours of these regions, i.e. the atoms of the corresponding side chains, differ greatly.

  14. Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae.

    Science.gov (United States)

    Hammerschmidt, S; Bethe, G; Remane, P H; Chhatwal, G S

    1999-04-01

    Lactoferrin (Lf), an iron-sequestering glycoprotein, predominates in mucosal secretions, where the level of free extracellular iron (10(-18) M) is not sufficient for bacterial growth. This represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. In this study we were able to show that Streptococcus pneumoniae specifically recognizes and binds the iron carrier protein human Lf (hLf). Pretreatment of pneumococci with proteases reduced hLf binding significantly, indicating that the hLf receptor is proteinaceous. Binding assays performed with 63 clinical isolates belonging to different serotypes showed that 88% of the tested isolates interacted with hLf. Scatchard analysis showed the existence of two hLf-binding proteins with dissociation constants of 5.7 x 10(-8) and 2.74 x 10(-7) M. The receptors were purified by affinity chromatography, and internal sequence analysis revealed that one of the S. pneumoniae proteins was homologous to pneumococcal surface protein A (PspA). The function of PspA as an hLf-binding protein was confirmed by the ability of purified PspA to bind hLf and to competitively inhibit hLf binding to pneumococci. S. pneumoniae may use the hLf-PspA interaction to overcome the iron limitation at mucosal surfaces, and this might represent a potential virulence mechanism.

  15. Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early

    Science.gov (United States)

    2013-07-01

    10-1-0422 TITLE: Targeting Cell Surface Proteins in Molecular Photoacoustic Imaging to Detect Ovarian Cancer Early PRINCIPAL...DATES COVERED 1 July 2010 - 30 June 2013 4. TITLE AND SUBTITLE Targeting Cell Surface Proteins in Molecular 5a. CONTRACT NUMBER Photoacoustic ...upon request). Aim 2) Prioritize ovarian cancer-associated surface proteins for their utility as molecular photoacoustic imaging targets and

  16. Proteomics and glycoproteomics of pluripotent stem-cell surface proteins.

    Science.gov (United States)

    Sun, Bingyun

    2015-03-01

    Pluripotent stem cells are a unique cell type with promising potential in regenerative and personalized medicine. Yet the difficulty to understand and coax their seemingly stochastic differentiation and spontaneous self-renewal have largely limited their clinical applications. A call has been made by numerous researchers for a better characterization of surface proteins on these cells, in search of biomarkers that can dictate developmental stages and lineage specifications, and can help formulate mechanistic insight of stem-cell fate choices. In the past two decades, proteomics has gained significant recognition in profiling surface proteins at high throughput. This review will summarize the impact of these studies on stem-cell biology, and discuss the used proteomic techniques. A systematic comparison of all the techniques and their results is also attempted here to help reveal pros, cons, and the complementarity of the existing methods. This awareness should assist in selecting suitable strategies for stem-cell related research, and shed light on technical improvements that can be explored in the future.

  17. Surface modification of graphene nanopores for protein translocation

    Science.gov (United States)

    Shan, Y. P.; Tiwari, P. B.; Krishnakumar, P.; Vlassiouk, I.; Li, W.Z.; Wang, X.W.; Darici, Y.; Lindsay, S.M.; Wang, H. D.; Smirnov, S.; He, J.

    2014-01-01

    Studies of DNA translocation through graphene nanopores have revealed their potential for DNA sequencing. Here we report a study of protein translocation through chemically modified graphene nanopores. A transmission electron microscope (TEM) was used to cut nanopores with diameters between 5-20 nm in multilayer graphene prepared by chemical vapor deposition (CVD). After oxygen plasma treatment, the dependence of the measured ionic current on salt concentration and pH was consistent with a small surface charge induced by the formation of carboxyl groups. While translocation of gold nanoparticles (10 nm) was readily detected through such treated pores of a larger diameter, translocation of protein ferritin was not observed either for oxygen plasma treated pores, or for pores modified with mercaptohexadecanoic acid. Ferritin translocation events were reliably observed after the pores were modified with the phospholipid-PEG (DPPE-PEG750) amphiphile. The ion current signature of translocation events was complex, suggesting that a series of interactions between the protein and pore occur during the process. PMID:24231385

  18. Surface proteins of bacteria of the genus Bifidobacterium 

    Directory of Open Access Journals (Sweden)

    Ewa Dylus

    2013-05-01

    Full Text Available Beneficial effects due to the presence of probiotic bacteria of the genus Bifidobacterium in the human intestinal tract are still an interesting object of study. So far activities have been confirmed of bifidobacteria in stimulation of the host immune system, stimulation of tumor cell apoptosis, improvement of bowel motility, alleviation of symptoms of lactose intolerance, cholesterol lowering capacity, prevention and treatment of diarrhea and irritable bowel syndrome, alleviation of allergy or atopic dermatitis, maintenance of homeostasis of the intestine, and stimulation of the development of normal intestinal microflora in infants. A multitude of therapeutic properties encourages researchers to investigate the possibility of using the potential of Bifidobacterium in the prevention and treatment of other conditions such as rheumatoid arthritis and depression. Although it is known that the beneficial effects are due to intestinal mucosal colonization by these bacteria, the cell components responsible for the colonization are still not determined. In addition to the beneficial effects of probiotic administration, there were also negative effects including sepsis. Therefore research has been directed to identify specific components of Bifidobacterium responsible for probiotic effects. Currently researchers are focused on identifying, isolating and evaluating the properties of surface proteins that are probably involved in the adhesion of bacterial cells to the intestinal epithelium, improving colonization. This paper is an overview of current knowledge on Bifidobacterium surface proteins. The ways of transport and anchoring proteins in Gram-positive bacterial cells, the assembly of cell wall, and a description of the genus Bifidobacterium are presented.

  19. Surface Proteins of Streptococcus agalactiae and Related Proteins in Other Bacterial Pathogens

    OpenAIRE

    Lindahl, Gunnar; Stålhammar-Carlemalm, Margaretha; Areschoug, Thomas

    2005-01-01

    Streptococcus agalactiae (group B Streptococcus) is the major cause of invasive bacterial disease, including meningitis, in the neonatal period. Although prophylactic measures have contributed to a substantial reduction in the number of infections, development of a vaccine remains an important goal. While much work in this field has focused on the S. agalactiae polysaccharide capsule, which is an important virulence factor that elicits protective immunity, surface proteins have received incre...

  20. Nanoporous titanium surfaces for sustained elution of proteins and antibiotics.

    Directory of Open Access Journals (Sweden)

    Amirhossein Ketabchi

    Full Text Available Current medically relevant metals for prosthetic reconstructions enjoy a relatively good success rate, but their performance drops significantly in patients with compromised health status, and post-surgical infections still remain an important challenge. To address these problems, different nanotechnology-based strategies have been exploited to create implantable metals with an enhanced bioactivity and antibacterial capacities. Among these, oxidative nanopatterning has emerged as a very effective approach to engender nanoporous surfaces that stimulate and guide the activity of adhering cells. The resulting nanoporosity is also attractive because it offers nanoconfined volumes that can be exploited to load bioactive compounds and modulate their release over time. Such extended elution is needed since a single exposure to growth factors and/or antibiotics, for instance, may not be adequate to further sustain bone regeneration and/or to counteract bacterial colonization. In this article, we assessed the capacities of nanoporous titanium surfaces generated by oxidative nanopatterning to provide controlled and sustained elution of proteins and antibiotic molecules. To this end, we have selected bovine serum albumin (BSA and vancomycin to reflect commonly used compounds, and investigated their adsorption and elution by Fourier-transform infrared (FT-IR and ultraviolet-visible (UV-VIS spectroscopy. Our results demonstrate that while the elution of albumin is not significantly affected by the nanoporosity, in the case of vancomycin, nanoporous surfaces provided an extended release. These findings were successively correlated to the establishment of interactions with the surface and physical-entrapment effects exerted by the nanopores, ultimately highlighting their synergistic contribution to the release profiles and thus their importance in the design of nanostructured eluting platforms for applications in medicine.

  1. Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.

    Science.gov (United States)

    Close, Devin W; Paul, Craig Don; Langan, Patricia S; Wilce, Matthew C J; Traore, Daouda A K; Halfmann, Randal; Rocha, Reginaldo C; Waldo, Geoffery S; Payne, Riley J; Rucker, Joseph B; Prescott, Mark; Bradbury, Andrew R M

    2015-07-01

    In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.

  2. SURFACE MODIFICATION OF TITANIUM FILMS WITH SODIUM ION IMPLANTATION: SURFACE PROPERTIES AND PROTEIN ADSORPTION

    Institute of Scientific and Technical Information of China (English)

    K. Y. Cai

    2007-01-01

    Sodium implanted titanium films with different ion doses were characterized to correlate their ion implantation parameters. Native titanium films and ion implanted titanium films were characterized with combined techniques of X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and light microscopy (LM). The surface presented increased sodium concentration on treated titanium films with ion dose increasing, except for the group with the highest ion dose of 4× 1017 ions/cm2. XPS depth profiling displayed that sodium entered titanium film around 25-50 nm depth depending on its implantation ion dose. AFM characterization showed that sodium ion implantation treatment changed the surface morphology from a relatively smooth titanium film to rough surfaces corresponding to different implantation doses.After sodium implantation, implanted titanium films presented big particles with island structure morphology. The surface morphology and particle growth displayed the corresponding trend.Fibrinogen adsorption on these titanium films was performed to correlate with the surface properties of treated titanium films. The results show that protein adsorption on ion-implanted samples with dose of 2 × 1017 and 4 × 1017 are statistically higher (p < 0. 01) than samples treated with dose of 5×1016 and 1 ×1017, as well as the control samples.

  3. Detection of multiscale pockets on protein surfaces using mathematical morphology.

    Science.gov (United States)

    Kawabata, Takeshi

    2010-04-01

    Detection of pockets on protein surfaces is an important step toward finding the binding sites of small molecules. In a previous study, we defined a pocket as a space into which a small spherical probe can enter, but a large probe cannot. The radius of the large probes corresponds to the shallowness of pockets. We showed that each type of binding molecule has a characteristic shallowness distribution. In this study, we introduced fundamental changes to our previous algorithm by using a 3D grid representation of proteins and probes, and the theory of mathematical morphology. We invented an efficient algorithm for calculating deep and shallow pockets (multiscale pockets) simultaneously, using several different sizes of spherical probes (multiscale probes). We implemented our algorithm as a new program, ghecom (grid-based HECOMi finder). The statistics of calculated pockets for the structural dataset showed that our program had a higher performance of detecting binding pockets, than four other popular pocket-finding programs proposed previously. The ghecom also calculates the shallowness of binding ligands, R(inaccess) (minimum radius of inaccessible spherical probes) that can be obtained from the multiscale molecular volume. We showed that each part of the binding molecule had a bias toward a specific range of shallowness. These findings will be useful for predicting the types of molecules that will be most likely to bind putative binding pockets, as well as the configurations of binding molecules. The program ghecom is available through the Web server (http://biunit.naist.jp/ghecom).

  4. Lacritin and other autophagy associated proteins in ocular surface health.

    Science.gov (United States)

    Karnati, Roy; Talla, Venu; Peterson, Katherine; Laurie, Gordon W

    2016-03-01

    Advantage may be taken of macroautophagy ('autophagy') to promote ocular health. Autophagy continually captures aged or damaged cellular material for lysosomal degradation and recyling. When autophagic flux is chronically elevated, or alternatively deficient, health suffers. Chronic elevation of flux and stress are the consequence of inflammatory cytokines or of dry eye tears but not normal tears invitro. Exogenous tear protein lacritin transiently accelerates flux to restore homeostasis invitro and corneal health invivo, and yet the monomeric active form of lacritin appears to be selectively deficient in dry eye. Tissue transglutaminase-dependent cross-linking of monomer decreases monomer quantity and monomer affinity for coreceptor syndecan-1 thereby abrogating activity. Tissue transglutaminase is elevated in dry eye. Mutation of arylsulfatase A, arylsulfatase B, ceroid-lipofuscinosis neuronal 3, mucolipin, or Niemann-Pick disease type C1 respectively underlie several diseases of apparently insufficient autophagic flux that affect the eye, including: metachromatic leukodystrophy, mucopolysaccharidosis type VI, juvenile-onset Batten disease, mucolipidosis IV, and Niemann-Pick type C associated with myelin sheath destruction of corneal sensory and ciliary nerves and of the optic nerve; corneal clouding, ocular hypertension, glaucoma and optic nerve atrophy; accumulation of 'ceroid-lipofuscin' in surface conjunctival cells, and in ganglion and neuronal cells; decreased visual acuity and retinal dystrophy; and neurodegeneration. For some, enzyme or gene replacement, or substrate reduction, therapy is proving to be successful. Here we discuss examples of restoring ocular surface homeostasis through alteration of autophagy, with particular attention to lacritin.

  5. Relevant uses of surface proteins – display on self‐organized biological structures

    OpenAIRE

    Jahns, Anika C.; Rehm, Bernd H. A.

    2012-01-01

    Summary Proteins are often found attached to surfaces of self‐assembling biological units such as whole microbial cells or subcellular structures, e.g. intracellular inclusions. In the last two decades surface proteins were identified that could serve as anchors for the display of foreign protein functions. Extensive protein engineering based on structure–function data enabled efficient display of technically and/or medically relevant protein functions. Small size, diversity of the anchor pro...

  6. Survey of surface proteins from the pathogenic Mycoplasma hyopneumoniae strain 7448 using a biotin cell surface labeling approach.

    Directory of Open Access Journals (Sweden)

    Luciano Antonio Reolon

    Full Text Available The characterization of the repertoire of proteins exposed on the cell surface by Mycoplasma hyopneumoniae (M. hyopneumoniae, the etiological agent of enzootic pneumonia in pigs, is critical to understand physiological processes associated with bacterial infection capacity, survival and pathogenesis. Previous in silico studies predicted that about a third of the genes in the M. hyopneumoniae genome code for surface proteins, but so far, just a few of them have experimental confirmation of their expression and surface localization. In this work, M. hyopneumoniae surface proteins were labeled in intact cells with biotin, and affinity-captured biotin-labeled proteins were identified by a gel-based liquid chromatography-tandem mass spectrometry approach. A total of 20 gel slices were separately analyzed by mass spectrometry, resulting in 165 protein identifications corresponding to 59 different protein species. The identified surface exposed proteins better defined the set of M. hyopneumoniae proteins exposed to the host and added confidence to in silico predictions. Several proteins potentially related to pathogenesis, were identified, including known adhesins and also hypothetical proteins with adhesin-like topologies, consisting of a transmembrane helix and a large tail exposed at the cell surface. The results provided a better picture of the M. hyopneumoniae cell surface that will help in the understanding of processes important for bacterial pathogenesis. Considering the experimental demonstration of surface exposure, adhesion-like topology predictions and absence of orthologs in the closely related, non-pathogenic species Mycoplasma flocculare, several proteins could be proposed as potential targets for the development of drugs, vaccines and/or immunodiagnostic tests for enzootic pneumonia.

  7. Characterization of the surface of protein-adsorbed dental materials by wetting and streaming potential measurements

    NARCIS (Netherlands)

    Matsumura, H.; Kawasaki, K.; Okumura, N.; Kambara, M.; Norde, W.

    2003-01-01

    In this study we have elucidated the water-wettability and the electrokinetic surface potential of protein-covered dental materials. The proteins used here as typical proteins were human serum albumin and lysozyme from hen*s egg. The wettability (hydrophobicity/hydrophilicity) and the surface potent

  8. Characterization of the surface of protein-adsorbed dental materials by wetting and streaming potential measurements

    NARCIS (Netherlands)

    Matsumura, H; Kawasaki, K; Okumura, N; Kambara, M; Norde, W

    2003-01-01

    In this study we have elucidated the water-wettability and the electrokinetic surface potential of protein-covered dental materials. The proteins used here as typical proteins were human serum albumin and lysozyme from hen's egg. The wettability (hydrophobicity/hydrophilicity) and the surface potent

  9. Cell surface hydrophobicity is conveyed by S-layer proteins - A study in recombinant lactobacilli

    NARCIS (Netherlands)

    Mei, H.C. van der; Belt-Gritter, B. van de; Pouwels, P.H.; Martinez, B.; Busscher, H.J.

    2003-01-01

    Cell surface hydrophobicity is one of the most important factors controlling adhesion of microorganisms to surfaces. In this paper, cell surface properties of lactobacilli and recombinant lactobacilli with and without a surface layer protein (SLP) associated with cell surface hydrophobicity were det

  10. Protein-protein interactions: general trends in the relationship between binding affinity and interfacial buried surface area.

    Science.gov (United States)

    Chen, Jieming; Sawyer, Nicholas; Regan, Lynne

    2013-04-01

    Protein-protein interactions play key roles in many cellular processes and their affinities and specificities are finely tuned to the functions they perform. Here, we present a study on the relationship between binding affinity and the size and chemical nature of protein-protein interfaces. Our analysis focuses on heterodimers and includes curated structural and thermodynamic data for 113 complexes. We observe a direct correlation between binding affinity and the amount of surface area buried at the interface. For a given amount of surface area buried, the binding affinity spans four orders of magnitude in terms of the dissociation constant (Kd ). Across the entire dataset, we observe no obvious relationship between binding affinity and the chemical composition of the interface. We also calculate the free energy per unit surface area buried, or "surface energy density," of each heterodimer. For interfacial surface areas between 500 and 2000 Å(2) , the surface energy density decreases as the buried surface area increases. As the buried surface area increases beyond about 2000 Å(2) , the surface energy density levels off to a constant value. We believe that these analyses and data will be useful for researchers with an interest in understanding, designing or inhibiting protein-protein interfaces.

  11. Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

    Institute of Scientific and Technical Information of China (English)

    Jian-lin Dou; Tao Jing; Jing-jing Fan; Zhi-ming Yuan

    2011-01-01

    A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection.Due to its ubiquitous ability to invade host cells,Salmonella typhimurium might be a good candidate for displaying viral antigens.We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S.typhimurium BRD509 using the ice nucleation protein.The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated.The results showed that display motifs in the protein can target integral foreign protein on the surface of S.typhimurium BRD509.Moreover,recombinant strains with surface displayed viral proteins retained their invasiveness,suggesting that the recombinant S.typhimurium can be used as live vaccine vector for eliciting complete immunogenicity.The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.

  12. Wolbachia surface protein induces innate immune responses in mosquito cells

    Directory of Open Access Journals (Sweden)

    Pinto Sofia B

    2012-01-01

    Full Text Available Abstract Background Wolbachia endosymbiotic bacteria are capable of inducing chronic upregulation of insect immune genes in some situations and this phenotype may influence the transmission of important insect-borne pathogens. However the molecules involved in these interactions have not been characterized. Results Here we show that recombinant Wolbachia Surface Protein (WSP stimulates increased transcription of immune genes in mosquito cells derived from the mosquito Anopheles gambiae, which is naturally uninfected with Wolbachia; at least two of the upregulated genes, TEP1 and APL1, are known to be important in Plasmodium killing in this species. When cells from Aedes albopictus, which is naturally Wolbachia-infected, were challenged with WSP lower levels of upregulation were observed than for the An. gambiae cells. Conclusions We have found that WSP is a strong immune elicitor in a naturally Wolbachia-uninfected mosquito species (Anopheles gambiae while a milder elicitor in a naturally-infected species (Aedes albopictus. Since the WSP of a mosquito non-native (nematode Wolbachia strain was used, these data suggest that there is a generalized tolerance to WSP in Ae. albopictus.

  13. Insights into molecular plasticity of choline binding proteins (pneumococcal surface proteins) by SAXS.

    Science.gov (United States)

    Buey, Rubén M; Monterroso, Begoña; Menéndez, Margarita; Diakun, Greg; Chacón, Pablo; Hermoso, Juan Antonio; Díaz, J Fernando

    2007-01-12

    Phosphocholine moieties decorating the pneumococcal surface are used as a docking station for a family of modular proteins, the so-called choline binding proteins or CBPs. Choline recognition is essential for CBPs function and may also be a determinant for their quaternary structure. There is little knowledge about modular arrangement or oligomeric structures in this family. Therefore, we have used the small angle X-ray scattering (SAXS) technique combined with analytical ultracentrifugation in order to model the three-dimensional envelope of two highly different CBPs: the phage encoded Cpl-1 lysozyme and the pneumococcal phosphorylcholine esterase Pce. Both enzymes have an N-terminal catalytic module and a C-terminal choline-binding module (CBM) that attaches them to the bacterial surface and comprises six and ten sequence repeats in Cpl-1 and Pce, respectively. SAXS experiments have shown an inherent conformational plasticity in Cpl-1 that accounts for the different relative position of these regions in the solution and crystal structures. Dimerization of Cpl-1 upon choline binding has been also visualised for the first time, and monomer-monomer interactions take place through the first CBR where a non-canonical choline binding site has now been identified. This mode of association seems to be independent of the absence or presence of the Cpl-1 catalytic module and reveals that the arrangement of the monomers differs from that previously found in the isolated CBM dimer of pneumococcal LytA amidase. In contrast, Pce displays the same modular disposition in the solution and crystal structures, and remains almost invariant upon choline binding. The present results suggest that protein dimerization and duplication of CBRs may be alternative but not equivalent ways of improving cell wall recognition by CBPs, since they provide different interaction geometries for choline residues present in (lipo)teichoic acids.

  14. Regulation of Macrophage Recognition through the Interplay of Nanoparticle Surface Functionality and Protein Corona.

    Science.gov (United States)

    Saha, Krishnendu; Rahimi, Mehran; Yazdani, Mahdieh; Kim, Sung Tae; Moyano, Daniel F; Hou, Singyuk; Das, Ridhha; Mout, Rubul; Rezaee, Farhad; Mahmoudi, Morteza; Rotello, Vincent M

    2016-04-26

    Using a family of cationic gold nanoparticles (NPs) with similar size and charge, we demonstrate that proper surface engineering can control the nature and identity of protein corona in physiological serum conditions. The protein coronas were highly dependent on the hydrophobicity and arrangement of chemical motifs on NP surface. The NPs were uptaken in macrophages in a corona-dependent manner, predominantly through recognition of specific complement proteins in the NP corona. Taken together, this study shows that surface functionality can be used to tune the protein corona formed on NP surface, dictating the interaction of NPs with macrophages.

  15. Photoswitchable method for the ordered attachment of proteins to surfaces

    Science.gov (United States)

    Camarero, Julio A.; DeYoreo, James J.; Kwon, Youngeun

    2011-07-05

    Described herein is a method for the attachment of proteins to any solid support with control over the orientation of the attachment. The method is extremely efficient, not requiring the previous purification of the protein to be attached, and can be activated by UV-light. Spatially addressable arrays of multiple protein components can be generated by using standard photolithographic techniques.

  16. Scanning electron microscopy study of protein immobilized on SIO2 Sol-gel surfaces

    Directory of Open Access Journals (Sweden)

    O.B.G. Assis

    2003-09-01

    Full Text Available Uniform attachment of enzymes to solid surfaces is essential in the development of bio and optical sensor devices. Immobilization by adsorption according to hydrophilic or hydrophobic nature is dependent on the charges and defects of the support surfaces. Sol-gel SiO2 densified glass surfaces, frequently used as supports for protein immobilization, are evaluated via scanning electron microscopy. The model protein is globular enzyme lysozyme, deposited by adsorption on functionalized surfaces. Formation of a protein layer is confirmed by FTIR spectroscopy, and the SEM images suggest discontinuous adsorption in areas where cracks predominate on the glass surface.

  17. Enhanced protein retention on poly(caprolactone) via surface initiated polymerization of acrylamide

    Science.gov (United States)

    Ma, Yuhao; Cai, Mengtan; He, Liu; Luo, Xianglin

    2016-01-01

    To enhance the biocompatibility or extend the biomedical application of poly(caprolactone) (PCL), protein retention on PCL surface is often required. In this study, poly(acrylamide) (PAAm) brushes were grown from PCL surface via surface-initiated atom transfer radical polymerization (SI-ATRP) and served as a protein-capturing platform. Grafted PAAm was densely packed on surface and exhibited superior protein retention ability. Captured protein was found to be resistant to washing under detergent environment. Furthermore, protein structure after being captured was investigated by circular dichroism (CD) spectroscopy, and the CD spectra verified that secondary structure of captured proteins was maintained, indicating no denaturation of protein happened for retention process.

  18. Surface proteins of Staphylococcus aureus play an important role in experimental skin infection.

    Science.gov (United States)

    Kwiecinski, Jakub; Jin, Tao; Josefsson, Elisabet

    2014-12-01

    Staphylococcus aureus is the most common cause of skin infections that range from mild diseases up to life-threatening conditions. Mechanisms of S. aureus virulence in those infections remain poorly studied. To investigate the impact of S. aureus surface proteins on skin infection, we used mouse models of skin abscess formation and skin necrosis, induced by a subcutaneous injection of bacteria. In the skin abscess model, a sortase-deficient S. aureus strain lacking all of its cell-wall anchored proteins was less virulent than its wild-type strain. Also, strains specifically lacking protein A, fibronecting binding proteins, clumping factor A or surface protein SasF were impaired in their virulence. When a model of dermonecrosis was studied, the S. aureus surface proteins could not be shown to be involved. In summary, surface proteins play an important role in virulence of S. aureus skin abscess infections, but not in formation of skin necrosis.

  19. Targeted Mutagenesis and Combinatorial Library Screening Enables Control of Protein Orientation on Surfaces and Increased Activity of Adsorbed Proteins.

    Science.gov (United States)

    Cruz-Teran, Carlos A; Carlin, Kevin B; Efimenko, Kirill; Genzer, Jan; Rao, Balaji M

    2016-08-30

    While nonspecific adsorption is widely used for immobilizing proteins on solid surfaces, the random nature of protein adsorption may reduce the activity of immobilized proteins due to occlusion of the active site. We hypothesized that the orientation a protein assumes on a given surface can be controlled by systematically introducing mutations into a region distant from its active site, thereby retaining activity of the immobilized protein. To test this hypothesis, we generated a combinatorial protein library by randomizing six targeted residues in a binding protein derived from highly stable, nonimmunoglobulin Sso7d scaffold; mutations were targeted in a region that is distant from the binding site. This library was screened to isolate binders that retain binding to its cognate target (chicken immunoglobulin Y, cIgY) as well as exhibit adsorption on unmodified silica at pH 7.4 and high ionic strength conditions. A single mutant, Sso7d-2B5, was selected for further characterization. Sso7d-2B5 retained binding to cIgY with an apparent dissociation constant similar to that of the parent protein; both mutant and parent proteins saturated the surface of silica with similar densities. Strikingly, however, silica beads coated with Sso7d-2B5 could achieve up to 7-fold higher capture of cIgY than beads coated with the parent protein. These results strongly suggest that mutations introduced in Sso7d-2B5 alter its orientation relative to the parent protein, when adsorbed on silica surfaces. Our approach also provides a generalizable strategy for introducing mutations in proteins so as to improve their activity upon immobilization, and has direct relevance to development of protein-based biosensors and biocatalysts.

  20. Sampling the conformation of protein surface residues for flexible protein docking

    Directory of Open Access Journals (Sweden)

    Amenta Nina

    2010-11-01

    Full Text Available Abstract Background The problem of determining the physical conformation of a protein dimer, given the structures of the two interacting proteins in their unbound state, is a difficult one. The location of the docking interface is determined largely by geometric complementarity, but finding complementary geometry is complicated by the flexibility of the backbone and side-chains of both proteins. We seek to generate candidates for docking that approximate the bound state well, even in cases where there is backbone and/or side-chain difference from unbound to bound states. Results We divide the surfaces of each protein into local patches and describe the effect of side-chain flexibility on each patch by sampling the space of conformations of its side-chains. Likely positions of individual side-chains are given by a rotamer library; this library is used to derive a sample of possible mutual conformations within the patch. We enforce broad coverage of torsion space. We control the size of the sample by using energy criteria to eliminate unlikely configurations, and by clustering similar configurations, resulting in 50 candidates for a patch, a manageable number for docking. Conclusions Using a database of protein dimers for which the bound and unbound structures of the monomers are known, we show that from the unbound patch we are able to generate candidates for docking that approximate the bound structure. In patches where backbone change is small (within 1 Å RMSD of bound, we are able to account for flexibility and generate candidates that are good approximations of the bound state (82% are within 1 Å and 98% are within 1.4 Å RMSD of the bound conformation. We also find that even in cases of moderate backbone flexibility our candidates are able to capture some of the overall shape change. Overall, in 650 of 700 test patches we produce a candidate that is either within 1 Å RMSD of the bound conformation or is closer to the bound state than the

  1. Flagellin and outer surface proteins from Borrelia burgdorferi are not glycosylated

    OpenAIRE

    Štěrba, Ján

    2012-01-01

    Glycosylation of four proteins from Borrelia burgdorferi s.s. was investigated ? flagellins FlaA, FlaB, and outer surface proteins OspA and OspB. Glycosylation of these four proteins was not proved by any of the used techniques. However, other glycan-staining positive proteins were present in the borrelia samples. These proteins were suggested to originate in the culture medium.

  2. Nonlinear surface dilatational rheology and foaming behavior of protein and protein fibrillar aggregates in the presence of natural surfactant

    NARCIS (Netherlands)

    Wan, Zhili; Yang, Xiaoquan; Sagis, L.M.C.

    2016-01-01

    The surface and foaming properties of native soy glycinin (11S) and its heat-induced fibrillar aggregates, in the presence of natural surfactant steviol glycoside (STE), were investigated and compared at pH 7.0 to determine the impact of protein structure modification on protein?surfactant interfaci

  3. Structural insights into the evolution of a non-biological protein: importance of surface residues in protein fold optimization.

    Directory of Open Access Journals (Sweden)

    Matthew D Smith

    Full Text Available Phylogenetic profiling of amino acid substitution patterns in proteins has led many to conclude that most structural information is carried by interior core residues that are solvent inaccessible. This conclusion is based on the observation that buried residues generally tolerate only conserved sequence changes, while surface residues allow more diverse chemical substitutions. This notion is now changing as it has become apparent that both core and surface residues play important roles in protein folding and stability. Unfortunately, the ability to identify specific mutations that will lead to enhanced stability remains a challenging problem. Here we discuss two mutations that emerged from an in vitro selection experiment designed to improve the folding stability of a non-biological ATP binding protein. These mutations alter two solvent accessible residues, and dramatically enhance the expression, solubility, thermal stability, and ligand binding affinity of the protein. The significance of both mutations was investigated individually and together, and the X-ray crystal structures of the parent sequence and double mutant protein were solved to a resolution limit of 2.8 and 1.65 A, respectively. Comparative structural analysis of the evolved protein to proteins found in nature reveals that our non-biological protein evolved certain structural features shared by many thermophilic proteins. This experimental result suggests that protein fold optimization by in vitro selection offers a viable approach to generating stable variants of many naturally occurring proteins whose structures and functions are otherwise difficult to study.

  4. Protein analysis in dissolved organic matter: what free proteins from soil leachate and surface water can tell us a perspective

    Science.gov (United States)

    Schulze, W.

    2004-12-01

    Mass spectrometry based analysis of proteins is widely used to study cellular processes in model organisms. However, it has not yet routinely been applied in environmental research. Based on observations that protein can readily be detected as a component of dissolved organic matter (DOM), this article gives an example about the possible use of protein analysis in ecology and environmental sciences focusing on different terrestrial ecosystems. At this stage, there are two areas of interest: (1) the identification of phylogenetic groups contributing to the DOM protein pool, and (2) identification of the organismic origin of specific enzymes that are important for ecosystem processes. In this paper, mass spectrometric protein analysis was applied to identify proteins from DOM and organism-free surface water samples derived from different environments. It is concluded, that mass spectrometric protein analysis is capable of distinguishing phylogenetic origin of proteins from leachates of different soil horizons, and from various sources of terrestrial surface water. Current limitation is imposed by the limited knowledge of complete genomes of soil organisms. The protein analysis allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific active enzymes. Further applications, such as in pollution research are conceivable. In summary, the analysis of proteins opens a new area of research between the fields of microbiology and biogeochemistry.

  5. Protein sequences bound to mineral surfaces persist into deep time

    OpenAIRE

    Demarchi, Beatrice; Hall, Shaun; Roncal-Herrero, Teresa; Freeman, Colin L.; Woolley, Jos; Crisp, Molly K; Wilson, Julie; Fotakis, Anna Katerina; Fischer, Roman; Kessler, Benedikt M; Jersie-Christensen, Rosa Rakownikow; Olsen, Jesper Velgaard; Haile, James; Thomas, Jessica; Marean, Curtis W.

    2016-01-01

    Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Mol...

  6. Detection of S-nitrosylated protein by surface plasmon resonance

    Directory of Open Access Journals (Sweden)

    Ruirui Wang

    2015-06-01

    Full Text Available S-Nitrosylation has recently emerged as an important posttranslational modification of proteins and is becoming an intensive field of research in plants. Protein S-nitrosation, a reversible post-translation modification of cysteine, affects many cell signaling pathways and plays critical roles in redox-sensitive cell signaling. Changes in protein function effectively transmit biological signals and thus provide a framework for elucidating signaling networks. This paper presented a new, universal immunosensor for detection of S-nitrosylated proteins. Electrochemical impedance spectroscopy (EIS and atomic force microscope (AFM were used to estimate the formation of self-assembled film. This method was based on the specific binding characteristics of biotin–streptavidin, using Biotin-HPDP labeled protein sulfhydryl group as the substrate to detect proteins. The sensor was used to detect bovine serum albumin (BSA, nitrosylated BSA and denitrosylated BSA. The results showed that 90.61% of nitrosylated BSA were reduced, verifying that protein S-nitrosylation is a reversible and effective post-translation modification. This method was successfully applied to detect S-nitrosylated protein in Feicheng peach. The results showed good repeatability and precision. This method provided a molecular basis for further exploring the mechanism of S-nitrosylation of proteins in plants.

  7. Pre-absorbed immunoproteomics: a novel method for the detection of Streptococcus suis surface proteins.

    Directory of Open Access Journals (Sweden)

    Wei Zhang

    Full Text Available Streptococcus suis serotype 2 (SS2 is a zoonotic pathogen that can cause infections in pigs and humans. Bacterial surface proteins are often investigated as potential vaccine candidates and biomarkers of virulence. In this study, a novel method for identifying bacterial surface proteins is presented, which combines immunoproteomic and immunoserologic techniques. Critical to the success of this new method is an improved procedure for generating two-dimensional electrophoresis gel profiles of S. suis proteins. The S. suis surface proteins identified in this study include muramidase-released protein precursor (MRP and an ABC transporter protein, while MRP is thought to be one of the main virulence factors in SS2 located on the bacterial surface. Herein, we demonstrate that the ABC transporter protein can bind to HEp-2 cells, which strongly suggests that this protein is located on the bacterial cell surface and may be involved in pathogenesis. An immunofluorescence assay confirmed that the ABC transporter is localized to the bacterial outer surface. This new method may prove to be a useful tool for identifying surface proteins, and aid in the development of new vaccine subunits and disease diagnostics.

  8. Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

    DEFF Research Database (Denmark)

    Koehler, JF; Birkelund, Svend; Stephens, RS

    1992-01-01

    The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis and cap...... the contribution of the MOMP variable segments to the topographical interactions which determine the antigenic structure responsible for human immune response....

  9. Surface-associated proteins of Staphylococcus aureus: their possible roles in virulence

    NARCIS (Netherlands)

    T.J. Foster (Timothy); D. McDevitt

    1994-01-01

    textabstractA class of proteins that are associated with the cell surface of Gram-positive bacteria has been recognised. Common structural features which are implicated in the proper secretion and attachment of these proteins to the cell surface occur in the C-termini. N-terminal domains interact wi

  10. Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

    DEFF Research Database (Denmark)

    Sundberg, Christina; Thodeti, Charles Kumar; Kveiborg, Marie;

    2004-01-01

    as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell...

  11. Development, characterization, and optimization of protein level in date bars using response surface methodology.

    Science.gov (United States)

    Nadeem, Muhammad; Salim-ur-Rehman; Muhammad Anjum, Faqir; Murtaza, Mian Anjum; Mueen-ud-Din, Ghulam

    2012-01-01

    This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM). Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD) with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables.

  12. How surface composition of high milk proteins powders is influenced by spray-drying temperature.

    Science.gov (United States)

    Gaiani, C; Morand, M; Sanchez, C; Tehrany, E Arab; Jacquot, M; Schuck, P; Jeantet, R; Scher, J

    2010-01-01

    High milk proteins powders are common ingredients in many food products. The surface composition of these powders is expected to play an essential role during their storage, handling and/or final application. Therefore, an eventual control of the surface composition by modifying the spray-drying temperature could be very useful in the improvement of powder quality and the development of new applications. For this purpose, the influence of five spray-drying temperatures upon the surface composition of the powders was investigated by X-ray photoelectron spectroscopy. The major milk proteins were studied: native micellar casein and native whey, both more or less enriched in lactose. The results show a surface enrichment in lipids for all the powders and in proteins for many powders. Whatever the drying temperature, lipids and proteins are preferentially located near the surface whereas lactose is found in the core. This surface enrichment is also highly affected by the spray-drying temperature. More lipids, more proteins and less lactose are systematically observed at the surface of powders spray-dried at lower outlet air temperatures. The nature of proteins is also found essential; surface enrichment in lipids being much stronger for whey proteins containing powders than for casein containing powders. Additionally, we found a direct correlation between the lipids surface concentration and the wetting ability for the 25 powders studied.

  13. Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution.

    Science.gov (United States)

    Shivange, Amol V; Hoeffken, Hans Wolfgang; Haefner, Stefan; Schwaneberg, Ulrich

    2016-12-01

    Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: (i) identification of conserved and highly variable regions; (ii) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; (iii) in vitro DNA recombination of synthetic genes; and (iv) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

  14. Expression of a hydrophilic surface protein in infective stages of Leishmania major.

    Science.gov (United States)

    Flinn, H M; Rangarajan, D; Smith, D F

    1994-06-01

    A family of differentially expressed genes from Leishmania major contains one sequence (Gene B) that encodes a novel, hydrophilic protein found on the surface of infective parasite stages. The 177-residue, acidic Gene B protein is characterised by an amino acid repetitive element, comprising 45% of the total molecule, that is related to the cell-wall binding domain of protein A from Staphylococcus aureus. No identifiable signal peptide, membrane-spanning domain or consensus for glycosylphosphatidylinositol anchor attachment to the cell surface is found elsewhere in the deduced protein sequence. In vitro, the Gene B protein fractionates with the parasite cell surface glycoconjugates, lipophosphoglycan and the glycoinositolphospholipids. This protein is the first characterised surface peptide marker for infective stages of the Leishmania life cycle.

  15. Protein sequences bound to mineral surfaces persist into deep time

    DEFF Research Database (Denmark)

    Demarchi, Beatrice; Hall, Shaun; Roncal-Herrero, Teresa

    2016-01-01

    Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laet...

  16. Straightforward protein immobilization on Sylgard 184 PDMS microarray surface.

    Science.gov (United States)

    Heyries, Kevin A; Marquette, Christophe A; Blum, Loïc J

    2007-04-10

    In this work, a straightforward technique for protein immobilization on Sylgard 184 is described. The method consists of a direct transfer of dried protein/salt solutions to the PDMS interface during the polymer curing. Such non-conventional treatment of proteins was found to have no major negative consequence on their integrity. The mechanisms of this direct immobilization were investigated using a lysine modified dextran molecule as a model. Clear experimental results suggested that both chemical bounding and molding effect were implicated. As a proof of concept study, three different proteins were immobilized on a single microarray (Arachis hypogaea lectin, rabbit IgG, and human IgG) and used as antigens for capture of chemiluminescent immunoassays. The proteins were shown to be easily recognized by their specific antibodies, giving antibody detection limits in the fmol range.

  17. New Tools for the site-specific attachment of proteins to surface

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kwon, Y; Coleman, M A

    2005-06-17

    Protein microarrays in which proteins are immobilized to a solid surface are ideal reagents for high-throughput experiments that require very small amounts of analyte. Such protein microarrays ('protein chips') can be used very efficiently to analyze all kind of protein interactions en masse. Although a variety of methods are available for attaching proteins on solid surfaces. Most of them rely on non-specific adsorption methods or on the reaction of chemical groups within proteins (mainly, amino and carboxylic acid groups) with complementary reactive groups. In both cases the protein is attached to the surface in random orientations. The use of recombinant affinity tags addresses the orientation issue, however in most of the cases the interaction of the tags are reversible (e.g., glutathione S-transferase, maltose binding protein and poly-His) and, hence, are not stable over the course of subsequent assays or require large mediator proteins (e.g., biotin-avidin and antigen antibody). The key for the covalent attachment of a protein to a solid support with a total control over the orientation is to introduce two unique and mutually reactive groups on both the protein and the surface. The reaction between these two groups should be highly selective thus behaving like a molecular 'Velcro'.

  18. New Tools for the site-specific attachment of proteins to surface

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Y; Coleman, M A; Camarero, J A

    2006-06-27

    Protein microarrays in which proteins are immobilized to a solid surface are ideal reagents for high-throughput experiments that require very small amounts of analyte. Such protein microarrays (''protein chips'') can be used very efficiently to analyze all kind of protein interactions en masse. Although a variety of methods are available for attaching proteins on solid surfaces. Most of them rely on non-specific adsorption methods or on the reaction of chemical groups within proteins (mainly, amino and carboxylic acid groups) with complementary reactive groups. In both cases the protein is attached to the surface in random orientations. The use of recombinant affinity tags addresses the orientation issue, however in most of the cases the interaction of the tags are reversible (e.g., glutathione S-transferase, maltose binding protein and poly-His) and, hence, are not stable over the course of subsequent assays or require large mediator proteins (e.g., biotin-avidin and antigen antibody). The key for the covalent attachment of a protein to a solid support with a total control over the orientation is to introduce two unique and mutually reactive groups on both the protein and the surface. The reaction between these two groups should be highly selective thus behaving like a molecular ''velcro''.

  19. Surface characterization of proteins using multi-fractal property of heat-denatured aggregates

    Science.gov (United States)

    Lahiri, Tapobrata; Mishra, Hrishikesh; Sarkar, Subrata; Misra, Krishna

    2008-01-01

    Multi-fractal property of heat-denatured protein aggregates (HDPA) is characteristic of its individual form. The visual similarity between digitally generated microscopic images of HDPA with that of surface-image of its individual X-ray structures in protein databank (PDB) displayed using Visual Molecular Dynamics (VMD) viewer is the basis of the study. We deigned experiments to view the fractal nature of proteins at different aggregate scales. Intensity based multi-fractal dimensions (ILMFD) extracted from various planes of digital microscopic images of protein aggregates were used to characterize HDPA into different classes. Moreover, the ILMFD parameters extracted from aggregates show similar classification pattern to digital images of protein surface displayed by VMD viewer using PDB entry. We discuss the use of irregular patterns of heat-denatured aggregate proteins to understand various surface properties in native proteins. PMID:18795110

  20. A highly stable nonbiofouling surface with well-packed grafted zwitterionic polysulfobetaine for plasma protein repulsion.

    Science.gov (United States)

    Chang, Yung; Liao, Shih-Chieh; Higuchi, Akon; Ruaan, Ruoh-Chyu; Chu, Chih-Wei; Chen, Wen-Yih

    2008-05-20

    An ideal nonbiofouling surface for biomedical applications requires both high-efficient antifouling characteristics in relation to biological components and long-term material stability from biological systems. In this study we demonstrate the performance and stability of an antifouling surface with grafted zwitterionic sulfobetaine methacrylate (SBMA). The SBMA was grafted from a bromide-covered gold surface via surface-initiated atom transfer radical polymerization to form well-packed polymer brushes. Plasma protein adsorption on poly(sulfobetaine methacrylate) (polySBMA) grafted surfaces was measured with a surface plasmon resonance sensor. It is revealed that an excellent stable nonbiofouling surface with grafted polySBMA can be performed with a cycling test of the adsorption of three model proteins in a wide range of various salt types, buffer compositions, solution pH levels, and temperatures. This work also demonstrates the adsorption of plasma proteins and the adhesion of platelets from human blood plasma on the polySBMA grafted surface. It was found that the polySBMA grafted surface effectively reduces the plasma protein adsorption from platelet-poor plasma solution to a level superior to that of adsorption on a surface terminated with tetra(ethylene glycol). The adhesion and activation of platelets from platelet-rich plasma solution were not observed on the polySBMA grafted surface. This work further concludes that a surface with good hemocompatibility can be achieved by the well-packed surface-grafted polySBMA brushes.

  1. Surface heterogeneity: a friend or foe of protein adsorption - insights from theoretical simulations.

    Science.gov (United States)

    Penna, Matthew; Ley, Kamron; Maclaughlin, Shane; Yarovsky, Irene

    2016-10-06

    A lack in the detailed understanding of mechanisms through which proteins adsorb or are repelled at various solid/liquid interfaces limits the capacity to rationally design and produce more sophisticated surfaces with controlled protein adsorption in both biomedical and industrial settings. To date there are three main approaches to achieve anti biofouling efficacy, namely chemically adjusting the surface hydrophobicity and introducing various degrees of surface roughness, or a combination of both. More recently, surface nanostructuring has been shown to have an effect on protein adsorption. However, the current resolution of experimental techniques makes it difficult to investigate these three phase systems at the molecular level. In this molecular dynamics study we explore in all-atom detail the adsorption process of one of the most surface active proteins, EAS hydrophobin, known for its versatile ability to self-assemble on both hydrophobic and hydrophilic surfaces forming stable monolayers that facilitate further biofilm growth. We model the adsorption of this protein on organic ligand protected silica surfaces with varying degrees of chemical heterogeneity and roughness, including fully homogenous hydrophobic and hydrophilic surfaces for comparison. We present a detailed characterisation of the functionalised surface structure and dynamics for each of these systems, and the effect the ligands have on interfacial water, the adsorption process and conformational rearrangements of the protein. Results suggest that the ligand arrangement that produces the highest hydrophilic chain mobility and the lack of significant hydrophobic patches shows the most promising anti-fouling efficacy toward hydrophobin. However, the presence on the protein surface of a flexible loop with amphipathic character (the Cys3-Cys4 loop) is seen to facilitate EAS adsorption on all surfaces by enabling the protein to match the surface pattern.

  2. In-cell thermodynamics and a new role for protein surfaces.

    Science.gov (United States)

    Smith, Austin E; Zhou, Larry Z; Gorensek, Annelise H; Senske, Michael; Pielak, Gary J

    2016-02-16

    There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

  3. Selective labelling of cell-surface proteins using CyDye DIGE Fluor minimal dyes.

    Science.gov (United States)

    Hagner-McWhirter, Asa; Winkvist, Maria; Bourin, Stephanie; Marouga, Rita

    2008-11-26

    Surface proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan DIGE technology and analysis of protein expression changes using DeCyder 2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

  4. Development, Characterization, and Optimization of Protein Level in Date Bars Using Response Surface Methodology

    OpenAIRE

    Muhammad Nadeem; Salim-ur-Rehman,; Faqir Muhammad Anjum; Mian Anjum Murtaza; Ghulam Mueen-ud-Din

    2012-01-01

    This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM). Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD) with 2 variables and 3 lev...

  5. Computational prediction and experimental assessment of secreted/surface proteins from Mycobacterium tuberculosis H37Rv.

    Directory of Open Access Journals (Sweden)

    Carolina Vizcaíno

    2010-06-01

    Full Text Available The mycobacterial cell envelope has been implicated in the pathogenicity of tuberculosis and therefore has been a prime target for the identification and characterization of surface proteins with potential application in drug and vaccine development. In this study, the genome of Mycobacterium tuberculosis H37Rv was screened using Machine Learning tools that included feature-based predictors, general localizers and transmembrane topology predictors to identify proteins that are potentially secreted to the surface of M. tuberculosis, or to the extracellular milieu through different secretory pathways. The subcellular localization of a set of 8 hypothetically secreted/surface candidate proteins was experimentally assessed by cellular fractionation and immunoelectron microscopy (IEM to determine the reliability of the computational methodology proposed here, using 4 secreted/surface proteins with experimental confirmation as positive controls and 2 cytoplasmic proteins as negative controls. Subcellular fractionation and IEM studies provided evidence that the candidate proteins Rv0403c, Rv3630, Rv1022, Rv0835, Rv0361 and Rv0178 are secreted either to the mycobacterial surface or to the extracellular milieu. Surface localization was also confirmed for the positive controls, whereas negative controls were located on the cytoplasm. Based on statistical learning methods, we obtained computational subcellular localization predictions that were experimentally assessed and allowed us to construct a computational protocol with experimental support that allowed us to identify a new set of secreted/surface proteins as potential vaccine candidates.

  6. New developments for the site-specific attachment of protein to surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2005-05-12

    Protein immobilization on surfaces is of great importance in numerous applications in biology and biophysics. The key for the success of all these applications relies on the immobilization technique employed to attach the protein to the corresponding surface. Protein immobilization can be based on covalent or noncovalent interaction of the molecule with the surface. Noncovalent interactions include hydrophobic interactions, hydrogen bonding, van der Waals forces, electrostatic forces, or physical adsorption. However, since these interactions are weak, the molecules can get denatured or dislodged, thus causing loss of signal. They also result in random attachment of the protein to the surface. Site-specific covalent attachment of proteins onto surfaces, on the other hand, leads to molecules being arranged in a definite, orderly fashion and uses spacers and linkers to help minimize steric hindrances between the protein surface. This work reviews in detail some of the methods most commonly used as well as the latest developments for the site-specific covalent attachment of protein to solid surfaces.

  7. Silica as a Matrix for Encapsulating Proteins: Surface Effects on Protein Structure Assessed by Circular Dichroism Spectroscopy

    Directory of Open Access Journals (Sweden)

    Genet H. Zemede

    2012-08-01

    Full Text Available The encapsulation of biomolecules in solid materials that retain the native properties of the molecule is a desired feature for the development of biosensors and biocatalysts. In the current study, protein entrapment in silica-based materials is explored using the sol-gel technique. This work surveys the effects of silica confinement on the structure of several model polypeptides, including apomyoglobin, copper-zinc superoxide dismutase, polyglutamine, polylysine, and type I antifreeze protein. Changes in the secondary structure of each protein following encapsulation are monitored by circular dichroism spectroscopy. In many cases, silica confinement reduces the fraction of properly-folded protein relative to solution, but addition of a secondary solute or modification of the silica surface leads to an increase in structure. Refinement of the glass surface by addition of a monosubstituted alkoxysilane during sol-gel processing is shown to be a valuable tool for testing the effects of surface chemistry on protein structure. Because silica entrapment prevents protein aggregation by isolating individual protein molecules in the pores of the glass material, one may monitor aggregation-prone polypeptides under solvent conditions that are prohibited in solution, as demonstrated with polyglutamine and a disease-related variant of superoxide dismutase.

  8. Site-specific immobilization of protein layers on gold surfaces via orthogonal sortases.

    Science.gov (United States)

    Raeeszadeh-Sarmazdeh, Maryam; Parthasarathy, Ranganath; Boder, Eric T

    2015-04-01

    We report a site-specific, sortase-mediated ligation to immobilize proteins layer-by-layer on a gold surface. Recombinant fluorescent proteins with a Sortase A recognition tag at the C-terminus were immobilized on peptide-modified gold surfaces. We used two sortases with different substrate specificities (Streptococcus pyogenes Sortase A and Staphylococcus aureus Sortase A) to immobilize layers of GFP and mCherry site-specifically on the gold surface. Surfaces were characterized using fluorescence and atomic force microscopy after immobilizing each layer of protein. Fluorescent micrographs showed that both protein immobilization on the modified gold surface and protein oligomerization are sortase-dependent. AFM images showed that either homogenous protein monolayers or layers of protein oligomers can be generated using appropriately tagged substrate proteins. Using Sortase A variants with orthogonal peptide substrate specificities, site-specific immobilization of appropriately tagged GFP onto a layer of immobilized mCherry was achieved without disruption of the underlying protein layer.

  9. Two methods for glass surface modification and their application in protein immobilization.

    Science.gov (United States)

    Qin, Ming; Hou, Sen; Wang, Likai; Feng, XiZeng; Wang, Rui; Yang, Yanlian; Wang, Chen; Yu, Lei; Shao, Bin; Qiao, MingQiang

    2007-11-15

    Protein immobilization is a crucial step in protein chip, biosensor, etc. Here, two methods to immobilize proteins on glass surface were analyzed, one is silanization method using 3-aminopropyltriethoxysilane (APTES), and the other is hydrophobin HFBI coating. The modified glass surfaces were characterized with X-ray photoelectron spectroscopy (XPS), water contact angle measurement (WCA) and immunoassay. The results of XPS and WCA illustrated that the surface property of glass can be changed by both the two methods. The following immunoassay using microcontact printing (microCP) verified that both methods could help protein immobilization effectively on glass slides. Compared with the amine treatment, it is concluded that hydrophobin self-assemblies is a simple and generic way for protein immobilization on glass slides, which has potential application in protein chips and biosensors.

  10. Identification of Posttranslational Modification-Dependent Protein Interactions Using Yeast Surface Displayed Human Proteome Libraries.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact specifically with posttranslational modifications such as phosphorylation is often necessary to understand cellular signaling pathways. Numerous methods for identifying proteins that interact with posttranslational modifications have been utilized, including affinity-based purification and analysis, protein microarrays, phage display, and tethered catalysis. Although these techniques have been used successfully, each has limitations. Recently, yeast surface-displayed human proteome libraries have been utilized to identify protein fragments with affinity for various target molecules, including phosphorylated peptides. When coupled with fluorescently activated cell sorting and high throughput methods for the analysis of selection outputs, yeast surface-displayed human proteome libraries can rapidly and efficiently identify protein fragments with affinity for any soluble ligand that can be fluorescently detected, including posttranslational modifications. In this review we compare the use of yeast surface display libraries to other methods for the identification of interactions between proteins and posttranslational modifications and discuss future applications of the technology.

  11. Cell surface expression of glycosylated, nonglycosylated, and truncated forms of a cytoplasmic protein pyruvate kinase.

    Science.gov (United States)

    Hiebert, S W; Lamb, R A

    1988-09-01

    The soluble cytoplasmic protein pyruvate kinase (PK) has been expressed at the cell surface in a membrane-anchored form (APK). The hybrid protein contains the NH2-terminal signal/anchor domain of a class II integral membrane protein (hemagglutinin/neuraminidase, of the paramyxovirus SV5) fused to the PK NH2 terminus. APK contains a cryptic site that is used for N-linked glycosylation but elimination of this site by site-specific mutagenesis does not prevent cell surface localization. Truncated forms of the APK molecule, with up to 80% of the PK region of APK removed, can also be expressed at the cell surface. These data suggest that neither the complete PK molecule nor its glycosylation are necessary for intracellular transport of PK to the cell surface, and it is possible that specific signals may not be needed in the ectodomain of this hybrid protein to specify cell surface localization.

  12. Multidimensional profiling of cell surface proteins and nuclear markers

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Helen Barcellos-Hoff, Mary; Parvin, Bahram

    2009-01-30

    Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables.

  13. Water-wettable polypropylene fibers by facile surface treatment based on soy proteins.

    Science.gov (United States)

    Salas, Carlos; Genzer, Jan; Lucia, Lucian A; Hubbe, Martin A; Rojas, Orlando J

    2013-07-24

    Modification of the wetting behavior of hydrophobic surfaces is essential in a variety of materials, including textiles and membranes that require control of fluid interactions, adhesion, transport processes, sensing, etc. This investigation examines the enhancement of wettability of an important class of textile materials, viz., polypropylene (PP) fibers, by surface adsorption of different proteins from soybeans, including soy flour, isolate,glycinin, and β-conglycinin. Detailed investigations of soy adsorption from aqueous solution (pH 7.4, 25 °C) on polypropylene thin films is carried out using quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). A significant amount of protein adsorbs onto the PP surfaces primarily due to hydrophobic interactions. We establish that adsorption of a cationic surfactant, dioctadecyldimethylammonium bromide (DODA) onto PP surfaces prior to the protein deposition dramatically enhances its adsorption. The adsorption of proteins from native (PBS buffer, pH 7.4, 25 °C) and denatured conditions (PBS buffer, pH 7.4, 95 °C) onto DODA-treated PP leads to a high coverage of the proteins on the PP surface as confirmed by a significant improvement in water wettability. A shift in the contact angle from 128° to completely wettable surfaces (≈0°) is observed and confirmed by imaging experiments conducted with fluorescence tags. Furthermore, the results from wicking tests indicate that hydrophobic PP nonwovens absorb a significant amount of water after protein treatment, i.e., the PP-modified surfaces become completely hydrophilic.

  14. Surface modification of diamond-like carbon films with protein via polydopamine inspired coatings

    Science.gov (United States)

    Tao, Caihong; Yang, Shengrong; Zhang, Junyan; Wang, Jinqing

    2009-10-01

    In this paper, we report a facile two-step approach to immobilize proteins onto DLC surfaces. The first step was a simple immersion of DLC in a solution of dopamine. Polydopamine was deposited on DLC as a stable anchor to present protein molecules. Then the protein ad-layer was deposited on it. The chemical components of the modified DLC surfaces were characterized by Fourier transform infrared spectra and X-ray photoelectron spectroscopy. The biocompatibility of it was evaluated in vitro by the tetrazolium salt method. And it was indicated that the BSA modified surface had good haemocompatibility properties, and was cytocompatible to PC-12 cells.

  15. Surface modification of diamond-like carbon films with protein via polydopamine inspired coatings

    Energy Technology Data Exchange (ETDEWEB)

    Tao Caihong [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China); China and Graduate University of Chinese Academy of Sciences, Beijing 100080 (China); Yang Shengrong [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China); Zhang Junyan, E-mail: zhangjunyan@lzb.ac.cn [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China); Wang Jinqing [State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Tianshui Middle Road 18th, Lanzhou 730000 (China)

    2009-10-15

    In this paper, we report a facile two-step approach to immobilize proteins onto DLC surfaces. The first step was a simple immersion of DLC in a solution of dopamine. Polydopamine was deposited on DLC as a stable anchor to present protein molecules. Then the protein ad-layer was deposited on it. The chemical components of the modified DLC surfaces were characterized by Fourier transform infrared spectra and X-ray photoelectron spectroscopy. The biocompatibility of it was evaluated in vitro by the tetrazolium salt method. And it was indicated that the BSA modified surface had good haemocompatibility properties, and was cytocompatible to PC-12 cells.

  16. Quantitative evaluation of interaction force between functional groups in protein and polymer brush surfaces.

    Science.gov (United States)

    Sakata, Sho; Inoue, Yuuki; Ishihara, Kazuhiko

    2014-03-18

    To understand interactions between polymer surfaces and different functional groups in proteins, interaction forces were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Various polymer brush surfaces were systematically prepared by surface-initiated atom transfer radical polymerization as well-defined model surfaces to understand protein adsorption behavior. The polymer brush layers consisted of phosphorylcholine groups (zwitterionic/hydrophilic), trimethylammonium groups (cationic/hydrophilic), sulfonate groups (anionic/hydrophilic), hydroxyl groups (nonionic/hydrophilic), and n-butyl groups (nonionic/hydrophobic) in their side chains. The interaction forces between these polymer brush surfaces and different functional groups (carboxyl groups, amino groups, and methyl groups, which are typical functional groups existing in proteins) were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Furthermore, the amount of adsorbed protein on the polymer brush surfaces was quantified by surface plasmon resonance using albumin with a negative net charge and lysozyme with a positive net charge under physiological conditions. The amount of proteins adsorbed on the polymer brush surfaces corresponded to the interaction forces generated between the functional groups on the cantilever and the polymer brush surfaces. The weakest interaction force and least amount of protein adsorbed were observed in the case of the polymer brush surface with phosphorylcholine groups in the side chain. On the other hand, positive and negative surfaces generated strong forces against the oppositely charged functional groups. In addition, they showed significant adsorption with albumin and lysozyme, respectively. These results indicated that the interaction force at the functional group level might be

  17. Surface-associated proteins of Staphylococcus aureus: their possible roles in virulence

    OpenAIRE

    Foster, Timothy; McDevitt, D

    1994-01-01

    textabstractA class of proteins that are associated with the cell surface of Gram-positive bacteria has been recognised. Common structural features which are implicated in the proper secretion and attachment of these proteins to the cell surface occur in the C-termini. N-terminal domains interact with the host by binding to soluble host proteins, to matrix proteins or to host cells. They probably have important roles in pathogenicity by allowing bacteria to avoid host defences and by acting a...

  18. Protein conformational transitions at the liquid-gas interface as studied by dilational surface rheology.

    Science.gov (United States)

    Noskov, Boris A

    2014-04-01

    Experimental results on the dynamic dilational surface elasticity of protein solutions are analyzed and compared. Short reviews of the protein behavior at the liquid-gas interface and the dilational surface rheology precede the main sections of this work. The kinetic dependencies of the surface elasticity differ strongly for the solutions of globular and non-globular proteins. In the latter case these dependencies are similar to those for solutions of non-ionic amphiphilic polymers and have local maxima corresponding to the formation of the distal region of the surface layer (type I). In the former case the dynamic surface elasticity is much higher (>60 mN/m) and the kinetic dependencies are monotonical and similar to the data for aqueous dispersions of solid nanoparticles (type II). The addition of strong denaturants to solutions of bovine serum albumin and β-lactoglobulin results in an abrupt transition from the type II to type I dependencies if the denaturant concentration exceeds a certain critical value. These results give a strong argument in favor of the preservation of the protein globular structure in the course of adsorption without any denaturants. The addition of cationic surfactants also can lead to the non-monotonical kinetic dependencies of the dynamic surface elasticity indicating destruction of the protein tertiary and secondary structures. The addition of anionic surfactants gives similar results only for the protein solutions of high ionic strength. The influence of cationic surfactants on the local maxima of the kinetic dependencies of the dynamic surface elasticity for solutions of a non-globular protein (β-casein) differs from the influence of anionic surfactants due to the heterogeneity of the charge distribution along the protein chain. In this case one can use small admixtures of ionic surfactants as probes of the adsorption mechanism. The effect of polyelectrolytes on the kinetic dependencies of the dynamic surface elasticity of protein

  19. Electrochemical Characterization of Protein Adsorption onto YNGRT-Au and VLGXE-Au Surfaces

    Directory of Open Access Journals (Sweden)

    Hanna Trzeciakiewicz

    2015-08-01

    Full Text Available The adsorption of the proteins CD13, mucin and bovine serum albumin on VLGXE-Au and YNGRT-Au interfaces was monitored by electrochemical impedance spectroscopy in the presence of [Fe(CN6]3−/4−. The hydrophobicity of the Au surface was tailored using specific peptides, blocking agents and diluents. The combination of blocking agents (ethanolamine or n-butylamine and diluents (hexanethiol or 2-mercaptoethanol was used to prepare various peptide-modified Au surfaces. Protein adsorption onto the peptide-Au surfaces modified with the combination of n-butylamine and hexanethiol produced a dramatic decrease in the charge transfer resistance, Rct, for all three proteins. In contrast, polar peptide-surfaces induced a minimal change in Rct for all three proteins. Furthermore, an increase in Rct was observed with CD13 (an aminopeptidase overexpressed in certain cancers in comparison to the other proteins when the VLGXE-Au surface was modified with n-butylamine as a blocking agent. The electrochemical data indicated that protein adsorption may be modulated by tailoring the peptide sequence on Au surfaces and that blocking agents and diluents play a key role in promoting or preventing protein adsorption. The peptide-Au platform may also be used for targeting cancer biomarkers with designer peptides.

  20. Shotgun proteomic analytical approach for studying proteins adsorbed onto liposome surface

    KAUST Repository

    Capriotti, Anna Laura

    2011-07-02

    The knowledge about the interaction between plasma proteins and nanocarriers employed for in vivo delivery is fundamental to understand their biodistribution. Protein adsorption onto nanoparticle surface (protein corona) is strongly affected by vector surface characteristics. In general, the primary interaction is thought to be electrostatic, thus surface charge of carrier is supposed to play a central role in protein adsorption. Because protein corona composition can be critical in modifying the interactive surface that is recognized by cells, characterizing its formation onto lipid particles may serve as a fundamental predictive model for the in vivo efficiency of a lipidic vector. In the present work, protein coronas adsorbed onto three differently charged cationic liposome formulations were compared by a shotgun proteomic approach based on nano-liquid chromatography-high-resolution mass spectrometry. About 130 proteins were identified in each corona, with only small differences between the different cationic liposome formulations. However, this study could be useful for the future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins into body fluids. © 2011 Springer-Verlag.

  1. Engineering and Characterization of Peptides and Proteins at Surfaces and Interfaces: A Case Study in Surface-Sensitive Vibrational Spectroscopy.

    Science.gov (United States)

    Ding, Bei; Jasensky, Joshua; Li, Yaoxin; Chen, Zhan

    2016-06-21

    Understanding molecular structures of interfacial peptides and proteins impacts many research fields by guiding the advancement of biocompatible materials, new and improved marine antifouling coatings, ultrasensitive and highly specific biosensors and biochips, therapies for diseases related to protein amyloid formation, and knowledge on mechanisms for various membrane proteins and their interactions with ligands. Developing methods for measuring such unique systems, as well as elucidating the structure and function relationship of such biomolecules, has been the goal of our lab at the University of Michigan. We have made substantial progress to develop sum frequency generation (SFG) vibrational spectroscopy into a powerful technique to study interfacial peptides and proteins, which lays a foundation to obtain unique and valuable insights when using SFG to probe various biologically relevant systems at the solid/liquid interface in situ in real time. One highlighting feature of this Account is the demonstration of the power of combining SFG with other techniques and methods such as ATR-FTIR, surface engineering, MD simulation, liquid crystal sensing, and isotope labeling in order to study peptides and proteins at interfaces. It is necessary to emphasize that SFG plays a major role in these studies, while other techniques and methods are supplemental. The central role of SFG is to provide critical information on interfacial peptide and protein structure (e.g., conformation and orientation) in order to elucidate how surface engineering (e.g., to vary the structure) can ultimately affect surface function (e.g., to optimize the activity). This Account focuses on the most significant recent progress in research on interfacial peptides and proteins carried out by our group including (1) the development of SFG analysis methods to determine orientations of regular as well as disrupted secondary structures, and the successful demonstration and application of an isotope

  2. ProtEx: a novel technology to display exogenous proteins on the cell surface for immunomodulation.

    Science.gov (United States)

    Singh, Narendra P; Yolcu, Esma S; Askenasy, Nadir; Shirwan, Haval

    2005-11-01

    Gene therapy as an immunomodulatory approach has the potential to treat various inherited and acquired immune-based human diseases. However, its clinical application has several challenges, varying from the efficiency of gene transfer, control of gene expression, cell and tissue targeting, and safety concerns associated with the introduction of exogenous DNA into cells/tissues. Gene therapy is also a time- and labor-intensive procedure. As an alternative, we recently developed a novel technology, ProtEx, that allows for rapid, efficient, and durable display of exogenous proteins on the surface of cells, tissues, and organs without detectable toxicity. This technology exploits the strong binding affinity (Kd = 10(-15) M) of streptavidin with biotin and involves generation of chimeric molecules composed of the extracellular portions of immunological proteins of interest and a modified form of streptavidin, biotinylation of biological surfaces, and decoration of the modified surface with chimeric proteins. Biotin persists on the cell surface for weeks both in vitro and in vivo, thereby providing a platform to display exogenous proteins with extended cell surface kinetics. Two chimeric proteins, rat FasL (SA-FasL) and human CD80 (CD80-SA), were generated and tested for cell surface display and immunomodulatory functions. SA-FasL and CD80-SA molecules persisted on the surface of various cell types for extended periods, varying from days to weeks in vitro and in vivo. The cell surface kinetics, however, were protein and cell type dependent. SA-FasL showed potent apoptotic activity against Fas+ cells as a soluble protein or displayed on the cell surface and effectively blocked alloreactive responses. The display of CD80-SA on the surface of tumor cells, however, converted them into antigen-presenting cells for effective stimulation of autologous and allogeneic T-cell responses. ProtEx technology, therefore, represents a practical and effective alternative to DNA

  3. Comparison of two different plasma surface-modification techniques for the covalent immobilization of protein monolayers.

    Science.gov (United States)

    Cifuentes, Anna; Borrós, Salvador

    2013-06-04

    The immobilization of biologically active species is crucial for the fabrication of smart bioactive surfaces. For this purpose, plasma polymerization is frequently used to modify the surface nature without affecting the bulk properties of the material. Thus, it is possible to create materials with surface functional groups that can promote the anchoring of all kinds of biomolecules. Different methodologies in protein immobilization have been developed in recent years, although some drawbacks are still not solved, such as the difficulties that some procedures involve and/or the denaturalization of the protein due to the immobilization process. In this work, two different strategies to covalently attach bovine serum albumin (BSA) protein are developed. Both techniques are compared in order to understand how the nature of the surface modification affects the conformation of the protein upon immobilization.

  4. Protein overexport in a Saccharomyces cerevisiae mutant is not due to facilitated release of cell-surface proteins.

    Science.gov (United States)

    Alexieva, K I; Venkov, P V

    2000-01-01

    Saccharomyces cerevisiae strain MW11 is a temperature-sensitive mutant which exports twenty times more proteins at 37 degrees C than parental or wild-type strains do. To understand the mechanism underlying the protein overexport in the mutant the possibility of an altered cell-wall structure leading to facilitated release of cell-surface proteins was studied. Data on calcofluor white and zymolyase sensitivities, resistance to killer 1 toxin and determination of exported acid phosphatase and invertase did not provide evidence for alterations in the cell-wall structure that could explain the protein overexport phenotype. The results were obtained in experiments when transcription of mutated gene was discontinued which permits the full expression of the protein overexport phenotype.

  5. Fate of the surface protein gp70 during entry of retrovirus into mouse fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, K.B.

    1985-04-15

    The kinetics of the viral surface protein gp70 and the viral core proteins p30 and p15C were followed during retrovirus entry into mouse fibroblasts. All three proteins were internalized, but whereas essentially all the gp70 was degraded, approximately one-third of the core proteins remained stable in the cells. These diverging routes of the different proteins are in agreement with the proposed route, that retrovirus enters the cells by endocytosis followed by a membrane fusion between the virus membrane and the vesicle membrane.

  6. Surface-enhanced infrared absorption spectroscopy (SEIRAS) to probe monolayers of membrane proteins.

    Science.gov (United States)

    Ataka, Kenichi; Stripp, Sven Timo; Heberle, Joachim

    2013-10-01

    Surface-enhanced infrared absorption spectroscopy (SEIRAS) represents a variation of conventional infrared spectroscopy and exploits the signal enhancement exerted by the plasmon resonance of nano-structured metal thin films. The surface enhancement decays in about 10nm with the distance from the surface and is, thus, perfectly suited to selectively probe monolayers of biomembranes. Peculiar to membrane proteins is their vectorial functionality, the probing of which requires proper orientation within the membrane. To this end, the metal surface used in SEIRAS is chemically modified to generate an oriented membrane protein film. Monolayers of uniformly oriented membrane proteins are formed by tethering His-tagged proteins to a nickel nitrilo-triacetic acid (Ni-NTA) modified gold surface and SEIRAS commands molecular sensitivity to probe each step of surface modification. The solid surface used as plasmonic substrate for SEIRAS, can also be employed as an electrode to investigate systems where electron transfer reactions are relevant, like e.g. cytochrome c oxidase or plant-type photosystems. Furthermore, the interaction of these membrane proteins with water-soluble proteins, like cytochrome c or hydrogenase, is studied on the molecular level by SEIRAS. The impact of the membrane potential on protein functionality is verified by monitoring light-dark difference spectra of a monolayer of sensory rhodopsin (SRII) at different applied potentials. It is demonstrated that the interpretations of all of these experiments critically depend on the orientation of the solid-supported membrane protein. Finally, future directions of SEIRAS including cellular systems are discussed. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

  7. Mapping lipid and detergent molecules at the surface of membrane proteins.

    Science.gov (United States)

    Cogdell, Richard J; Gardiner, Alastair T; Roszak, Aleksander W; Stončius, Sigitas; Kočovský, Pavel; Isaacs, Neil W

    2011-06-01

    Electron-density maps for the crystal structures of membrane proteins often show features suggesting binding of lipids and/or detergent molecules on the hydrophobic surface, but usually it is difficult to identify the bound molecules. In our studies, heavy-atom-labelled phospholipids and detergents have been used to unequivocally identify these binding sites at the surfaces of test membrane proteins, the reaction centres from Rhodobacter sphaeroides and Blastochloris viridis. The generality of this method is discussed in the present article.

  8. Proteomic analysis of cell surface-associated proteins from probiotic Lactobacillus plantarum

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Madsen, Søren M; Glenting, Jacob;

    2009-01-01

    In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel...... of probiotics in the gastrointestinal tract. The results provide the basis for future studies on the molecular mechanisms of probiotics....

  9. Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells.

    Science.gov (United States)

    Jin, Takashi; Tiwari, Dhermendra K; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M

    2010-11-01

    To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells.

  10. Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

    Directory of Open Access Journals (Sweden)

    Xiuchun Ge

    Full Text Available BACKGROUND: Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. METHODS AND FINDINGS: We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. CONCLUSIONS: The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

  11. Surface selective binding of nanoclay particles to polyampholyte protein chains

    Science.gov (United States)

    Pawar, Nisha; Bohidar, H. B.

    2009-07-01

    Binding of nanoclay (Laponite) to gelatin-A and gelatin-B (both polyampholytes) molecules was investigated at room temperature (25 °C) both experimentally and theoretically. The stoichiometric binding ratio between gelatin and Laponite was found to be strongly dependent on the solution ionic strength. Large soluble complexes were formed at higher ionic strengths of the solution, a result supported by data obtained from light scattering, viscosity, and zeta potential measurements. The binding problem was theoretically modeled by choosing a suitable two-body screened Coulomb potential, U(R+)=(q-/2ɛ)[(Q-/R-)e-kR--(Q+/R+)e-kR+], where the protein dipole has charges Q+ and Q_ that are located at distances R+ and R_ from the point Laponite charge q- and the dispersion liquid has dielectric constant (ɛ). U(R+) accounted for electrostatic interactions between a dipole (protein molecule) and an effective charge (Laponite particle) located at an angular position θ. Gelatin-A and Laponite association was facilitated by a strong attractive interaction potential that led to preferential binding of the biopolymer chains to negatively charged face of Laponite particles. In the case of gelatin-B selective surf ace patch binding dominated the process where the positively charged rim and negatively charged face of the particles were selectively bound to the oppositely charged segments of the biopolymer. The equilibrium separation (Re) between the protein and nanoclay particle revealed monovalent salt concentration dependence given by Re˜[NaCl]α where α =0.6±0.2 for gelatin-A and α =0.4±0.2 for gelatin-B systems. The equilibrium separations were ≈30% less compared to the gelatin-A system implying preferential short-range ordering of the gelatin-B-nanoclay pair in the solvent.

  12. Extraction of cell surface-associated proteins from living yeast cells.

    NARCIS (Netherlands)

    F.M. Klis; M. de Jong; S. Brul; P.W.J. de Groot

    2007-01-01

    To extract cell surface-associated proteins from living fungal cells, reducing agents such as beta-mercaptoethanol and dithiothreitol are often used. We show here that both compounds are moderately lipophilic and may perturb the plasma membrane, thus causing the release of cytosolic proteins, especi

  13. Surface-tethered polymers to influence protein adsorption and microbial adhesion

    NARCIS (Netherlands)

    Norde, Willem

    2007-01-01

    In various applications it is desired that biological cells or protein molecules are immobilized at surfaces. Examples are enzymes or cells in bioreactors and biosensors, immuno-proteins in solid-state diagnostics and proteinaceous farmacons in drug delivery systems. In order to retain biological ac

  14. Functionalization of SU-8 Photoresist Surfaces with IgG Proteins

    DEFF Research Database (Denmark)

    Blagoi, Gabriela; Keller, Stephan Urs; Johansson, Alicia;

    2008-01-01

    The negative epoxy-based photoresist SU-8 has a variety of applications within microelectromechanical systems (MEMS) and lab-on-a-chip systems. Here, several methods to functionalize SU-8 surfaces with IgG proteins were investigated. Fluorescent labeled proteins and fluorescent sandwich immunoass...

  15. Protein adsorption on the poly(L-lactic acid) surface modified by chitosan and its derivatives

    Institute of Scientific and Technical Information of China (English)

    JIAO YanPeng; ZHOU ChangRen; LI LiHua; DING Shan; LU Lu; LUO BingHong; LI Hong

    2009-01-01

    Surface modification of biomaterials has been adopted over the years to improve their biocompatibility.In this study,aiming to promote hydrophilicity and to introduce natural recognition sites onto poly(L-lactic acid) (PLLA) films,chitosan and its derivatives,carboxymethyl chitosan(CMC) and N-methylene phosphonic chitosan (NPC),were used to modify the surface of PLLA films by an entrapment method. Radiolabeled (12SI) proteins were used to measure the amount of protein adsorbed to PLLA surfaces. Fibronectin (Fn) was used to study the protein adsorption on the modified PLLA surfaces,including isotherm adsorption and adsorption kinetics of single protein,competitive adsorption of binary proteins system and serum multi-proteins and the desorption behavior in serum solution. The results showed that in the isotherm adsorption,Fn had a larger adsorption capacity on the CS-modified surface at lower concentrations,but had a high adsorption capacity at CMC-modified surface at higher concentrations. In the study of absorption kinetics,Fn had a fastest adsorption equilibrium and a highest equilibrium adsorption capacity at the CS-modified surface,while it was opposite at the PCS-modified surface. When BSA and serum were added,it had the greatest effect on the adsorption of Fn on the PCS-modified surface. After 6 hours soaking in the desorption study,Fn had reached desorption equilibrium on all the modified surfaces,which had different effects on the desorption rate and the remaining percentage of Fn.

  16. Surface characterization of proteins using multi-fractal property of heat-denatured aggregates

    OpenAIRE

    Lahiri, Tapobrata; Mishra, Hrishikesh; Sarkar, Subrata; Misra, Krishna

    2008-01-01

    Multi-fractal property of heat-denatured protein aggregates (HDPA) is characteristic of its individual form. The visual similarity between digitally generated microscopic images of HDPA with that of surface-image of its individual X-ray structures in protein databank (PDB) displayed using Visual Molecular Dynamics (VMD) viewer is the basis of the study. We deigned experiments to view the fractal nature of proteins at different aggregate scales. Intensity based multi-fractal dimensions (ILMFD)...

  17. Gold nanoparticles: role of size and surface chemistry on blood protein adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Benetti, F., E-mail: filippo.benetti@unitn.it; Fedel, M. [BIOtech Research Centre (Italy); Minati, L.; Speranza, G. [Fondazione Bruno Kessler (Italy); Migliaresi, C. [BIOtech Research Centre (Italy)

    2013-06-15

    Material interaction with blood proteins is a critical issue, since it could influence the biological processes taking place in the body following implantation/injection. This is particularly important in the case of nanoparticles, where innovative properties, such as size and high surface to volume ratio can lead to a behavioral change with respect to bulk macroscopic materials and could be responsible for a potential risk for human health. The aim of this work was to compare gold nanoparticles (AuNP) and planar surfaces to study the role of surface curvature moving from the macro- to the nano-size in the process of blood protein adsorption. In the course of the study, different protocols were tested to optimize the analysis of protein adsorption on gold nanoparticles. AuNP with different size (10, 60 and 200 nm diameter) and surface coatings (citrate and polyethylene glycol) were carefully characterized. The stabilizing action of blood proteins adsorbed on AuNP was studied measuring the variation of size and solubility of the nanoparticles following incubation with single protein solutions (human serum albumin and fibrinogen) and whole blood plasma. In addition, we developed a method to elute proteins from AuNP to study the propensity of gold materials to adsorb plasma proteins in function of dimensional characteristics and surface chemistry. We showed a different efficacy of the various eluting media tested, proving that even the most aggressive agent cannot provide a complete detachment of the protein corona. Enhanced protein adsorption was evidenced on AuNP if compared to gold laminae (bare and PEGylated) used as macroscopic control, probably due to the superior AuNP surface reactivity.

  18. Microscopic Investigation of Reversible Nanoscale Surface Size Dependent Protein Conjugation

    Directory of Open Access Journals (Sweden)

    Michael A. Carpenter

    2009-05-01

    Full Text Available Aβ1-40 coated 20 nm gold colloidal nanoparticles exhibit a reversible color change as pH is externally altered between pH 4 and 10. This reversible process may contain important information on the initial reversible step reported for the fibrillogenesis of Aβ (a hallmark of Alzheimer’s disease. We examined this reversible color change by microscopic investigations. AFM images on graphite surfaces revealed the morphology of Aβ aggregates with gold colloids. TEM images clearly demonstrate the correspondence between spectroscopic features and conformational changes of the gold colloid.

  19. Chemical imaging of protein adsorption and crystallization on a wettability gradient surface.

    Science.gov (United States)

    Glassford, Stefanie; Chan, K L Andrew; Byrne, Bernadette; Kazarian, Sergei G

    2012-02-14

    The use of self-assembled monolayers is an established method to study the effect of surface properties on proteins and other biological materials. The generation of a monolayer with a gradient of chemical properties allows for the study of multiple surface properties simultaneously in a high throughput manner. Typically, in order to detect the presence of proteins or biological material on a surface, the use of additional dyes or tags is required. Here we present a novel method of studying the effect of gradient surface properties on protein adsorption and crystallization in situ through the use of ATR-FTIR spectroscopic imaging, which removes the need for additional labeling. We describe the successful application of this technique to the measurement of the growth of a gradient monolayer of octyltrichlorosilane across the surface of a silicon ATR element. ATR-FTIR imaging was also used to study the adsorption of lysozyme, as a model protein, onto the modified surface. The sensitivity of measurements obtained with a focal plane array (FPA) detector were improved though the use of pixel averaging which allowed small absorption bands to be detected with minimal effect on the spatial resolution along the gradient. Study of the effect of surface hydrophobicity on both adsorption of lysozyme to the element and lysozyme crystallization revealed that more lysozyme adsorbed to the hydrophobic side of the ATR element and more lysozyme crystals formed in the same region. These findings strongly suggest a correlation exists between surface protein adsorption and protein crystallization. This method could be applied to the study of other proteins and whole cells.

  20. Chemical surface modification of parylene C for enhanced protein immobilization and cell proliferation.

    Science.gov (United States)

    Zhang, Changhong; Thompson, Mark E; Markland, Frank S; Swenson, Steve

    2011-10-01

    To introduce the adhesion site of proteins and/or cells on parylene C (PC)-coated medical devices that can be used as implantable biosensors or drug delivery capsules, the PC surfaces were initially modified by the Friedel-Crafts acylation reaction to generate active chlorines. These chlorines were then employed to initiate the atom transfer radical polymerization of tert-butyl acrylate (TBA) and form a polymer brush layer of polyTBA on PC; the acrylate groups in the polymer brushes were hydrolyzed to carboxylic acid groups and further activated into succinimidyl ester groups via the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide coupling reaction. The PC surface grafted with polymer brushes and activated by succinimide showed efficient attachment of proteins, including gelatin, contortrostatin (CN) and bovine serum albumin (BSA), all at high density on the PC surface. The CN density on the surface was evaluated for both monolayer and polymer brush-based coatings. Based on fluorescence measurements, the polymer brush gives a 60-fold higher surface protein density than the monolayer-based system. Gelatin was used as a model protein and covalently coated onto the modified PC surface for cell culture study. Substrates with gelatin coating showed a significantly higher cell attachment and proliferation in 7 days cultures as compared to the uncoated substrates. In addition, a conventional photolithography technique was coupled with the surface chemistry to successfully pattern the BSA labeled with fluorescein isothiocyanate on the modified PC surfaces.

  1. Nonlinear Surface Dilatational Rheology and Foaming Behavior of Protein and Protein Fibrillar Aggregates in the Presence of Natural Surfactant.

    Science.gov (United States)

    Wan, Zhili; Yang, Xiaoquan; Sagis, Leonard M C

    2016-04-19

    The surface and foaming properties of native soy glycinin (11S) and its heat-induced fibrillar aggregates, in the presence of natural surfactant steviol glycoside (STE), were investigated and compared at pH 7.0 to determine the impact of protein structure modification on protein-surfactant interfacial interactions. The adsorption at, and nonlinear dilatational rheological behavior of, the air-water interface were studied by combining drop shape analysis tensiometry, ellipsometry, and large-amplitude oscillatory dilatational rheology. Lissajous plots of surface pressure versus deformation were used to analyze the surface rheological response in terms of interfacial microstructure. The heat treatment generates a mixture of long fibrils and unconverted peptides. The presence of small peptides in 11S fibril samples resulted in a faster adsorption kinetics than that of native 11S. The addition of STE affected the adsorption of 11S significantly, whereas no apparent effect on the adsorption of the 11S fibril-peptide system was observed. The rheological response of interfaces stabilized by 11S-STE mixtures also differed significantly from the response for 11S fibril-peptide-STE mixtures. For 11S, the STE reduces the degree of strain hardening in extension and increases strain hardening in compression, suggesting the interfacial structure may change from a surface gel to a mixed phase of protein patches and STE domains. The foams generated from the mixtures displayed comparable foam stability to that of pure 11S. For 11S fibril-peptide mixtures STE only significantly affects the response in extension, where the degree of strain softening is decreased compared to the pure fibril-peptide system. The foam stability of the fibril-peptide system was significantly reduced by STE. These findings indicate that fibrillization of globular proteins could be a potential strategy to modify the complex surface and foaming behaviors of protein-surfactant mixtures.

  2. Exploring the Plant–Microbe Interface by Profiling the Surface-Associated Proteins of Barley Grains

    DEFF Research Database (Denmark)

    Sultan, Abida; Andersen, Birgit; Svensson, Birte

    2016-01-01

    -associated proteins and xylanolytic activities of two barley cultivars. The surface-associated proteome was dominated by plant proteins with roles in defense and stress-responses, while the relatively less abundant microbial (bacterial and fungal) proteins were involved in cell-wall and polysaccharide degradation...... and included xylanases. The surface-associated proteomes showed elevated xylanolytic activity and contained several xylanases. Integration of proteomics with enzyme assays is a powerful tool for analysis and characterization of the interaction between microbial consortia and plants in their natural environment....

  3. The effect of polymer surface modification on polymer-protein interaction via interfacial polymerization and hydrophilic polymer grafting

    Science.gov (United States)

    Protein membrane separation is prone to fouling on the membrane surface resulting from protein adsorption onto the surface. Surface modification of synthetic membranes is one way to reduce fouling. We investigated surface modification of polyethersulfone (PES) as a way of improving hydrophilicity ...

  4. Deposition at glancing angle, surface roughness, and protein adsorption: Monte Carlo simulations.

    Science.gov (United States)

    Zhdanov, Vladimir P; Rechendorff, Kristian; Hovgaard, Mads B; Besenbacher, Flemming

    2008-06-19

    To generate rough surfaces in Monte Carlo simulations, we use the 2 + 1 solid-on-solid model of deposition with rapid transient diffusion of newly arrived atoms supplied at glancing angle. The surfaces generated are employed to scrutinize the effect of surface roughness on adsorption of globular and anisotropic rodlike proteins. The obtained results are compared with the available experimental data for Ta deposition at glancing angle and for the bovine serum albumin and fibrinogen uptake on the corresponding Ta films.

  5. Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

    Science.gov (United States)

    Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2013-11-01

    Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

  6. Identification and characterization of the surface-layer protein of Clostridium tetani.

    Science.gov (United States)

    Qazi, Omar; Brailsford, Alan; Wright, Anne; Faraar, Jeremy; Campbell, Jim; Fairweather, Neil

    2007-09-01

    Many bacterial species produce a paracrystalline layer, the surface layer, which completely surrounds the exterior of the cell. In some bacteria, the surface layer is implicated in pathogenesis. Two proteins present in cell wall extracts from Clostridium tetani have been investigated and identified one of these has been unambiguously as the surface-layer protein (SLP). The gene, slpA, has been located in the genome of C. tetani E88 that encodes the SLP. The molecular mass of the protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is considerably larger than that predicted from the gene; however the protein does not appear to be glycosylated. Furthermore, analysis of five C. tetani strains, including three recent clinical isolates, shows considerable variation in the sizes of the SLP.

  7. High throughput atmospheric pressure plasma-induced graft polymerization for identifying protein-resistant surfaces.

    Science.gov (United States)

    Gu, Minghao; Kilduff, James E; Belfort, Georges

    2012-02-01

    Three critical aspects of searching for and understanding how to find highly resistant surfaces to protein adhesion are addressed here with specific application to synthetic membrane filtration. They include the (i) discovery of a series of previously unreported monomers from a large library of monomers with high protein resistance and subsequent low fouling characteristics for membrane ultrafiltration of protein-containing fluids, (ii) development of a new approach to investigate protein-resistant mechanisms from structure-property relationships, and (iii) adaptation of a new surface modification method, called atmospheric pressure plasma-induced graft polymerization (APP), together with a high throughput platform (HTP), for low cost vacuum-free synthesis of anti-fouling membranes. Several new high-performing chemistries comprising two polyethylene glycol (PEG), two amines and one zwitterionic monomers were identified from a library (44 commercial monomers) of five different classes of monomers as strong protein-resistant monomers. Combining our analysis here, using the Hansen solubility parameters (HSP) approach, and data from the literature, we conclude that strong interactions with water (hydrogen bonding) and surface flexibility are necessary for producing the highest protein resistance. Superior protein-resistant surfaces and subsequent anti-fouling performance was obtained with the HTP-APP as compared with our earlier HTP-photo graft-induced polymerization (PGP).

  8. Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

    Directory of Open Access Journals (Sweden)

    Han Lanlan

    2011-10-01

    Full Text Available Abstract Background Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB. In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. Results Multiple sequence alignment revealed that the C-terminal region (LcsB of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP or beta-galactosidase (Gal was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. Conclusion The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological

  9. Protein arrangement on modified diamond-like carbon surfaces – An ARXPS study

    Energy Technology Data Exchange (ETDEWEB)

    Oosterbeek, Reece N., E-mail: reece.oosterbeek@auckland.ac.nz [Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019 (New Zealand); Seal, Christopher K. [Light Metals Research Centre, The University of Auckland, Private Bag 92019 (New Zealand); Hyland, Margaret M. [Department of Chemical and Materials Engineering, The University of Auckland, Private Bag 92019 (New Zealand)

    2014-12-01

    Highlights: • DLC coatings were modified by Ar{sup +} ion sputtering and laser graphitisation. • The surface properties of the coatings were measured, and it was found that the above methods increased sp{sup 2} content and altered surface energy. • ARXPS was used to observe protein arrangement on the surface. • Polar CO/CN groups were seen to be segregated towards the interface, indicating they play an important role in bonding. • This segregation increased with increasing polar surface energy, indicating an increased net attraction between polar groups. - Abstract: Understanding the nature of the interface between a biomaterial implant and the biological fluid is an essential step towards creating improved implant materials. This study examined a diamond-like carbon coating biomaterial, the surface energy of which was modified by Ar{sup +} ion sputtering and laser graphitisation. The arrangement of proteins was analysed by angle resolved X-ray photoelectron spectroscopy, and the effects of the polar component of surface energy on this arrangement were observed. It was seen that polar groups (such as CN, CO) are more attracted to the coating surface due to the stronger polar interactions. This results in a segregation of these groups to the DLC–protein interface; at increasing takeoff angle (further from to DLC–protein interface) fewer of these polar groups are seen. Correspondingly, groups that interact mainly by dispersive forces (CC, CH) were found to increase in intensity as takeoff angle increased, indicating they are segregated away from the DLC–protein interface. The magnitude of the segregation was seen to increase with increasing polar surface energy, this was attributed to an increased net attraction between the solid surface and polar groups at higher polar surface energy (γ{sub S}{sup p})

  10. An Investigation of Freezing of Supercooled Water on Anti-Freeze Protein Modified Surfaces

    Institute of Scientific and Technical Information of China (English)

    Thibaut V J Charpentier; Anne Neville; Paul Millner; Rob Hewson; Ardian Morina

    2013-01-01

    This work investigates how functionalization ofaluminium surfaces with natural type Ⅲ Anti-Freeze Protein (AFP) affects the mechanism of heterogeneous ice nucleation.First the bulk ice nucleation properties of distilled water and aqueous solution of AFP were evaluated by differential scanning calorimetry.Then the modified surface was characterized by Secondary Ions Mass Spectroscopy (SIMS),Fourier Transform InfraRed (FTIR) spectroscopy and contact angle measurement.Freezing experiments were then conducted in which water droplets underwent a slow controlled cooling.This study shows that compared to uncoated aluminium,the anti-freeze proteins functionalized surfaces exhibit a higher and narrower range of freezing temperature.It was found that these proteins that keep living organisms from freezing in cold environment act in the opposite way once immobilized on surfaces by promoting ice nucleation.Some suggestions regarding the mechanism of action of the observed phenomena were proposed based on the Classical Nucleation Theory (CNT).

  11. Isolation of two biologically active cell surface proteins from Brucella abortus by chromatofocusing

    Energy Technology Data Exchange (ETDEWEB)

    Tabatabai, L.B.; Deyoe, B.L.

    1983-01-01

    Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pI 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized (/sup 125/I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis.

  12. Monte Carlo study of the molecular mechanisms of surface-layer protein self-assembly

    Science.gov (United States)

    Horejs, Christine; Mitra, Mithun K.; Pum, Dietmar; Sleytr, Uwe B.; Muthukumar, Murugappan

    2011-03-01

    The molecular mechanisms guiding the self-assembly of proteins into functional or pathogenic large-scale structures can be only understood by studying the correlation between the structural details of the monomer and the eventual mesoscopic morphologies. Among the myriad structural details of protein monomers and their manifestations in the self-assembled morphologies, we seek to identify the most crucial set of structural features necessary for the spontaneous selection of desired morphologies. Using a combination of the structural information and a Monte Carlo method with a coarse-grained model, we have studied the functional protein self-assembly into S(surface)-layers, which constitute the crystallized outer most cell envelope of a great variety of bacterial cells. We discover that only few and mainly hydrophobic amino acids, located on the surface of the monomer, are responsible for the formation of a highly ordered anisotropic protein lattice. The coarse-grained model presented here reproduces accurately many experimentally observed features including the pore formation, chemical description of the pore structure, location of specific amino acid residues at the protein-protein interfaces, and surface accessibility of specific amino acid residues. In addition to elucidating the molecular mechanisms and explaining experimental findings in the S-layer assembly, the present work offers a tool, which is chemical enough to capture details of primary sequences and coarse-grained enough to explore morphological structures with thousands of protein monomers, to promulgate design rules for spontaneous formation of specific protein assemblies.

  13. Mechanistic aspects of protein corona formation: insulin adsorption onto gold nanoparticle surfaces

    Science.gov (United States)

    Grass, Stefan; Treuel, Lennart

    2014-02-01

    In biological fluids, an adsorption layer of proteins, a "protein corona" forms around nanoparticles (NPs) largely determining their biological identity. In many interactions with NPs proteins can undergo structural changes. Here, we study the adsorption of insulin onto gold NPs (mean hydrodynamic particle diameter 80 ± 18 nm), focusing on the structural consequences of the adsorption process for the protein. We use surface enhanced Raman scattering (SERS) spectroscopy to study changes in the protein's secondary structure as well as the impact on integrity and conformations of disulfide bonds immediately on the NP surface. A detailed comparison to SERS spectra of cysteine and cystine provides first mechanistic insights into the causes for these conformational changes. Potential biological and toxicological implications of these findings are also discussed.

  14. Phagocytosis escape by a Staphylococcus aureus protein that connects complement and coagulation proteins at the bacterial surface.

    Science.gov (United States)

    Ko, Ya-Ping; Kuipers, Annemarie; Freitag, Claudia M; Jongerius, Ilse; Medina, Eva; van Rooijen, Willemien J; Spaan, András N; van Kessel, Kok P M; Höök, Magnus; Rooijakkers, Suzan H M

    2013-01-01

    Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a 'capsule'-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.

  15. Shear rheology of mixed protein adsorption layers vs their structure studied by surface force measurements.

    Science.gov (United States)

    Danov, Krassimir D; Kralchevsky, Peter A; Radulova, Gergana M; Basheva, Elka S; Stoyanov, Simeon D; Pelan, Eddie G

    2015-08-01

    The hydrophobins are proteins that form the most rigid adsorption layers at liquid interfaces in comparison with all other investigated proteins. The mixing of hydrophobin HFBII with other conventional proteins is expected to reduce the surface shear elasticity and viscosity, E(sh) and η(sh), proportional to the fraction of the conventional protein. However, the experiments show that the effect of mixing can be rather different depending on the nature of the additive. If the additive is a globular protein, like β-lactoglobulin and ovalbumin, the surface rigidity is preserved, and even enhanced. The experiments with separate foam films indicate that this is due to the formation of a bilayer structure at the air/water interface. The more hydrophobic HFBII forms the upper layer adjacent to the air phase, whereas the conventional globular protein forms the lower layer that faces the water phase. Thus, the elastic network formed by the adsorbed hydrophobin remains intact, and even reinforced by the adjacent layer of globular protein. In contrast, the addition of the disordered protein β-casein leads to softening of the HFBII adsorption layer. Similar (an even stronger) effect is produced by the nonionic surfactant Tween 20. This can be explained with the penetration of the hydrophobic tails of β-casein and Tween 20 between the HFBII molecules at the interface, which breaks the integrity of the hydrophobin interfacial elastic network. The analyzed experimental data for the surface shear rheology of various protein adsorption layers comply with a viscoelastic thixotropic model, which allows one to determine E(sh) and η(sh) from the measured storage and loss moduli, G' and G″. The results could contribute for quantitative characterization and deeper understanding of the factors that control the surface rigidity of protein adsorption layers with potential application for the creation of stable foams and emulsions with fine bubbles or droplets.

  16. Insights into cellulase-lignin non-specific binding revealed by computational redesign of the surface of green fluorescent protein: Protein Redesign to Lower Protein-lignin Binding

    Energy Technology Data Exchange (ETDEWEB)

    Haarmeyer, Carolyn N. [Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing Michigan 48824; Smith, Matthew D. [Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing Michigan 48824; Chundawat, Shishir P. S. [Great Lakes Bioenergy Research Center (GLBRC), Michigan State University, East Lansing Michigan; Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway New Jersey; Sammond, Deanne [Biosciences Center, National Renewable Energy Laboratory, Golden Colorado; Whitehead, Timothy A. [Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing Michigan 48824; Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing Michigan 48824

    2016-11-07

    Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active

  17. Investigation of cellular and protein interactions with model self-assembled monolayer surfaces

    Science.gov (United States)

    Tegoulia, Vassiliki Apostolou

    Self-assembled monolayers (SAMs) of alkanethiolates on gold have been used to investigate the effect of substrate surface properties on bacterial and blood cell adhesion in the presence and absence of blood proteins. Protein adsorption and binding strength on SAMs as well as complement activation by these model surfaces were also studied. It is hoped that information gained, regarding factors that influence biological processes, will lead to strategies for designing materials and surfaces that specifically inhibit cell adhesion and protein adsorption. Single component SAMs of the general formula HS(CH2) 10X, where X = CH3, CH2OH. COOH and CH2(OCH 2CH2)3OH, and two component mixed SAMs created from binary solutions of HS(CH2), OCH3 and HS(CH 2)10CH2OH, were used. Adhesion was investigated under well-defined flow conditions. Adhesion was found to be higher for the hydrophobic methyl and minimal for the tri(ethyleneoxide) terminated SAM. Preincubation of the SAMs with fibrinogen led to an increase in cell adhesion for bacteria and a decrease for leukocyte adhesion. The effect of surface chemistry on protein adsorption was studied for three blood proteins, fibrinogen, fibronectin and albumin. Adsorption was found to be higher on the hydrophobic CH3 surface and lower but comparable for the other surfaces while proteins adsorbed strongly on all surfaces. SAMs were also used to evaluate complement activation by foreign surfaces. The hydroxyl rich SAMs were found to activate complement more significantly than the anionic carboxyl and the hydrophobic methyl terminated SAMs. A surface modification was introduced to incorporate a zwitterionic phosphorylcholine (PC) group on a hydroxyl monolayer in an effort to create a biomimetic surface that could minimize cell adhesion and protein adsorption. The good antifouling properties of the phosphorylcholine modified surface led to the synthesis of a novel phosphorylcholine functionalized thiol. Single component and two component

  18. Protein-nanoparticle interactions: the effects of surface compositional and structural heterogeneity are scale dependent

    Science.gov (United States)

    Huang, Rixiang; Carney, Randy P.; Stellacci, Francesco; Lau, Boris L. T.

    2013-07-01

    Nanoparticles (NPs) in the biological environment are exposed to a large variety and concentration of proteins. Proteins are known to adsorb in a `corona' like structure on the surface of NPs. In this study, we focus on the effects of surface compositional and structural heterogeneity on protein adsorption by examining the interaction of self-assembled monolayer coated gold NPs (AuNPs) with two types of proteins: ubiquitin and fibrinogen. This work was designed to systematically investigate the role of surface heterogeneity in nanoparticle-protein interaction. We have chosen the particles as well as the proteins to provide different types (in distribution and length-scale) of heterogeneity. The goal was to unveil the role of heterogeneity and of its length-scale in the particle-protein interaction. Dynamic light scattering and circular dichroism spectroscopy were used to reveal different interactions at pH above and below the isoelectric points of the proteins, which is related to the charge heterogeneity on the protein surface. At pH 7.4, there was only a monolayer of proteins adsorbed onto the NPs and the secondary structure of proteins remained intact. At pH 4.0, large aggregates of nanoparticle-protein complexes were formed and the secondary structures of the proteins were significantly disrupted. In terms of interaction thermodynamics, results from isothermal titration calorimetry showed that ubiquitin adsorbed differently onto (1) AuNPs with charged and nonpolar terminals organized into nano-scale structure (66-34 OT), (2) AuNPs with randomly distributed terminals (66-34 brOT), and (3) AuNPs with homogeneously charged terminals (MUS). This difference in adsorption behavior was not observed when AuNPs interacted with fibrinogen. The results suggested that the interaction between the proteins and AuNPs was influenced by the surface heterogeneity on the AuNPs, and this influence depends on the scale of surface heterogeneity and the size of the proteins

  19. Maintaining solvent accessible surface area under rotamer substitution for protein design.

    Science.gov (United States)

    Leaver-Fay, Andrew; Butterfoss, Glenn L; Snoeyink, Jack; Kuhlman, Brian

    2007-06-01

    Although quantities derived from solvent accessible surface areas (SASA) are useful in many applications in protein design and structural biology, the computational cost of accurate SASA calculation makes SASA-based scores difficult to integrate into commonly used protein design methodologies. We demonstrate a method for maintaining accurate SASA during a Monte Carlo search of sequence and rotamer space for a fixed protein backbone. We extend the fast Le Grand and Merz algorithm (Le Grand and Merz, J Comput Chem, 14, 349), which discretizes the solvent accessible surface for each atom by placing dots on a sphere and combines Boolean masks to determine which dots are exposed. By replacing semigroup operations with group operations (from Boolean logic to counting dot coverage) we support SASA updates. Our algorithm takes time proportional to the number of atoms affected by rotamer substitution, rather than the number of atoms in the protein. For design simulations with a one hundred residue protein our approach is approximately 145 times faster than performing a Le Grand and Merz SASA calculation from scratch following each rotamer substitution. To demonstrate practical effectiveness, we optimize a SASA-based measure of protein packing in the complete redesign of a large set of proteins and protein-protein interfaces.

  20. Characteristics of surface layer proteins from two new and native strains of Lactobacillus brevis.

    Science.gov (United States)

    Mobarak Qamsari, Elahe; Kasra Kermanshahi, Rouha; Erfan, Mohammad; Ghadam, Parinaz; Sardari, Soroush; Eslami, Neda

    2017-02-01

    In this work, some important characteristics of surface layer (S-layer) proteins extracted from two new and native Lactobacillus strains, L.brevis KM3 and L.brevis KM7, were investigated. The presence of S-layer on the external surface of L.brevis KM3 was displayed by thin sectioning and negative staining. SDS-PAGE analysis were shown same dominant protein bands approximately around 48kDa for both S-layer proteins. Moreover, the S-layer reappeared when LiCl treated cells were allowed to grow again. Protein secondary structure and thermal behavior were evaluated by using circular dichroism (CD) and differential scanning calorimetry (DSC), respectively. Both S-layer proteins had high content of β-sheet and low amount of α-helix. The thermograms of lyophilized S-layer proteins of L.brevis KM3 and L.brevis KM7 showed one transition peak at 67.9°C and 59.14°C, respectively. To determine monodispersity of extracted S-layer proteins, dynamic light scattering (DLS) was used. The results indicated that the main population of S-layer molecules in two tested lactobacillus strains were composed of monomer with an expected diameter close to 10nm. Furthermore, Zeta potential measurements were showed positive potential for both S-layer proteins, as expected. Our results could be used as the basis for biotechnological applications of these two new S-layer proteins.

  1. Correlation of average hydrophobicity, water/air interface surface rheological properties and foaming properties of proteins.

    Science.gov (United States)

    Medrano, A; Abirached, C; Araujo, A C; Panizzolo, L A; Moyna, P; Añón, M C

    2012-04-01

    A comparative study on the behavior in the air-water interface of β-lactoglobulin, α-lactoalbumin, glycinin and β-conglycinin was performed. The behavior at the interface was evaluated by equilibrium surface tension and surface rheological properties of adsorbed films. There were significant differences (α ≤ 0.05) in the values of the constants of adsorption to the interface of the four proteins. The glycinin had the slowest rate of adsorption, due to its low average hydrophobicity, low molecular flexibility and large molecular size. Smaller proteins like β-lactoglobulin and α-lactoalbumin tended to greater equilibrium pressure values than the larger proteins because of its higher rate of adsorption to the interface. The foam capacity of proteins showed a positive correlation with the average hydrophobicity; the maximal retained liquid volume or the initial rate of passage of liquid to foam were significantly lower (α ≤ 0.05) when protein was glycinin. The dilatational modulus of glycinin was the lowest, which implies lowest resistance to disruption of the film. Glycinin protein has lower proportion of gravitational drainage and higher disproportionation having perhaps a less resistant film. In conclusion, β-conglycinin and whey proteins showed a similar behavior, so β-conglycinin might be the best soybean protein to replace milk proteins in food formulations.

  2. Development, Characterization, and Optimization of Protein Level in Date Bars Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Muhammad Nadeem

    2012-01-01

    Full Text Available This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM. Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables.

  3. Isolation of cell surface proteins for mass spectrometry-based proteomics.

    Science.gov (United States)

    Elschenbroich, Sarah; Kim, Yunee; Medin, Jeffrey A; Kislinger, Thomas

    2010-02-01

    Defining the cell surface proteome has profound importance for understanding cell differentiation and cell-cell interactions, as well as numerous pathogenic abnormalities. Owing to their hydrophobic nature, plasma membrane proteins that reside on the cell surface pose analytical challenges and, despite efforts to overcome difficulties, remain under-represented in proteomic studies. Limitations in the classically employed ultracentrifugation-based approaches have led to the invention of more elaborate techniques for the purification of cell surface proteins. Three of these methods--cell surface coating with cationic colloidal silica beads, biotinylation and chemical capture of surface glycoproteins--allow for marked enrichment of this subcellular proteome, with each approach offering unique advantages and characteristics for different experiments. In this article, we introduce the principles of each purification method and discuss applications from the recent literature.

  4. Wang-Landau sampling of the interplay between surface adsorption and folding of HP lattice proteins

    Energy Technology Data Exchange (ETDEWEB)

    Li, Ying Wai [ORNL; Wuest, Thomas [Swiss Federal Research Institute, Switzerland; Landau, David P [University of Georgia, Athens, GA

    2014-01-01

    Generic features associated with the adsorption of proteins on solid surfaces are reviewed within the framework of the hydrophobic-polar (HP) lattice protein model. The thermodynamic behavior and structural properties of various HP protein sequences interacting with attractive surfaces have been studied using extensive Wang-Landau sampling with different types of surfaces, each of which attracts either: all monomers, only hydrophobic (H) monomers, or only polar (P) monomers, respectively. Consequently, different types of folding behavior occur for varied surface strengths. Analysis of the combined patterns of various structural observables, e.g., the derivatives of the numbers of interaction contacts, together with the specific heat, leads to the identification of fundamental categories of folding and transition hierarchies. We also inferred a connection between the transition categories and the relative surface strengths, i.e., the ratios of the surface attractive strengths to the intra-chain attraction among H monomers. We thus believe that the folding hierarchies and identification scheme are generic for different HP sequences interacting with attractive surfaces, regardless of the chain length, sequence, or surface attraction.

  5. Detecting local ligand-binding site similarity in nonhomologous proteins by surface patch comparison.

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2012-04-01

    Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations.

  6. The ability of IgY to recognize surface proteins of Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    Basri A. Gani

    2009-12-01

    Full Text Available Background: Streptococcus mutans are gram positive bacteria classified into viridians group, and have a role in pathogenesis of dental caries. It’s adhesion to the tooth surface is mediated by cell surface proteins, which interact with specific receptor located in tooth pellicle. Glucan binding protein, Glukosyltransferase, and antigen I/II are basic proteins of S. mutans, which have a role in initiating the interaction. A previous study showed that chicken’s IgY can interfere the interaction. Purpose: The objective of this study was to assess the ability of IgY in recognizing the surface molecule of Streptococcus mutans expressed by various serotypes (c, d, e, f and a strain derived from IPB, Bogor. Method: Western blot was used as a method to determine such capability. Result: The result showed that IgY has a potency to recognize antigen I/II, but not the other proteins on the cell surface of all bacteria tested. Conclusion: The ability of IgY to bind the surface protein, antigen I/II, indicates that this avian antibody could be used as a candidate for anti-adhesion in preventing dental caries.

  7. Mitogen-activated protein kinases mediate Mycobacterium tuberculosis–induced CD44 surface expression in monocytes

    Indian Academy of Sciences (India)

    Natarajan Palaniappan; S Anbalagan; Sujatha Narayanan

    2012-03-01

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.

  8. Interaction of Moringa oleifera seed protein with a mineral surface and the influence of surfactants.

    Science.gov (United States)

    Kwaambwa, Habauka M; Hellsing, Maja S; Rennie, Adrian R; Barker, Robert

    2015-06-15

    The paper describes the adsorption of purified protein from seeds of Moringa oleifera to a sapphire interface and the effects of addition of the anionic surfactant sodium dodecylsulfate (SDS) and the cationic surfactant hexadecyltrimethylammonium bromide (CTAB). Neutron reflection was used to determine the structure and composition of interfacial layers adsorbed at the solid/solution interface. The maximum surface excess of protein was found to be about 5.3 mg m(-2). The protein does not desorb from the solid/liquid interface when rinsed with water. Addition of SDS increases the reflectivity indicating co-adsorption. It was observed that CTAB is able to remove the protein from the interface. The distinct differences to the behavior observed previously for the protein at the silica/water interface are identified. The adsorption of the protein to alumina in addition to other surfaces has shown why it is an effective flocculating agent for the range of impurities found in water supplies. The ability to tailor different surface layers in combination with various surfactants also offers the potential for adsorbed protein to be used in separation technologies.

  9. Selective radiolabeling of cell surface proteins to a high specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, J.A.; Lau, A.L.; Cunningham, D.D.

    1987-02-10

    A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with /sup 125/I-(hydroxyphenyl)propionyl groups via /sup 125/I-sulfosuccinimidyl (hydroxyphenyl)propionate (/sup 125/II-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-(/sup 125/I)iodide labeling technique revealed that /sup 125/I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with /sup 125/I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spacial orientations on erythroctyes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane.

  10. Binding of Solvent Molecules to a Protein Surface in Binary Mixtures Follows a Competitive Langmuir Model.

    Science.gov (United States)

    Kulschewski, Tobias; Pleiss, Jürgen

    2016-09-06

    The binding of solvent molecules to a protein surface was modeled by molecular dynamics simulations of of Candida antarctica (C. antarctica) lipase B in binary mixtures of water, methanol, and toluene. Two models were analyzed: a competitive Langmuir model which assumes identical solvent binding sites with a different affinity toward water (KWat), methanol (KMet), and toluene (KTol) and a competitive Langmuir model with an additional interaction between free water and already bound water (KWatWat). The numbers of protein-bound molecules of both components of a binary mixture were determined for different compositions as a function of their thermodynamic activities in the bulk phase, and the binding constants were simultaneously fitted to the six binding curves (two components of three different mixtures). For both Langmuir models, the values of KWat, KMet, and KTol were highly correlated. The highest binding affinity was found for methanol, which was almost 4-fold higher than the binding affinities of water and toluene (KMet ≫ KWat ≈ KTol). Binding of water was dominated by the water-water interaction (KWatWat). Even for the three protein surface patches of highest water affinity, the binding affinity of methanol was 2-fold higher than water and 8-fold higher than toluene (KMet > KWat > KTol). The Langmuir model provides insights into the protein destabilizing mechanism of methanol which has a high binding affinity toward the protein surface. Thus, destabilizing solvents compete with intraprotein interactions and disrupt the tertiary structure. In contrast, benign solvents such as water or toluene have a low affinity toward the protein surface. Water is a special solvent: only few water molecules bind directly to the protein; most water molecules bind to already bound water molecules thus forming water patches. A quantitative mechanistic model of protein-solvent interactions that includes competition and miscibility of the components contributes a robust basis

  11. Influence of surface modification and static pressure on microdialysis protein extraction efficiency.

    Science.gov (United States)

    Chu, Jiangtao; Undin, Torgny; Lind, Sara Bergström; Hjort, Klas; Dahlin, Andreas P

    2015-10-01

    There is growing interest in using microdialysis (MD) for monitoring larger and more complex molecules such as neuropeptides and proteins. This promotes the use of MD membranes with molecular weight cut off (MWCO) of 100 kDa or above. The hydrodynamic property of the membrane goes to ultrafiltration or beyond, making the MD catheters more sensitive to pressure. In the meantime, despite the large pore size, studies have shown that membrane biofouling still lead to unstable catheter performance. The objective is to study in vitro how 500 kDa dextran and Poloxamer 407 surface modification affect the fluid recovery (FR) and extraction efficiency (EE) of 100 kDa MWCO MD catheters. A pressure chamber was designed to facilitate the tests, using as MD sample a protein standard with similar concentrations as in human cerebral spinal fluid, comparing native and Poloxamer 407 modified MD catheters. The collected dialysate fractions were examined for FR and protein EE, employing Dot-it Spot-it Protein Assay for total protein EE and targeted mass spectrometry (MS) for EE of individual proteins and peptides. The FR results suggested that the surface modified catheters were less sensitive to the pressure and provide higher precision, and provided a FR closer to 100%. The surface modification did not show a significant effect on the protein EE. The average total protein EE of surface modified catheters was slightly higher than that of the native ones. The MS EE data of individual proteins showed a clear trend of complex response in EE with pressure.

  12. Droplet evaporation of pure water and protein solution on nanostructured superhydrophobic surfaces of varying heights.

    Science.gov (United States)

    Choi, Chang-Hwan; Kim, Chang-Jin C J

    2009-07-07

    Evaporation of liquids on substrates is important for many applications including lab-on-a-chip, especially when they are in droplets. Unlike on planar substrates, droplet evaporation on micropatterned substrates has been studied only recently and none so far on nanopatterns. Driven by the applicability of nanostructured surfaces to biomaterials and tissue engineering, we report on the evaporative process of sessile droplets of pure water and a protein solution on superhydrophobic surfaces of sharp-tip post structures in a submicrometer pitch (230 nm) and varying heights (100-500 nm). We find that the nanotopographical three-dimensionalities such as structural height and sidewall profile affect the surface superhydrophobicity in such a way that only tall and slender nanostructures provide the surface with great superhydrophobicity (a contact angle more than 170 degrees). The evaporation process was different between the pure water and the protein solution; unlike pure water, a significant contact-line spreading and pinning effect was observed in a droplet of a protein solution with an intermediate transition from a dewetting (Cassie) to a wetting (Wenzel) state. Enabled by well-defined nanostructures, our results highlight that the surface superhydrophobicity and the droplet evaporation are significantly affected by the three-dimensional nanometric topography and the surface fouling such as protein adsorption.

  13. The surface-associated protein of Staphylococcus saprophyticus is a lipase.

    Science.gov (United States)

    Sakinc, Türkan; Woznowski, Magdalena; Ebsen, Michael; Gatermann, Sören G

    2005-10-01

    Staphylococcus saprophyticus surface-associated protein (Ssp) was the first surface protein described for this organism. Ssp-positive strains display a fuzzy layer of surface-associated material in electron micrographs, whereas Ssp-negative strains appear to be smooth. The physiologic function of Ssp, however, has remained elusive. To clone the associated gene, we determined the N-terminal sequence, as well as an internal amino acid sequence, of the purified protein. We derived two degenerate primers from these peptide sequences, which we used to identify the ssp gene from genomic DNA of S. saprophyticus 7108. The gene was cloned by PCR techniques and was found to be homologous to genes encoding staphylococcal lipases. In keeping with this finding, strains 7108 and 9325, which are Ssp positive, showed lipase activity on tributyrylglycerol agar plates, whereas the Ssp-negative strain CCM883 did not. Association of enzyme activity with the cloned DNA was proven by introducing the gene into Staphylococcus carnosus TM300. When wild-type strain 7108 and an isogenic mutant were analyzed by transmission electron microscopy, strain 7108 exhibited the fuzzy surface layer, whereas the mutant appeared to be smooth. Lipase activity and the surface appendages could be restored by reintroduction of the cloned gene into the mutant. Experiments using immobilized collagen type I did not provide evidence for the involvement of Ssp in adherence to this matrix protein. Our experiments thus provided evidence that Ssp is a surface-associated lipase of S. saprophyticus.

  14. Surfing the Protein-Protein Interaction Surface Using Docking Methods: Application to the Design of PPI Inhibitors

    Directory of Open Access Journals (Sweden)

    Rushikesh Sable

    2015-06-01

    Full Text Available Blocking protein-protein interactions (PPI using small molecules or peptides modulates biochemical pathways and has therapeutic significance. PPI inhibition for designing drug-like molecules is a new area that has been explored extensively during the last decade. Considering the number of available PPI inhibitor databases and the limited number of 3D structures available for proteins, docking and scoring methods play a major role in designing PPI inhibitors as well as stabilizers. Docking methods are used in the design of PPI inhibitors at several stages of finding a lead compound, including modeling the protein complex, screening for hot spots on the protein-protein interaction interface and screening small molecules or peptides that bind to the PPI interface. There are three major challenges to the use of docking on the relatively flat surfaces of PPI. In this review we will provide some examples of the use of docking in PPI inhibitor design as well as its limitations. The combination of experimental and docking methods with improved scoring function has thus far resulted in few success stories of PPI inhibitors for therapeutic purposes. Docking algorithms used for PPI are in the early stages, however, and as more data are available docking will become a highly promising area in the design of PPI inhibitors or stabilizers.

  15. Nanometer polymer surface features: the influence on surface energy, protein adsorption and endothelial cell adhesion

    Science.gov (United States)

    Carpenter, Joseph; Khang, Dongwoo; Webster, Thomas J.

    2008-12-01

    Current small diameter (collagen type IV) adsorption. Furthermore, this study provided evidence that the vertical dimension of nanometer surface features, rather than the lateral dimension, is largely responsible for these increases. In this manner, this study provides key design parameters that may promote vascular graft efficacy.

  16. In various protein complexes, disordered protomers have large per-residue surface areas and area of protein-, DNA- and RNA-binding interfaces.

    Science.gov (United States)

    Wu, Zhonghua; Hu, Gang; Yang, Jianyi; Peng, Zhenling; Uversky, Vladimir N; Kurgan, Lukasz

    2015-09-14

    We provide first large scale analysis of the peculiarities of surface areas of 5658 dissimilar (below 50% sequence similarity) proteins with known 3D-structures that bind to proteins, DNA or RNAs. We show here that area of the protein surface is highly correlated with the protein length. The size of the interface surface is only modestly correlated with the protein size, except for RNA-binding proteins where larger proteins are characterized by larger interfaces. Disordered proteins with disordered interfaces are characterized by significantly larger per-residue areas of their surfaces and interfaces when compared to the structured proteins. These result are applicable for proteins involved in interaction with DNA, RNA, and proteins and suggest that disordered proteins and binding regions are less compact and more likely to assume extended shape. We demonstrate that disordered protein binding residues in the interfaces of disordered proteins drive the increase in the per residue area of these interfaces. Our results can be used to predict in silico whether a given protomer from the DNA, RNA or protein complex is likely to be disordered in its unbound form.

  17. Truncation of merozoite surface protein 3 disrupts its trafficking and that of acidic-basic repeat protein to the surface of Plasmodium falciparum merozoites.

    Science.gov (United States)

    Mills, Kerry E; Pearce, J Andrew; Crabb, Brendan S; Cowman, Alan F

    2002-03-01

    Merozoite surface protein 3 (MSP3), an important vaccine candidate, is a soluble polymorphic antigen associated with the surface of Plasmodium falciparum merozoites. The MSP3 sequence contains three blocks of heptad repeats that are consistent with the formation of an intramolecular coiled-coil. MSP3 also contains a glutamic acid-rich region and a putative leucine zipper sequence at the C-terminus. We have disrupted the msp3 gene by homologous recombination, resulting in the expression of a truncated form of MSP3 that lacks the putative leucine zipper sequence but retains the glutamic acid-rich region and the heptad repeats. Here, we show that truncated MSP3, lacking the putative leucine zipper region, does not localize to the parasitophorous vacuole or interact with the merozoite surface. Furthermore, the acidic-basic repeat antigen (ABRA), which is present on the merozoite surface, also was not localized to the merozoite surface in parasites expressing the truncated form of MSP3. The P. falciparum merozoites lacking MSP3 and ABRA on the surface show reduced invasion into erythrocytes. These results suggest that MSP3 is not absolutely essential for blood stage growth and that the putative leucine zipper region is required for the trafficking of both MSP3 and ABRA to the parasitophorous vacuole.

  18. The influence of the surface properties of silicon-fluorine hydrogel on protein adsorption.

    Science.gov (United States)

    Xie, Haijiao; Zhao, Zhengbai; An, Shuangshuang; Jiang, Yong

    2015-12-01

    A range of fluorinated hydrogels were synthesized using the copolymerization of 1, 1, 1, 3, 3, 3-hexafluoroisopropyl methacrylate (HFMA) or 1H, 1H, 7H-dodecafluoroheptyl methacrylate (DFMA) with hydrophilic monomers. Bovine serum albumin (BSA) and Lysozyme (LZM) were chosen as model proteins to investigate the performance of protein adsorption on the surface of these fluorinated hydrogels. It was found that the performance of the fluorinated hydrogels toward protein adsorption was different for different proteins; simultaneously, the amount of protein adsorption was related to but not linear with the fluorine content on the hydrogel surface. With increasing HFMA content, the mass of BSA adsorption increased in the first stage and then decreased, meanwhile the mass of LZM adsorption exhibited an upward trend in general. In addition, the amount of protein adsorption was also related to the type and length of the fluorinated groups. The hydrogels made from DFMA behaved better than HFMA hydrogels in terms of reducing protein adsorption. This study might provide further reference in choosing fluorine monomer to prepare protein-repelling hydrogels.

  19. Predicting Cell Association of Surface-Modified Nanoparticles Using Protein Corona Structure - Activity Relationships (PCSAR).

    Science.gov (United States)

    Kamath, Padmaja; Fernandez, Alberto; Giralt, Francesc; Rallo, Robert

    2015-01-01

    Nanoparticles are likely to interact in real-case application scenarios with mixtures of proteins and biomolecules that will absorb onto their surface forming the so-called protein corona. Information related to the composition of the protein corona and net cell association was collected from literature for a library of surface-modified gold and silver nanoparticles. For each protein in the corona, sequence information was extracted and used to calculate physicochemical properties and statistical descriptors. Data cleaning and preprocessing techniques including statistical analysis and feature selection methods were applied to remove highly correlated, redundant and non-significant features. A weighting technique was applied to construct specific signatures that represent the corona composition for each nanoparticle. Using this basic set of protein descriptors, a new Protein Corona Structure-Activity Relationship (PCSAR) that relates net cell association with the physicochemical descriptors of the proteins that form the corona was developed and validated. The features that resulted from the feature selection were in line with already published literature, and the computational model constructed on these features had a good accuracy (R(2)LOO=0.76 and R(2)LMO(25%)=0.72) and stability, with the advantage that the fingerprints based on physicochemical descriptors were independent of the specific proteins that form the corona.

  20. Molecular kinetics of proteins at the surface of porcine sperm before and during fertilization.

    Science.gov (United States)

    Tsai, P S; Gadella, B M

    2009-01-01

    Fertilization is a decisive moment in life and enables the combination of the DNA from two gametes to ultimately form a new organism. The sperm surface, especially the head area, has distinguishable subdomains that are involved in distinct fertilization processes. It is known that the sperm head surface undergoes constant remodelling during epididymal maturation and migration in the male and female genital tract. But intriguingly, the identity, origin and spatial ordering of proteins at the sperm surface that are involved in mammalian fertilization are essentially unknown. This review deals with sperm surface protein modifications that are under somatic cell control. As soon as the sperm is released from the seminiferous tubules it is subjected to these modifications. These surface reorganisations continue until the sperm reside in the fallopian tube where they meet the oocyte and may fertilize it. Most likely, a selective process allows only functionally mature and intact sperm to optimally interact and fertilize the oocyte. Recent data suggest that even the perivitelline fluid is involved in sperm surface remodelling as it contains factors which could facilitate the first penetrating sperm to fertilize the oocyte. In this contribution, the kinetics of proteins at the sperm surface will be overviewed. Better understanding of this would help to design strategies to improve male fertility or to devise novel contraceptives.

  1. Protein adsorption at polymer-grafted surfaces : Comparison between a mixture of saliva proteins and some well-defined model proteins

    NARCIS (Netherlands)

    Kawasaki, K; Kambara, M; Matsumura, H; Norde, W

    2003-01-01

    Grafting a dense layer of soluble polymers onto a surface is a well-established method for controlling protein adsorption. In the present study, polyethylene oxide (PEO) layers of three different grafting densities were prepared, i.e. 10-15 nm(2), 5.5 nm(2) and 4 nm(2) per polymer chain, respectivel

  2. Protein adsorption at polymer-grafted surfaces: Comparison between a mixture of saliva proteins and some well-defined model proteins

    NARCIS (Netherlands)

    Kawasaki, K.; Kambara, M.; Matsumura, H.; Norde, W.

    2003-01-01

    Grafting a dense layer of soluble polymers onto a surface is a well-established method for controlling protein adsorption. In the present study, polyethylene oxide (PEO) layers of three different grafting densities were prepared, i.e. 10-15 nm2, 5.5 nm2 and 4 nm2 per polymer chain, respectively. The

  3. Surface Modification of Poly(dimethylsiloxane) Using Ionic Complementary Peptides to Minimize Nonspecific Protein Adsorption.

    Science.gov (United States)

    Yu, Xiaoling; Xiao, Junzhu; Dang, Fuquan

    2015-06-02

    Poly(dimethylsiloxane) (PDMS) has become a widely used material for microfluidic and biological applications. However, PDMS has unacceptably high levels of nonspecific protein adsorption, which significantly lowers the performance of PDMS-based microfluidic chips. Most existing methods to reduce protein fouling of PDMS are to make the surface more hydrophilic by surface oxidization, polymer grafting, and physisorbed coatings. These methods suffer from the relatively short-term stability, the multistep complex treatment procedure, or the insufficient adsorption reduction. Herein, we developed a novel and facile modification method based on self-assembled peptides with well-tailored amino acid composition and sequence, which can also interact strongly with the PDMS surface in the same way as proteins, for suppressing the nonspecific protein fouling and improving the biocompatibility of PDMS-based microfluidic chips. We first demonstrated that an ionic complementary peptide, EAR16-II with a sequence of [(Ala-Glu-Ala-Glu-Ala-Arg-Ala-Arg)2], can readily self-assemble into an amphipathic film predominantly composed of tightly packed β-sheets on the native hydrophobic and plasma-oxidized hydrophilic PDMS surfaces upon low concentrations of carbohydrates. The self-assembled EAR16-II amphipathic film exposed its hydrophobic side to the solution and thus rendered the PDMS surface hydrophobic with water contact angles (WCAs) of around 110.0°. However, the self-assembled EAR16-II amphipathic film exhibited excellent protein-repelling and blood compatibility properties comparable to or better than those obtained with previously reported methods. A schematic model has been proposed to explain the interactions of EAR16-II with the PDMS surface and the antifouling capability of EAR16-II coatings at a molecular level. The current work will pave the way to the development of novel coating materials to address the nonspecific protein adsorption on PDMS, thereby broadening the

  4. Display of recombinant proteins at the surface of lactic acid bacteria: strategies and applications.

    Science.gov (United States)

    Michon, C; Langella, P; Eijsink, V G H; Mathiesen, G; Chatel, J M

    2016-05-03

    Lactic acid bacteria (LAB) are promising vectors of choice to deliver active molecules to mucosal tissues. They are recognized as safe by the World Health Organization and some strains have probiotic properties. The wide range of potential applications of LAB-driven mucosal delivery includes control of inflammatory bowel disease, vaccine delivery, and management of auto-immune diseases. Because of this potential, strategies for the display of proteins at the surface of LAB are gaining interest. To display a protein at the surface of LAB, a signal peptide and an anchor domain are necessary. The recombinant protein can be attached to the membrane layer, using a transmembrane anchor or a lipoprotein-anchor, or to the cell wall, by a covalent link using sortase mediated anchoring via the LPXTG motif, or by non-covalent liaisons employing binding domains such as LysM or WxL. Both the stability and functionality of the displayed proteins will be affected by the kind of anchor used. The most commonly surfaced exposed recombinant proteins produced in LAB are antigens and antibodies and the most commonly used LAB are lactococci and lactobacilli. Although it is not necessarily so that surface-display is the preferred localization in all cases, it has been shown that for certain applications, such as delivery of the human papillomavirus E7 antigen, surface-display elicits better biological responses, compared to cytosolic expression or secretion. Recent developments include the display of peptides and proteins targeting host cell receptors, for the purpose of enhancing the interactions between LAB and host. Surface-display technologies have other potential applications, such as degradation of biomass, which is of importance for some potential industrial applications of LAB.

  5. Optimization of the Preparation of Fish Protein Anti-Obesity Hydrolysates Using Response Surface Methodology

    OpenAIRE

    2013-01-01

    The enzymatic condition for producing the anti-obesity hydrolysates from fish water-soluble protein was optimized with the aid of response surface methodology, which also derived a statistical model for experimental validation. Compared with neutral protease, papain and protamex, the porcine pancreas lipase inhibitory rate of hydrolysates from fish water-soluble protein was higher with alkaline protease. Results showed that the model terms were significant, the terms of lack of fit were not s...

  6. The impact of a carbon nanotube on the cholesterol domain localized on a protein surface

    CERN Document Server

    Gburski, Zygmunt; Raczynski, Przemyslaw; 10.1016/j.ssc.2009.12.005

    2011-01-01

    The influence of a single walled carbon nanotube on the structure of a cholesterol cluster (domain) developed over the surface of the endothelial protein 1LQV has been investigated using the classical molecular dynamics (MD) simulation technique. We have observed a substantial impact of carbon nanotube on the arrangement of the cholesterol domain. The carbon nanotube can drag out cholesterol molecules, remarkable reducing the volume of the domain settled down on the protein.

  7. Kinetic Control of Histidine-Tagged Protein Surface Density on Supported Lipid Bilayers

    Energy Technology Data Exchange (ETDEWEB)

    Nye, Jeffrey A. [Univ. of California, Berkeley, CA (United States); Groves, Jay T. [Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2008-02-28

    Nickel-chelating lipids are general tools for anchoring polyhistidine-tagged proteins to supported lipid bilayers (SLBs), but controversy exists over the stability of the protein-lipid attachment. In this study, we show that chelator lipids are suitable anchors for building stable, biologically active surfaces but that a simple Langmuirian model is insufficient to describe their behavior. Desorption kinetics from chelator lipids are governed by the valency of surface binding: monovalently bound proteins desorb within minutes (t1/2 ≈ 6 min), whereas polyvalently bound species remain bound for hours (t1/2 ≈ 12 h). Evolution between surface states is slow, so equilibrium is unlikely to be reached on experimental timescales. However, by tuning incubation conditions, the populations of each species can be kinetically controlled, providing a wide range of protein densities on SLBs with a single concentration of chelator lipid. In conclusion, we propose guidelines for the assembly of SLB surfaces functionalized with specific protein densities and demonstrate their utility in the formation of hybrid immunological synapses.

  8. Surface layer proteins isolated from Clostridium difficile induce clearance responses in macrophages.

    Science.gov (United States)

    Collins, Laura E; Lynch, Mark; Marszalowska, Izabela; Kristek, Maja; Rochfort, Keith; O'Connell, Mary; Windle, Henry; Kelleher, Dermot; Loscher, Christine E

    2014-05-01

    Clostridium difficile is the leading cause of hospital-acquired diarrhoea worldwide, and if the bacterium is not cleared effectively it can pose a risk of recurrent infections and complications such as colitis, sepsis and death. In this study we demonstrate that surface layer proteins from the one of the most frequently acquired strains of C. difficile, activate mechanisms in murine macrophage in vitro that are associated with clearance of bacterial infection. Surface layer proteins (SLPs) isolated from C. difficile induced the production of pro-inflammatory cytokines and chemokines and increased macrophage migration and phagocytotic activity in vitro. Furthermore, we also observed up-regulation of a number of cell surface markers on the macrophage, which are important in pathogen recognition and antigen presentation. The effects of SLPs on macrophages were reversed in the presence of a p38 inhibitor, indicating the potential importance of this signalling protein in how SLP activates the immune system. In conclusion this study shows that surface layer proteins from a common strain of C. difficile can activate a clearance response in macrophage and suggests that these proteins are important in clearance of C. difficile infection. Understanding how the immune system clears C. difficile infection could offer important insights for new treatment strategies.

  9. Antigenicity and Immunogenicity of Rotavirus VP6 Protein Expressed on the Surface of Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    L. E. Esteban

    2013-01-01

    Full Text Available Group A rotaviruses are the major etiologic agents of acute gastroenteritis worldwide in children and young animals. Among its structural proteins, VP6 is the most immunogenic and is highly conserved within this group. Lactococcus lactis is a food-grade, Gram-positive, and nonpathogenic lactic acid bacteria that has already been explored as a mucosal delivery system of heterologous antigens. In this work, the nisin-controlled expression system was used to display the VP6 protein at the cell surface of L. lactis. Conditions for optimal gene expression were established by testing different nisin concentrations, cell density at induction, and incubation times after induction. Cytoplasmic and cell wall protein extracts were analyzed by Western blot and surface expression was confirmed by flow cytometry. Both analysis provided evidence that VP6 was efficiently expressed and displayed on the cell surface of L. lactis. Furthermore, the humoral response of mice immunized with recombinant L. lactis was evaluated and the displayed recombinant VP6 protein proved to be immunogenic. In conclusion, this is the first report of displaying VP6 protein on the surface of L. lactis to induce a specific immune response against rotavirus. These results provide the basis for further evaluation of this VP6-displaying L. lactis as a mucosal delivery vector in a mouse model of rotavirus infection.

  10. Group B Streptococcus surface proteins as major determinants for meningeal tropism.

    Science.gov (United States)

    Tazi, Asmaa; Bellais, Samuel; Tardieux, Isabelle; Dramsi, Shaynoor; Trieu-Cuot, Patrick; Poyart, Claire

    2012-02-01

    Streptococcus agalactiae (group B Streptococcus, GBS), a normal constituent of the intestinal microbiota is the major cause of human neonatal infections and a worldwide spread 'hypervirulent' clone, GBS ST-17, is strongly associated with neonatal meningitis. Adhesion to epithelial and endothelial cells constitutes a key step of the infectious process. Therefore GBS surface-anchored proteins are obvious potential adhesion mediators of barrier crossing and determinant of hypervirulence. This review addresses the most recent molecular insights gained from studies on GBS surface proteins proven to be involved in the crossing of the brain-blood barrier and emphasizes on the specificity of a hypervirulent clone that displays meningeal tropism.

  11. Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to G protein-coupled receptor oligomerization.

    Science.gov (United States)

    Comps-Agrar, Laëtitia; Maurel, Damien; Rondard, Philippe; Pin, Jean-Philippe; Trinquet, Eric; Prézeau, Laurent

    2011-01-01

    G protein-coupled receptors (GPCRs) are key players in cell-cell communication, the dysregulation of which has often deleterious effects leading to pathologies such as psychiatric and neurological diseases. Consequently, GPCRs represent excellent drug targets, and as such are the object of intense research in drug discovery for therapeutic application. Recently, the GPCR field has been revolutionized by the demonstration that GPCRs are part of large protein complexes that control their pharmacology, activity, and signaling. Moreover, in these complexes, one GPCR can either associate with itself, forming homodimers or homooligomers, or with other receptor types, forming heterodimeric or heterooligomeric receptor entities that display new receptor features. These features include alterations in ligand cooperativity and selectivity, the activation of novel signaling pathways, and novel processes of desensitization. Thus, it has become necessary to identify GPCR-associated protein complexes of interest at the cell surface, and to determine the state of oligomerization of these receptors and their interactions with their partner proteins. This is essential to understand the function of GPCRs in their native environment, as well as ways to either modulate or control receptor activity with appropriate pharmacological tools, and to develop new therapeutic strategies. This requires the development of technologies to precisely address protein-protein interactions between oligomers at the cell surface. In collaboration with Cisbio Bioassay, we have developed such a technology, which combines TR-FRET detection with a new labeling method called SnapTag. This technology has allowed us to address the oligomeric state of many GPCRs.

  12. Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile

    Directory of Open Access Journals (Sweden)

    Ali Nazari Shirvan

    2016-06-01

    Full Text Available Abstract Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291—a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.

  13. Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG.

    Science.gov (United States)

    Formosa-Dague, Cécile; Speziale, Pietro; Foster, Timothy J; Geoghegan, Joan A; Dufrêne, Yves F

    2016-01-12

    Staphylococcus aureus surface protein SasG promotes cell-cell adhesion during the accumulation phase of biofilm formation, but the molecular basis of this interaction remains poorly understood. Here, we unravel the mechanical properties of SasG on the surface of living bacteria, that is, in its native cellular environment. Nanoscale multiparametric imaging of living bacteria reveals that Zn(2+) strongly increases cell wall rigidity and activates the adhesive function of SasG. Single-cell force measurements show that SasG mediates cell-cell adhesion via specific Zn(2+)-dependent homophilic bonds between β-sheet-rich G5-E domains on neighboring cells. The force required to unfold individual domains is remarkably strong, up to ∼500 pN, thus explaining how SasG can withstand physiological shear forces. We also observe that SasG forms homophilic bonds with the structurally related accumulation-associated protein of Staphylococcus epidermidis, suggesting the possibility of multispecies biofilms during host colonization and infection. Collectively, our findings support a model in which zinc plays a dual role in activating cell-cell adhesion: adsorption of zinc ions to the bacterial cell surface increases cell wall cohesion and favors the projection of elongated SasG proteins away from the cell surface, thereby enabling zinc-dependent homophilic bonds between opposing cells. This work demonstrates an unexpected relationship between mechanics and adhesion in a staphylococcal surface protein, which may represent a general mechanism among bacterial pathogens for activating cell association.

  14. Identification of novel surface-exposed proteins of Rickettsia rickettsii by affinity purification and proteomics.

    Science.gov (United States)

    Gong, Wenping; Xiong, Xiaolu; Qi, Yong; Jiao, Jun; Duan, Changsong; Wen, Bohai

    2014-01-01

    Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

  15. Identification of novel surface-exposed proteins of Rickettsia rickettsii by affinity purification and proteomics.

    Directory of Open Access Journals (Sweden)

    Wenping Gong

    Full Text Available Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs, which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

  16. Generic folding and transition hierarchies for surface adsorption of hydrophobic-polar lattice model proteins

    Science.gov (United States)

    Li, Ying Wai; Wüst, Thomas; Landau, David P.

    2013-01-01

    The thermodynamic behavior and structural properties of hydrophobic-polar (HP) lattice proteins interacting with attractive surfaces are studied by means of Wang-Landau sampling. Three benchmark HP sequences (48mer, 67mer, and 103mer) are considered with different types of surfaces, each of which attract either all monomers, only hydrophobic (H) monomers, or only polar (P) monomers, respectively. The diversity of folding behavior in dependence of surface strength is discussed. Analyzing the combined patterns of various structural observables, such as, e.g., the derivatives of the numbers of surface contacts, together with the specific heat, we are able to identify generic categories of folding and transition hierarchies. We also infer a connection between these transition categories and the relative surface strengths, i.e., the ratio of the surface attractive strength to the interchain attraction among H monomers. The validity of our proposed classification scheme is reinforced by the analysis of additional benchmark sequences. We thus believe that the folding hierarchies and identification scheme are generic for HP proteins interacting with attractive surfaces, regardless of chain length, sequence, or surface attraction.

  17. Anti-fouling surfaces by combined molecular self-assembly and surface-initiated ATRP for micropatterning active proteins.

    Science.gov (United States)

    Xiu, K M; Cai, Q; Li, J S; Yang, X P; Yang, W T; Xu, F J

    2012-02-01

    A simple method by combined molecular self assembly and surface-initiated atom transfer radical polymerization (SI-ATRP) was proposed to prepare a biologically inert surface for micropatterning active proteins. The MPEG microdomains having a short terminal poly(ethylene glycol) (PEG) unit were prepared by self assembly of 2-(methyoxy(polyethylenoxy) propyl)trimethoxy silane (MPEG-silane). The remaining local regions or poly(poly(ethylene glycol)methyl ether methacrylate-co-glycidyl methacrylate) (P(PEGMEMA-co-GMA)) microdomains were produced via SI-ATRP of PEGMEMA and GMA comonomers. The epoxy groups of the P(PEGMEMA-co-GMA) microdomains were used directly for covalent coupling of an active protein (human immunoglobulin or IgG) via the ring-opening reaction to produce the IgG-coupled microdomains. The IgG-coupled microdomains interact only and specifically with target anti-IgG, while the other antifouling microregions from self-assembled monolayers with short terminal PEG units effectively prevent specific and non-specific protein fouling. When extended to other active biomolecules, microarrays for specific and non-specific analyte interactions with a high signal-to-noise ratio could be readily tailored.

  18. Using extremely halophilic bacteria to understand the role of surface charge and surface hydration in protein evolution, folding, and function

    Science.gov (United States)

    Hoff, Wouter; Deole, Ratnakar; Osu Collaboration

    2013-03-01

    Halophilic Archaea accumulate molar concentrations of KCl in their cytoplasm as an osmoprotectant, and have evolved highly acidic proteomes that only function at high salinity. We examine osmoprotection in the photosynthetic Proteobacteria Halorhodospira halophila. We find that H. halophila has an acidic proteome and accumulates molar concentrations of KCl when grown in high salt media. Upon growth of H. halophila in low salt media, its cytoplasmic K + content matches that of Escherichia coli, revealing an acidic proteome that can function in the absence of high cytoplasmic salt concentrations. These findings necessitate a reassessment of two central aspects of theories for understanding extreme halophiles. We conclude that proteome acidity is not driven by stabilizing interactions between K + ions and acidic side chains, but by the need for maintaining sufficient solvation and hydration of the protein surface at high salinity through strongly hydrated carboxylates. We propose that obligate protein halophilicity is a non-adaptive property resulting from genetic drift in which constructive neutral evolution progressively incorporates weakly stabilizing K + binding sites on an increasingly acidic protein surface.

  19. Relevant uses of surface proteins--display on self-organized biological structures.

    Science.gov (United States)

    Jahns, Anika C; Rehm, Bernd H A

    2012-03-01

    Proteins are often found attached to surfaces of self-assembling biological units such as whole microbial cells or subcellular structures, e.g. intracellular inclusions. In the last two decades surface proteins were identified that could serve as anchors for the display of foreign protein functions. Extensive protein engineering based on structure-function data enabled efficient display of technically and/or medically relevant protein functions. Small size, diversity of the anchor protein as well as support structure, genetic manipulability and controlled cultivation of phages, bacterial cells and yeasts contributed to the establishment of designed and specifically functionalized tools for applications as sensors, catalysis, biomedicine, vaccine development and library-based screening technologies. Traditionally, phage display is employed for library screening but applications in biomedicine and vaccine development are also perceived. For some diagnostic purposes phages are even too small in size so other carrier materials where needed and gave way for cell and yeast display. Only recently, intracellular inclusions such as magnetosomes, polyhydroxyalkanoate granules and lipid bodies were conceived as stable subcellular structures enabling the display of foreign protein functions and showing potential as specific and tailor-made devices for medical and biotechnological applications.

  20. A comparison of surface proteins in embryonal carcinoma cells and their differentiated derivatives.

    Science.gov (United States)

    Keil-Dlouha, V; Paulin, D; Bagilet, L K; Keil, B

    1980-03-27

    Surface proteins from five cell lines (three embryonal carcinoma cell lines (F9, PCC4 and PCC3), teratocarcinoma-derived endodermal cells (PYS) and fibroblasts (line 3/A/1-D-3 differentiated from PCC3) were compared by two-dimensional polyacrylamide gel electrophoresis after selective iodination with 125I in the presence of lactoperoxidase. The labeled proteins were solubilized either in Nonidet P40/urea/ampholyte/mercaptoethanol solution or in Nonidet P40 only. In total, about thirty major 125I-labeled surface proteins were identified by their isoelectric point and molecular weight. 14 proteins are present in all five cell types, although their quantity or accessibility for labeling differs between differentiated and undifferentiated cells. Three proteins (200, 160 and 150 kilodaltons) are present in undifferentiated cells only. Two of them (160 and 150 kilodaltons) were solubilized by Nonidet P40/urea/ampholyte/mercaptoethanol, but not by Nonidet P40. One protein (50 kilodaltons) was found in nullipotent F9 cells only. About 14--15 proteins (including fibronectin) were released by Nonidet P40/urea/ampholyte/mercaptoethanol but not by Nonidet P40. They are presumably bound to submembrane or cytoskeleton structures by non-covalent bonds.

  1. Dynamics of putative raft-associated proteins at the cell surface.

    Science.gov (United States)

    Kenworthy, Anne K; Nichols, Benjamin J; Remmert, Catha L; Hendrix, Glenn M; Kumar, Mukesh; Zimmerberg, Joshua; Lippincott-Schwartz, Jennifer

    2004-06-07

    Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (> 4 microm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.

  2. Testing the Coulomb/Accessible Surface Area solvent model for protein stability, ligand binding, and protein design

    Directory of Open Access Journals (Sweden)

    Bathelt Christine

    2008-03-01

    Full Text Available Abstract Background Protein structure prediction and computational protein design require efficient yet sufficiently accurate descriptions of aqueous solvent. We continue to evaluate the performance of the Coulomb/Accessible Surface Area (CASA implicit solvent model, in combination with the Charmm19 molecular mechanics force field. We test a set of model parameters optimized earlier, and we also carry out a new optimization in this work, using as a target a set of experimental stability changes for single point mutations of various proteins and peptides. The optimization procedure is general, and could be used with other force fields. The computation of stability changes requires a model for the unfolded state of the protein. In our approach, this state is represented by tripeptide structures of the sequence Ala-X-Ala for each amino acid type X. We followed an iterative optimization scheme which, at each cycle, optimizes the solvation parameters and a set of tripeptide structures for the unfolded state. This protocol uses a set of 140 experimental stability mutations and a large set of tripeptide conformations to find the best tripeptide structures and solvation parameters. Results Using the optimized parameters, we obtain a mean unsigned error of 2.28 kcal/mol for the stability mutations. The performance of the CASA model is assessed by two further applications: (i calculation of protein-ligand binding affinities and (ii computational protein design. For these two applications, the previous parameters and the ones optimized here give a similar performance. For ligand binding, we obtain reasonable agreement with a set of 55 experimental mutation data, with a mean unsigned error of 1.76 kcal/mol with the new parameters and 1.47 kcal/mol with the earlier ones. We show that the optimized CASA model is not inferior to the Generalized Born/Surface Area (GB/SA model for the prediction of these binding affinities. Likewise, the new parameters perform

  3. Dewetting of polystyrene thin films on poly(ethylene glycol)-modified surfaces as a simple approach for patterning proteins.

    Science.gov (United States)

    Cai, Yangjun; Newby, Bi-min Zhang

    2008-05-20

    A simple technique for patterning proteins utilizing dewetted polystyrene (PS) droplets is demonstrated. A polystyrene thin film was spin coated on a poly(ethylene glycol) (PEG) silane-modified surface. As the PS film dewets from the surface, upon annealing, to form droplets, the PEG-silane-modified surface is exposed, which retains its capability to resist protein adsorption, and the PS droplets allow the selective adsorption of proteins. In contrast to the undewetted flat PS film, the droplet surface had a greater amount of adsorbed proteins. Atomic force microscopy scans reveal that the roughness of the droplet surface is higher, and a multilayer of proteins results on the droplet surface. Moreover, micro- and nanoscale droplet patterns can easily be achieved by tuning the thickness of PS thin films. Because dewetting approaches for generating ordered dewetting droplets have been successfully generated by others, those approaches could be easily combined with this technique to fabricate ordered protein patterns.

  4. Baculovirus superinfection: a probable restriction factor on the surface display of proteins for library screening.

    Science.gov (United States)

    Xu, Xiaodong; Chen, Yuanrong; Zhao, Yu; Liu, Xiaofen; Dong, Beitao; Jones, Ian M; Chen, Hongying

    2013-01-01

    In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells co-expressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the re-infection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides.

  5. Competitive Protein Adsorption of Albumin and Immunoglobulin G from Human Serum onto Polymer Surfaces

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hou, Xiaolin

    2010-01-01

    Competitive protein adsorption from human serum onto unmodified polyethylene terephthalate (PET) surfaces and plasma-polymerized PET surfaces, using the monomer diethylene glycol vinyl ether (DEGVE), has been investigated using radioactive labeling. Albumin and immunoglobulin G (IgG) labeled...... with two different iodine isotopes have been added to human serum solutions of different concentrations, and adsorption has been performed using adsorption times from approximately 5 s to 24 h. DEGVE surfaces showed indications of being nonfouling regarding albumin and IgG adsorption during competitive...

  6. Bacterial Surface-Displayed GII.4 Human Norovirus Capsid Proteins Bound to HBGA-Like Molecules in Romaine Lettuce.

    Science.gov (United States)

    Wang, Ming; Rong, Shaofeng; Tian, Peng; Zhou, Yue; Guan, Shimin; Li, Qianqian; Wang, Dapeng

    2017-01-01

    Human Noroviruses (HuNoVs) are the main cause of non-bacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein on bacterial surface and used it as a new strategy to explore interaction between HuNoV protein and receptor candidates from romaine lettuce. The surface-displayed HuNoV proteins were confirmed on the surface of the transformed bacteria by an immunofluorescence assay. The distribution patterns of the surface-displayed HuNoV proteins in romaine lettuce were identified through a confocal immunofluorescence assay. The surface-displayed HuNoV proteins could be found in the stomata, and the surfaces of vein and leaf of romaine lettuce. The surface-displayed HuNoV proteins could be captured by an ELISA assay utilizing extract from leaf (LE) or vein (VE). The binding of the surface-displayed HuNoV proteins to LE or VE could be competitively blocked by histo-blood group antigens from human saliva. In addition, the binding of the surface-displayed HuNoV proteins to LE or VE could also be attenuated by heat denaturation of lettuce proteins, and abolished by oxidation of lettuce carbohydrates. The results indicated that histo-blood group antigen-like molecules in LE or VE were involved in the binding of the surface-displayed HuNoV proteins to romaine lettuce. All data demonstrated that the surface-displayed HuNoV proteins could be utilized in a new and simple system for investigation of the interaction between the HuNoVs and their candidate ligands.

  7. Direct site-directed photocoupling of proteins onto surfaces coated with β-cyclodextrins

    DEFF Research Database (Denmark)

    Städe, Lars W; Wimmer, Reinhard; Stensballe, Allan

    2010-01-01

    photoimmobilization of N27pBpa-cutinase on quartz slides coated with beta-CD was achieved from liquid or dry films by total internal reflection fluorescence (TIRF). The Dock'n'Flash method offers a solution for direct photocoupling and patterning of recombinant proteins onto surfaces with site-specific attachment....

  8. Novel surface display system for proteins on non-genetically modified gram-positive bacteria

    NARCIS (Netherlands)

    Bosma, T; Kanninga, R; Neef, J; Audouy, SAL; van Roosmalen, ML; Steen, A; Buist, G; Kok, J; Kuipers, OP; Robillard, G; Leenhouts, K

    2006-01-01

    A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial

  9. Dynamics of Agglutinin-Like Sequence (ALS) Protein Localization on the Surface of Candida Albicans

    Science.gov (United States)

    Coleman, David Andrew

    2009-01-01

    The ALS gene family encodes large cell-surface glycoproteins associated with "C. albicans" pathogenesis. Als proteins are thought to act as adhesin molecules binding to host tissues. Wide variation in expression levels among the ALS genes exists and is related to cell morphology and environmental conditions. "ALS1," "ALS3," and "ALS4" are three of…

  10. Evidence by EIS of the interaction between proteins and tin oxide electrode surface

    Energy Technology Data Exchange (ETDEWEB)

    Cachet, Hubert [Laboratoire Interfaces et Systemes Electrochimiques, UPR 15 du CNRS, Universite Pierre et Marie Curie, Case courrier no133-4 place Jussieu 75005 Paris (France); Debiemme-Chouvy, Catherine, E-mail: catherine.debiemme-chouvy@upmc.f [Laboratoire Interfaces et Systemes Electrochimiques, UPR 15 du CNRS, Universite Pierre et Marie Curie, Case courrier no133-4 place Jussieu 75005 Paris (France)

    2010-08-30

    Macromolecules like proteins are able to adhere to tin oxide electrodes at open circuit potential as proved by electrogravimetry experiments. In this work, electrochemical impedance studies were performed at aqueous electrolyte/F- or Sb-doped semiconducting tin oxide interfaces, including natural seawater. By this way, it was possible to characterize the potential dependence of the interfacial capacitance in various physicochemical conditions, without or in the presence of bovine serum albumin (BSA). In the potential range where tin oxide is in the depletion regime (blocking interface), a capacitance excess is evidenced which can be attributed to the formation of surface states which are the signature of chemical bonding. By simulating the so-called surface state capacitance, three states have been pointed out. They are centred at 0.7, 0.9 and 1.1 eV in the tin oxide bandgap. On the basis of experimental arguments, the state at 1.1 eV was ascribed to the OH-terminated tin oxide surface, the two other states were found to be specific of the interaction of organic matter with the oxide surface. In the presence of BSA, the density of surface atoms (about 10{sup 13} cm{sup -2}) involved in bonding is of the order of magnitude of the surface concentration of one BSA monolayer. The lasting character of these bonds was also shown. This finding shows the definitive protein immobilisation at the SnO{sub 2} surface.

  11. Identification of sporozoite surface proteins and antigens of Eimeria nieschulzi (Apicomplexa)

    Energy Technology Data Exchange (ETDEWEB)

    Tilley, M.; Upton, S.J. (Kansas State Univ., Manhattan (USA))

    1990-03-01

    Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, lectin binding, and {sup 125}I surface labeling of sporozoites were used to probe sporozoites of the rat coccidian, Eimeria nieschulzi. Analysis of silver stained gels revealed greater than 50 bands. Surface iodination revealed about 14 well labeled, and about 10 weakly labeled but potential, surface proteins. The most heavily labeled surface proteins had molecular masses of 60, 53-54, 45, 28, 23-24, 17, 15, 14, 13, and 12 kD. Following electrophoresis and Western blotting, 2 of the 12 125I labeled lectin probes bound to two bands on the blots, which collectively indicated that two bands were glycosylated. Concanavalin A (ConA) specifically recognized a band at 53 kD, which may represent a surface glycoprotein, and a lectin derived from Osage orange (MPA) bound to a single band at 82-88 kD, that may also be a surface molecule. Immunoblotting using sera collected from rats inoculated orally with oocysts, as well as sera from mice hyperimmunized with sporozoites, revealed that many surface molecules appear to be immunogenic.

  12. Tailoring the nanostructured surfaces of hydroxyapatite bioceramics to promote protein adsorption, osteoblast growth, and osteogenic differentiation.

    Science.gov (United States)

    Lin, Kaili; Xia, Lunguo; Gan, Jingbo; Zhang, Zhiyuan; Chen, Hong; Jiang, Xinquan; Chang, Jiang

    2013-08-28

    To promote and understand the biological responses of the implant via nanostructured surface design is essential for the development of bioactive bone implants. However, the control of the surface topography of the bioceramics in nanoscale is a big challenge because of their brittle property. Herein, the hydroxyapatite (HAp) bioceramics with distinct nanostructured topographies were fabricated via hydrothermal treatment using α-tricalcium phosphate ceramic as hard-template under different reaction conditions. HAp bioceramics with nanosheet, nanorod and micro-nanohybrid structured surface in macroscopical size were obtained by controlling the composition of the reaction media. Comparing with the traditional sample with flat and dense surface, the fabricated HAp bioceramics with hierarchical 3D micro-nanotextured surfaces possessed higher specific surface area, which selectively enhanced adsorption of specific proteins including Fn and Vn in plasma, and stimulated osteoblast adhesion, growth, and osoteogenic differentiation. In particular, the biomimetic features of the hierarchical micro-nanohybrid surface resulted in the best ability for simultaneous enhancement of protein adsorption, osteoblast proliferation, and differentiation. The results suggest that the hierarchical micro-nanohybrid topography might be one of the critical factors to be considered in the design of functional bone grafts.

  13. A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jarmander Johan

    2012-09-01

    Full Text Available Abstract Background The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H:gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His6-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H:gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His6-tag facing the medium. Conclusions Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His6-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent

  14. Comparison of five methods for direct extraction of surface proteins from Listeria monocytogenes for proteomic analysis by orbitrap mass spectrometry.

    Science.gov (United States)

    Tiong, Hung King; Hartson, Steven; Muriana, Peter M

    2015-03-01

    Extracts of surface proteins, with minimal artifacts from contaminating cytosolic components, are highly desirable for investigating surface factors involved in the attachment and formation of biofilms by bacteria that are problematic in commercial food processing facilities. In this study, we compared the protein profiles of the food pathogen, Listeria monocytogenes, recovered after applying different surface protein extraction methods compiled from the literature: trypsin-enzymatic shaving with BICAM/sucrose or Tris/sucrose buffers (Tryp B+S, Tryp T+S), Tris-buffered urea (UB), lithium chloride (LiCl) and Tris-buffered urea applied with hypotonic-stressed cells (UB-Ghost), and subjected them to liquid chromatography tandem mass spectrometry and protein identification. The data indicate that the UB-Ghost extraction method provides a cleaner extract of surface proteins including the predicted (this study and the literature) or validated members (literature) from L. monocytogenes. This was determined by an accumulative lower unique peptide number exhibited by mass spectrometry for total cytoplasmic proteins among different surface extracts, with a majority of proteins demonstrating hydrophilic properties. The extracted proteins were from different functional categories and have associations with the cell surface, intermediary metabolism, information pathways, or functionally unknown proteins as suggested by in silico analyses performed by other groups (Leger and ListiList). The utilization of an optimized method for surface protein extraction should greatly facilitate identification by LC-MS/MS that could be useful to anyone working on molecular proteomics of bacterial surfaces.

  15. Lipid and protein composition of the surface tegument from larvae of Taenia taeniaeformis.

    Science.gov (United States)

    Mills, G L; Coley, S C; Williams, J F

    1984-04-01

    A tegumental fraction from fully developed larvae of Taenia taeniaeformis was recovered by low speed centrifugation following incubation of the parasites in a 0.1% solution of digitonin. Scanning electron microscopy of the parasite carcass revealed no surface microtrichs, and transmission electron microscopy indicated that the subtegumental layer was undamaged. The tegumental fraction, judging from the distribution of 3H-Concanavalin A, was enriched for surface components, exhibited low succinic dehydrogenase activity, and an electron microscopic examination of the pellet showed a slightly expanded but intact distal tegumental layer. The fraction, which made up 3.0% of the dry weight of the parasite, consisted of 52% protein and 32% lipid. Thirty-three proteins, ranging in Mr from 9,000 to 276,000 daltons, were detected after sodium dodecyl sulfate solubilization and polyacrylamide gel electrophoresis. Seven of these proteins were glycoproteins. Cholesterol, phosphatidylethanolamine, phosphatidylserine, and glycosphingolipids were the major lipids.

  16. Chemoselective Attachment of Biologically Active Proteins to Surfaces by Native Chemical Ligation

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, C L; de Yoreo, J J; Coleman, M; Camarero, J A

    2003-11-22

    The present work describes our ongoing efforts towards the creation of micro and nanoscaled ordered arrays of protein covalently attached to site-specific chemical linkers patterned by different microlithographic techniques. We present a new and efficient solid-phase approach for the synthesis of chemically modified long alkyl-thiols. These compounds can be used to introduce chemoselective reacting groups onto silicon-based surfaces. We show that these modified thiols can be used for creating nano- and micrometric chemical patterns by using different lithographic techniques. We show that these patterns can react chemoselectively with proteins which have been recombinantly modified to contain complementary chemical groups at specific positions thus resulting in the oriented attachment of the protein to the surface.

  17. The Surface of Ice in the presence of Antifreeze Proteins studied by Atomic Force Microscopy

    Science.gov (United States)

    Zepeda, Salvador; Orme, Christine; Yeh, Yin

    2002-03-01

    The surface of ice has been a topic of interest for centuries. In particular, the surface structure and properties have been explored with the advent of new surface techniques. Several groups have convincingly shown a surface transition layer to exist between the solid-vapor interface as well as the solid-liquid interface. In addition, the characteristics of this region may be directly correlated with growth morphologies of ice. Certain peptide molecules have the ability to significantly alter the growth morphology of an ice crystal. Do these molecules simply disrupt this transition region? Or do they anchor themselves deep into it reaching the bulk-ice phase? And is there a similar mechanism by which they function? We use AFM to study the morphological changes to the true ice surface due to the presence antifreeze proteins. We will discuss the implications of our results on the longstanding debate to the above questions.

  18. THE EFFECTS OF SURFACE CHEMISTRY ON THE PROPERTIES OF PROTEINS CONFINED IN NANO-POROUS MATERIALS

    Energy Technology Data Exchange (ETDEWEB)

    Garrett, L. M.; O' Neill, H.

    2007-01-01

    The entrapment of proteins using the sol-gel route provides a means to retain its native properties and artifi cially reproduce the molecular crowding and confi nement experienced by proteins in the cell allowing investigation of the physico-chemical and structural properties of biomolecules at the biotic/abiotic interface. The biomolecules are spatially separated and ‘caged’ in the gel structure but solutes can freely permeate the matrix. Thus, properties such as the folding of ensembles of individual molecules can be examined in the absence of aggregation effects that can occur in solution studies. Green fl uorescent protein from Aequorea coerulescens was used as a model protein to examine the unfolding/re-folding properties of protein in silica gels. The recombinant protein was isolated and purifi ed from Escherichia coli extracts by cell lysis, three-phase partitioning, dialysis, and anion exchange chromatography. The purity of the protein was greater than 90% as judged by SDS PAGE gel analysis. Sol-gels were synthesized using tetramethylorthosilicate (TMOS) in combination with, methyltrimethoxyorthosilane (MTMOS), ethyltrimethoxyorthosilane (ETMOS), 3-aminopropyltriethoxysilane (APTES), and 3-glycidoxypropyltrimethoxysilane (GPTMS). The acid induced denaturation and renaturation of GFP was analyzed by UV-visible, fl uorescence, and circular dichroism (CD) spectroscopies. No renaturation was observed in gels that were made with TMOS only, and in the presence of APTES, MTMOS, and ETMOS. However, in gels that were made with GPTMS, the CD and UV-visible spectra indicated that the protein had refolded. The fl uorescence emission spectrum indicated that approximately 20% of fl uorescence had returned. This study highlights the importance of the surface chemistry of the silica gels for the refolding properties of the entrapped GFP. Future studies will investigate the effect of surface chemistry on the thermal and solvent stability of the entrapped protein.

  19. Merozoite surface proteins in red blood cell invasion, immunity and vaccines against malaria

    Science.gov (United States)

    Beeson, James G.; Drew, Damien R.; Boyle, Michelle J.; Feng, Gaoqian; Fowkes, Freya J.I.; Richards, Jack S.

    2016-01-01

    Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria, and P. vivax being a second important cause, especially in Asia, the Americas and the Pacific. During infection with Plasmodium spp., the merozoite form of the parasite invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs and, therefore, understanding merozoite invasion, host immune responses to merozoite surface antigens, and targeting merozoite surface proteins and invasion ligands by novel vaccines and therapeutics have been important areas of research. Merozoite invasion involves multiple interactions and events, and substantial processing of merozoite surface proteins occurs before, during and after invasion. The merozoite surface is highly complex, presenting a multitude of antigens to the immune system. This complexity has proved challenging to our efforts to understand merozoite invasion and malaria immunity, and to developing merozoite antigens as malaria vaccines. In recent years, there has been major progress in this field, and several merozoite surface proteins show strong potential as malaria vaccines. Our current knowledge on this topic is reviewed, highlighting recent advances and research priorities. PMID:26833236

  20. Protein adsorption on a hydrophobic surface: a molecular dynamics study of lysozyme on graphite.

    Science.gov (United States)

    Raffaini, Giuseppina; Ganazzoli, Fabio

    2010-04-20

    Adsorption of human lysozyme on hydrophobic graphite is investigated through atomistic computer simulations with molecular mechanics (MM) and molecular dynamics (MD) techniques. The chosen strategy follows a simulation protocol proposed by the authors to model the initial and the final adsorption stage on a bare surface. Adopting an implicit solvent and considering 10 starting molecular orientations so that all the main sides of the protein can face the surface, we first carry out an energy minimization to investigate the initial adsorption stage, and then long MD runs of selected arrangements to follow the surface spreading of the protein maximizing its adsorption strength. The results are discussed in terms of the kinetics of surface spreading, the interaction energy, and the molecular size, considering both the footprint and the final thickness of the adsorbed protein. The structural implications of the final adsorption geometry for surface aggregation and nanoscale structural organization are also pointed out. Further MD runs are carried out in explicit water for the native structure and the most stable adsorption state to assess the local stability of the geometry obtained in implicit solvent, and to calculate the statistical distribution of the water molecules around the whole lysozyme and its backbone.

  1. Surface (glyco-)proteins: primary structure and crystallization under microgravity conditions

    Science.gov (United States)

    Claus, H.; Akca, E.; Schultz, N.; Karbach, G.; Schlott, B.; Debaerdemaeker, T.; De Clercq, J.-P.; König, H.

    2001-08-01

    The Archaea comprise microorganisms that live under environmental extremes, like high temperature, low pH value or high salt concentration. Their cells are often covered by a single layer of (glyco)protein subunits (S-layer) in hexagonal arrangement. In order to get further hints about the molecular mechanisms of protein stabilization we compared the primary and secondary structures of archaeal S-layer (glyco)proteins. We found an increase of charged amino acids in the S-layer proteins of the extreme thermophilic species compared to their mesophilic counterparts. Our data and those of other authors suggest that ionic interactions, e.g., salt bridges seem to be played a major role in protein stabilization at high temperatures. Despite the differences in the growth optima and the predominance of some amino acids the primary structures of S-layers revealed also a significant degree of identity between phylogenetically related archaea. These obervations indicate that protein sequences of S-layers have been conserved during the evolution from extremely thermophilic to mesophilic life. To support these findings the three-dimensional structure of the S-layer proteins has to be elucidated. Recently, we described the first successful crystallization of an extreme thermophilic surface(glyco)protein under microgravity conditions.

  2. Plasma graft of poly(ethylene glycol) methyl ether methacrylate (PEGMA) on RGP lens surface for reducing protein adsorption

    Science.gov (United States)

    Shiheng, Yin; Li, Ren; Yingjun, Wang

    2017-01-01

    Poly(ethylene glycol) methyl ether methacrylate (PEGMA) was grafted on fluorosilicone acrylate rigid gas permissible contact lens surface by means of argon plasma induced polymerization to improve surface hydrophilicity and reduce protein adsorption. The surface properties were characterized by contact angle measurement, x-ray photoelectron spectroscopy (XPS) and atomic force microscopy respectively. The surface protein adsorption was evaluated by lysozyme solution immersion and XPS analysis. The results indicated that a thin layer of PEGMA was successfully grafted. The surface hydrophilicity was bettered and surface free energy increased. The lysozyme adsorption on the lens surface was reduced greatly. The study was supported by National Natural Science Foundation of China (No. 51273072).

  3. Exhaustive comparison and classification of ligand-binding surfaces in proteins

    Science.gov (United States)

    Murakami, Yoichi; Kinoshita, Kengo; Kinjo, Akira R; Nakamura, Haruki

    2013-01-01

    Many proteins function by interacting with other small molecules (ligands). Identification of ligand-binding sites (LBS) in proteins can therefore help to infer their molecular functions. A comprehensive comparison among local structures of LBSs was previously performed, in order to understand their relationships and to classify their structural motifs. However, similar exhaustive comparison among local surfaces of LBSs (patches) has never been performed, due to computational complexity. To enhance our understanding of LBSs, it is worth performing such comparisons among patches and classifying them based on similarities of their surface configurations and electrostatic potentials. In this study, we first developed a rapid method to compare two patches. We then clustered patches corresponding to the same PDB chemical component identifier for a ligand, and selected a representative patch from each cluster. We subsequently exhaustively as compared the representative patches and clustered them using similarity score, PatSim. Finally, the resultant PatSim scores were compared with similarities of atomic structures of the LBSs and those of the ligand-binding protein sequences and functions. Consequently, we classified the patches into ∼2000 well-characterized clusters. We found that about 63% of these clusters are used in identical protein folds, although about 25% of the clusters are conserved in distantly related proteins and even in proteins with cross-fold similarity. Furthermore, we showed that patches with higher PatSim score have potential to be involved in similar biological processes. PMID:23934772

  4. A facile approach for imprinting protein on the surface of multi-walled carbon nanotubes.

    Science.gov (United States)

    Liu, Ren; Sha, Mo; Jiang, Sisi; Luo, Jing; Liu, Xiaoya

    2014-03-01

    This study describes a green, facile and low cost approach for imprinting protein on the surface of multi-walled carbon nanotubes (MWNTs) using papain as the template, dopamine as the functional monomer. By simply mixing MWNTs, dopamine, template protein in weak alkaline aqueous solution, a thin adherent polydopamine (PDA) film imprinted with protein was spontaneously obtained on the surface of MWNTs to produce the imprinted nanomaterials (MWNTs@MIPs). The obtained MWNTs@MIPs were characterized with Fourier transform infrared spectrometer (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The adsorption process of the MWNTs@MIPs towards template protein was investigated in detail. The effects of the concentration of the monomer and template, polymerization time, extraction process were optimized. The prepared MWNTs@MIPs show fast binding kinetics, high binding capacity and acceptable specific recognition behavior towards template proteins. Furthermore, the stability and regeneration were also investigated, which indicated that the MWNTs@MIPs had good reusability. The good recognizing behavior coupled to the low cost and facile one-step preparation make the MWNTs@MIPs attractive for separation and specific protein recognition.

  5. Limitations of yeast surface display in engineering proteins of high thermostability.

    Science.gov (United States)

    Park, Sheldon; Xu, Yao; Stowell, Xiaoran Fu; Gai, Feng; Saven, Jeffery G; Boder, Eric T

    2006-05-01

    Engineering proteins that can fold to unique structures remains a challenge. Protein stability has previously been engineered via the observed correlation between thermal stability and eukaryotic secretion level. To explore the limits of an expression-based approach, variants of the highly thermostable three-helix bundle protein alpha3D were studied using yeast surface display. A library of alpha3D mutants was created to explore the possible correlation of protein stability and fold with expression level. Five efficiently expressed mutants were then purified and further studied biochemically. Despite their differences in stability, most mutants expressed at levels comparable with that of wild-type alpha3D. Two other related sequences (alpha3A and alpha3B) that form collapsed, stable molten globules but lack a uniquely folded structure were similarly expressed at high levels by yeast display. Together these observations suggest that the quality control system in yeast is unable to discriminate between well-folded proteins of high stability and molten globules. The present study, therefore, suggests that an optimization of the surface display efficiency on yeast may yield proteins that are thermally and chemically stable yet are poorly folded.

  6. THE SURFACE-MEDIATED UNFOLDING KINETICS OF GLOBULAR PROTEINS IS DEPENDENT ON MOLECULAR WEIGHT AND TEMPERATURE

    Energy Technology Data Exchange (ETDEWEB)

    Patananan, A.N.; Goheen, S.C.

    2008-01-01

    The adsorption and unfolding pathways of proteins on rigid surfaces are essential in numerous complex processes associated with biomedical engineering, nanotechnology, and chromatography. It is now well accepted that the kinetics of unfolding are characterized by chemical and physical interactions dependent on protein deformability and structure, as well as environmental pH, temperature, and surface chemistry. Although this fundamental process has broad implications in medicine and industry, little is known about the mechanism because of the atomic lengths and rapid time scales involved. Therefore, the unfolding kinetics of myoglobin, β-glucosidase, and ovalbumin were investigated by adsorbing the globular proteins to non-porous cationic polymer beads. The protein fractions were adsorbed at different residence times (0, 9, 10, 20, and 30 min) at near-physiological conditions using a gradient elution system similar to that in high-performance liquid chromatography. The elution profi les and retention times were obtained by ultraviolet/visible spectrophotometry. A decrease in recovery was observed with time for almost all proteins and was attributed to irreversible protein unfolding on the non-porous surfaces. These data, and those of previous studies, fi t a positively increasing linear trend between percent unfolding after a fi xed (9 min) residence time (71.8%, 31.1%, and 32.1% of myoglobin, β-glucosidase, and ovalbumin, respectively) and molecular weight. Of all the proteins examined so far, only myoglobin deviated from this trend with higher than predicted unfolding rates. Myoglobin also exhibited an increase in retention time over a wide temperature range (0°C and 55°C, 4.39 min and 5.74 min, respectively) whereas ovalbumin and β-glucosidase did not. Further studies using a larger set of proteins are required to better understand the physiological and physiochemical implications of protein unfolding kinetics. This study confi rms that surface

  7. Combinatorial synthesis with high throughput discovery of protein-resistant membrane surfaces.

    Science.gov (United States)

    Gu, Minghao; Vegas, Arturo J; Anderson, Daniel G; Langer, Robert S; Kilduff, James E; Belfort, Georges

    2013-08-01

    Using combinatorial methods, we synthesized a series of new vinyl amide monomers and graft-polymerized them to light-sensitive poly(ether sulfone) (PES) porous films for protein resistance. To increase the discovery rate and statistical confidence, we developed high throughput surface modification methods (HTP) that allow synthesis, screening and selection of desirable monomers from a large library in a relatively short time (days). A series of amide monomers were synthesized by amidation of methacryloyl chloride with amines and grafted onto commercial poly(ether sulfone) (PES) membranes using irradiation from atmospheric pressure plasma (APP). The modified PES membrane surfaces were then tested and screened for static protein adhesion using HTP. Hydroxyl amide monomers N-(3-hydroxypropyl)methacrylamide (A3), N-(4-hydroxybutyl)methacrylamide (A4), and N-(4-hydroxybutyl)methacrylamide (A6), ethylene glycol (EG) monomer N-(3-methoxypropyl)methacrylamide (A7), and N-(2-(dimethylamino)ethyl)-N-methylmethacrylamide (A8), and N-(2-(diethylamino)ethyl)-N-methylmethacrylamide (A9) all terminated with tertiary amines and were shown to have protein resistance. The PES membranes modified with these monomers exhibited both low protein adhesion (i.e. membrane plugging or fouling) and high flux. Their performance is comparable with previously identified best performing PEG and zwitterionic monomers, i.e. the so-called gold-standard for protein resistance. Combining a Hansen solubility parameter (HSP) analysis of the amide monomers and the HTP filtration results, we conclude that monomer solubility in water correlates with protein-resistant surfaces, presumably through its effects on surface-water interactions.

  8. Effect of surface attractive strength on structural transitions of a confined HP lattice protein

    Energy Technology Data Exchange (ETDEWEB)

    Pattanasiri, Busara [University of Georgia, Athens, GA; Li, Ying Wai [ORNL; Wuest, Thomas [ETH Zurich, Switzerland; Landau, David P [University of Georgia, Athens, GA

    2015-01-01

    We investigate the influence of surface attractive strength on structural transitions of a hydrophobic-polar (HP) lattice protein confined in a slit formed by two parallel, attractive walls. We apply Wang-Landau sampling together with efficient Monte Carlo updates to estimate the density of states of the system. The conformational transitions, namely, the debridging process and hydrophobic core formation, can be identified by analyzing the specific heat together with several structural observables, such as the numbers of surface contacts, the number of hydrophobic pairs, and radii of gyration in different directions. As temperature decreases, we find that the occurrence of the debridging process is conditional depending on the surface attractive strength. This, in turn, affects the nature of the hydrophobic core formation that takes place at a lower temperature. We illustrate these observations with the aid of a HP protein chain with 48 monomers.

  9. Templating Biomineralization: Surface Directed Protein Self-assembly and External Magnetic Field Stimulation of Osteoblasts

    Science.gov (United States)

    Ba, Xiaolan

    biomineralization is investigated by SEM, GIXRD and energy dispersive X-ray spectroscopy (EDXS). Gene expression during the exposure of SMF is also studies by RT-PCR. The results indicated that exposure to SMF induces osteoblasts to produce larger quantities of HA, with higher degree of crystalline order. The controlling and understanding of protein on the surface is of great interest in biomedical application such as implant medicine, biosensor design, food processing, and chromatographic separations. The adsorbed protein onto the surface significantly determines the performance of biomaterials in a biological environment. Recent studies have suggested that the preservation of the native secondary structure of protein adsorbed is essential for biological application. In order to manipulate protein adsorption and design biocompatible materials, the mechanisms underlying protein-surface interactions, especially how surface properties of materials induce conformational changes of adsorbed proteins, needs to be well understood. Here we demonstrated that even though SPS is a necessary condition, it is not sufficient. We show that low substrate conductivity as well as proper salt concentration are also critical in sustained protein adsorption continuously. These factors allow one to pattern regions of different conducting properties and for the first time patterns physiologically relevant protein structures. Here we show that we can achieve patterned biomineralized regimes, both with plasma proteins in a simple and robust manner without additional functionalization or application of electrochemical gradients. Since the data indicate that the patterns just need to differ in electrical conductivity, rather than surface chemistry, we propose that the creation of transient image charges, due to incomplete charge screening, may be responsible for sustain the driving force for continual protein absorption.

  10. Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations

    Energy Technology Data Exchange (ETDEWEB)

    Moreno-Gordaliza, Estefanía, E-mail: emorenog@ucm.es [Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden (Netherlands); Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040, Madrid (Spain); Stigter, Edwin C.A. [Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden (Netherlands); Department of Molecular Cancer Research, Universitair Medisch Centrum Utrecht, Wilhelmina Kinder Ziekenhuis, Lundlaan 6, 3584, EA Utrecht (Netherlands); Lindenburg, Petrus W.; Hankemeier, Thomas [Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden (Netherlands)

    2016-06-07

    A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10{sup −9} m{sup 2} V{sup −1} s{sup −1}) when compared with unmodified fused silica (5.9 ± 0.1 10{sup −8} m{sup 2} V{sup −1} s{sup −1}). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1–1.8% coefficient-of-variation (CV) within a day) and 2–3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use. - Highlights: • New coating using recrystallized surface-layer proteins on

  11. Modulating protein behaviors on responsive surface by external electric fields: A molecular dynamics study

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yun, E-mail: xieyunxx@gdpu.edu.cn; Pan, Yufang; Zhang, Rong; Liang, Ying; Li, Zhanchao

    2015-01-30

    Graphical abstract: The adsorption of Cyt c on phosphorylcholine self-assembled monolayers (N atoms of the choline groups are colored in blue while the P atoms of the phosphate groups in orange). - Highlights: • PC-SAM could sensitively adjust its charge distribution to applied electric fields. • Adsorption of Cyt c on the PC-SAM is promoted or retarded as the charge distribution of the SAM changes. • Orientations of Cyt c on the PC-SAM are regulated by the structural changes of the SAM. • The structural changes of the SAM cause little deformation in Cyt c. - Abstract: Molecular dynamics simulations were employed to investigate the modulation of protein behaviors on the electrically responsive zwitterionic phosphorylcholine self-assembled monolayers (PC-SAMs). Results show that PC-SAMs could sensitively respond to the applied electric fields and exhibit three states with different charge distributions, namely both the negatively charged phosphate groups and the positively charged choline groups are exposed to the solution in the absence of electric fields (state 1), phosphate groups exposed in the presence of positive electric fields (state 2), and choline groups exposed in the presence of negative electric fields (state 3). Under state 1, the adsorption of Cyt c on the PC-SAM is reversible and the orientations of Cyt c are randomly distributed. Under state 2, the adsorption of Cyt c is enhanced due to the electrostatic attractions between the exposed phosphate groups and the positively charged protein; when adsorbed on the PC-SAMs, Cyt c tends to adopt the orientation with the heme plane perpendicular to the surface plane, and the percentage of this orientation increases as the field strength rises up. Under state 3, the adsorption of Cyt c is retarded because of the electrostatic repulsions between the exposed choline groups and the protein; however, if the gaps between PC chains are large enough, Cyt c could insert into the PC-SAM and access the

  12. Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study

    Directory of Open Access Journals (Sweden)

    Mamgain Hitesh

    2009-10-01

    Full Text Available Abstract Background Imaging tools such as scanning electron microscope (SEM and atomic force microscope (AFM can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections. Methods We validated knockdown and overexpression of SMAR1 using RT-PCR as well as Western blotting in human embryonic kidney (HEK 293, human breast cancer (MCF-7 and mouse melanoma (B16F1 cell lines. The samples were then processed for cell surface roughness studies using atomic force microscopy (AFM and scanning electron microscopy (SEM. The same samples were used for microarray analysis as well. Tumors sections from control and SMAR1 treated mice as well as tissues sections from different grades of human breast cancer on poly L-lysine coated slides were used for AFM and SEM studies. Results Tumor sections from mice injected with melanoma cells showed pronounced surface roughness. In contrast, tumor sections obtained from nude mice that were first injected with melanoma cells followed by repeated injections of SMAR1-P44 peptide, exhibited relatively smoother surface profile. Interestingly, human breast cancer tissue sections that showed reduced SMAR1 expression exhibited increased surface roughness compared to the adjacent normal breast tissue. Our AFM data establishes that treatment of cells with SMAR1-P44 results into increase in cytoskeletal volume that is supported by comparative gene expression data showing an increase in the expression of specific cytoskeletal proteins compared to the control cells. Altogether, these findings indicate that tumor suppressor function of SMAR1 might be exhibited through smoothening of cell surface by regulating expression of cell surface proteins. Conclusion Tumor suppressor

  13. Changes in the surface protein of the fat globules during homogenization and heat treatment of concentrated milk.

    Science.gov (United States)

    Ye, Aiqian; Anema, Skelte G; Singh, Harjinder

    2008-08-01

    The changes in milk fat globules and fat globule surface proteins of both low-preheated and high-preheated concentrated milks, which were homogenized at low or high pressure, were examined. The average fat globule size decreased with increasing homogenization pressure. The total surface protein (mg m-2) of concentrated milk increased after homogenization, the extent of the increase being dependent on the temperature and the pressure of homogenization, as well as on the preheat treatment. The concentrates obtained from high-preheated milks had higher surface protein concentration than the concentrates obtained from low-preheated milks after homogenization. Concentrated milks heat treated at 79 degrees C either before or after homogenization had greater amounts of fat globule surface protein than concentrated milks heat treated at 50 or 65 degrees C. This was attributed to the association of whey protein with the native MFGM (milk fat globule membrane) proteins and the adsorbed skim milk proteins. Also, at the same homogenization temperature and pressure, the amount of whey protein on the fat globule surface of the concentrated milk that was heated after homogenization was greater than that of the concentrated milk that was heated before homogenization. The amounts of the major native MFGM proteins did not change during homogenization, indicating that the skim milk proteins did not displace the native MFGM proteins but adsorbed on to the newly formed surface.

  14. Visualisation of variable binding pockets on protein surfaces by probabilistic analysis of related structure sets

    Directory of Open Access Journals (Sweden)

    Ashford Paul

    2012-03-01

    Full Text Available Abstract Background Protein structures provide a valuable resource for rational drug design. For a protein with no known ligand, computational tools can predict surface pockets that are of suitable size and shape to accommodate a complementary small-molecule drug. However, pocket prediction against single static structures may miss features of pockets that arise from proteins' dynamic behaviour. In particular, ligand-binding conformations can be observed as transiently populated states of the apo protein, so it is possible to gain insight into ligand-bound forms by considering conformational variation in apo proteins. This variation can be explored by considering sets of related structures: computationally generated conformers, solution NMR ensembles, multiple crystal structures, homologues or homology models. It is non-trivial to compare pockets, either from different programs or across sets of structures. For a single structure, difficulties arise in defining particular pocket's boundaries. For a set of conformationally distinct structures the challenge is how to make reasonable comparisons between them given that a perfect structural alignment is not possible. Results We have developed a computational method, Provar, that provides a consistent representation of predicted binding pockets across sets of related protein structures. The outputs are probabilities that each atom or residue of the protein borders a predicted pocket. These probabilities can be readily visualised on a protein using existing molecular graphics software. We show how Provar simplifies comparison of the outputs of different pocket prediction algorithms, of pockets across multiple simulated conformations and between homologous structures. We demonstrate the benefits of use of multiple structures for protein-ligand and protein-protein interface analysis on a set of complexes and consider three case studies in detail: i analysis of a kinase superfamily highlights the

  15. Comparative assessment of different histidine-tags for immobilization of protein onto surface plasmon resonance sensorchips.

    Science.gov (United States)

    Fischer, Marcus; Leech, Andrew P; Hubbard, Roderick E

    2011-03-01

    Surface plasmon resonance (SPR) is widely used to assess the kinetics and thermodynamics of binding of two molecules. The major challenge is immobilization of one molecule onto the sensorchip for robust detection of binding of the other molecule. We have compared a number of immobilization strategies for noncovalent attachment of an example protein (the substrate binding protein SiaP) by hexa-histidine (His), deca-His, and double-His tags to a nickel-nitrilotriacetic acid (NTA) surface. The stability of immobilization was assessed, and the binding of two low molecular weight ligands, Neu5Ac and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), at different temperatures studied. The hexa-His tagged SiaP washed off from the surface too rapidly for ligand binding to be measured reliably. Systematic variation of chip loading identified conditions under which the deca-His tagged SiaP could generate reliable results. The double-His tagged protein performed as well as covalently attached deca-His tagged protein at 15, 25, and 35 °C. The observed ligand binding kinetics were comparable for all immobilization strategies, and thermodynamic values calculated from SPR are in agreement with solution-based isothermal titration calorimetry measurements. Extended trials suggest that covalent attachment is preferable for screening campaigns, whereas the double-His-tag strategy allows rapid regeneration of the chip, for example, when tight binding compounds are assessed.

  16. Influence of surface wettability on competitive protein adsorption and initial attachment of osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wei Jianhua; Liu Baolin [Department of Oral and Maxillofacial Surgery, School of Stomatology, Fourth Military Medical University, 145 West Changle Road, Xi' an 710032 (China); Igarashi, Toshio [Department of Oral and Maxillofacial Surgery, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580 (Japan); Okumori, Naoto; Igarashi, Takayasu; Maetani, Takashi [Japan Institute for Advanced Dentistry, 1-8-25 Shiba, Minato-ku, Tokyo 105-0014 (Japan); Yoshinari, Masao, E-mail: yosinari@tdc.ac.j [Division of Oral Implants Research and HRC7, Oral Health Science Center, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502 (Japan)

    2009-08-01

    This study investigated the influence of surface wettability on competitive protein adsorption and the initial attachment of osteoblasts. A thin-film coating of hexamethyldisiloxane (HMDSO) and subsequent O{sub 2}-plasma treatment was carried out on substrates with a mirror surface in order to create a wide range of wettabilities. The adsorption behavior of fibronectin (Fn) and albumin (Alb) in both individual and competitive mode, and the initial attachment of mouse osteoblastic cells (MC3T3-E1) over a wide range of wettabilities were investigated. The contact angle of HMDSO coatings without O{sub 2}-plasma treatment against double-distilled water was more than 100{sup 0}, whereas it dramatically decreased after the O{sub 2}-plasma treatment to almost 0{sup 0}, resulting in super-hydrophilicity. Individually, Fn adsorption showed a biphasic inclination, whereas Alb showed greater adsorption to hydrophobic surfaces. In the competitive mode, in a solution containing both Fn and Alb, Fn showed greater adsorption on hydrophilic surfaces, whereas Alb predominantly adsorbed on hydrophobic surfaces. The initial attachment of osteoblastic cells increased with an increase in surface wettability, in particular, on a super-hydrophilic surface, which correlated well with Fn adsorption in the competitive mode. These results suggest that Fn adsorption may be responsible for increasing cell adhesion on hydrophilic surfaces in a body fluid or culture media under physiological conditions.

  17. Fixation and stabilization of Escherichia coli cells displaying genetically engineered cell surface proteins.

    Science.gov (United States)

    Freeman, A; Abramov, S; Georgiou, G

    1996-12-05

    A large biotechnological potential is inherent in the display of proteins (e.g., enzymes, single-chain antibodies, on the surface of bacterial cells) (Georgiou et al., 1993). Applications such as immobilized whole-cell biocatalysts or cellular adsorbents require cell fixation to prevent disintegration, stabilization of the anchored protein from leakage, denaturation or proteolysis, and total loss of cell viability, preventing medium and potential product contamination with cells. In this article we describe the adaptation of a simple two-stage chemical crosslinking procedure based on "bi-layer encagement" (Tor et al., 1989) for stabilizing Escherichia coli cells expressing an Lpp-OmpA (46-159)-beta-lactamase fusion that displays beta-lactamase on the cell surface. Bilayer crosslinking and coating the bacteria with a polymeric matrix is accomplished by treating the cells first with either glutaraldehyde or polyglutaraldehyde, followed by secondary crosslinking with polyacrylamide hydrazide. These treatments resulted in a 5- to 25-fold reduction of the thermal inactivation rate constant at 55 degrees C of surface anchored beta-lactamase and completely prevented the deterioration of the cells for at least a week of storage at 4 degrees C. The stabilization procedure developed paves the way to scalable biotechnological applications of E. coli displaying surface anchored proteins as whole-cell biocatalysts and adsorbents.

  18. Impact of surface coating and food-mimicking media on nanosilver-protein interaction

    Energy Technology Data Exchange (ETDEWEB)

    Burcza, Anna, E-mail: anna.burcza@mri.bund.de; Gräf, Volker; Walz, Elke; Greiner, Ralf [Max Rubner-Institute, Department of Food Technology and Bioprocess Engineering (Germany)

    2015-11-15

    The application of silver nanoparticles (AgNPs) in food contact materials has recently become a subject of dispute due to the possible migration of silver in nanoform into foods and beverages. Therefore, the analysis of the interaction of AgNPs with food components, especially proteins, is of high importance in order to increase our knowledge of the behavior of nanoparticles in food matrices. AgPURE™ W10 (20 nm), an industrially applied nanomaterial, was compared with AgNPs of similar size frequently investigated for scientific purposes differing in the surface capping agent (spherical AgNP coated with either PVP or citrate). The interactions of the AgNPs with whey proteins (BSA, α-lactalbumin and β-lactoglobulin) at different pH values (4.2, 7 or 7.4) were investigated using surface plasmon resonance, SDS-PAGE, and asymmetric flow field-flow fractionation. The data obtained by the three different methods correlated well. Besides the nature of the protein and the nanoparticle coating, the environment was shown to affect the interaction significantly. The strongest interaction was obtained with BSA and AgNPs in an acidic environment. Neutral and slightly alkaline conditions however, seemed to prevent the AgNP-protein interaction almost completely. Furthermore, the interaction of whey proteins with AgPURE™ W10 was found to be weaker compared to the interaction with the other two AgNPs under all conditions investigated.

  19. Effects of Proteins from Culture Medium on Surface Property of Silanes- Functionalized Magnetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Chen ZP

    2008-01-01

    Full Text Available Abstract Monodisperse magnetic nanoparticles (MNPs were synthesized by thermal decomposition of iron-oleate and functionalized with silanes bearing various functional groups such as amino group (NH2, short-chain poly(ethylene glycol (PEG, and carboxylic group (COOH. Then, silanes-functionalized magnetic nanoparticles (silanes-MNPs were incubated in cell culture medium plus fetal calf serum to investigate the effects of proteins from culture medium on surface property of MNPs. Zeta potential measurements showed that although surface charges of silanes-MNPs were different, they exhibited negative charges at neutral pH and approximate isoelectric points after they were incubated in cell culture medium. The reason was that silanes-MNPs could easily adsorb proteins from culture medium via non-covalent binding, resulting in the formation of protein-silanes-MNPs conjugates. Moreover, silanes-MNPs with various functional groups had different adsorption capacity to proteins, as confirmed by Coomassie blue fast staining method. The in vitro cell experiments showed that protein-silanes-MNPs had higher cellular uptake by cancer cells than silanes-MNPs.

  20. Impact of surface coating and food-mimicking media on nanosilver-protein interaction

    Science.gov (United States)

    Burcza, Anna; Gräf, Volker; Walz, Elke; Greiner, Ralf

    2015-11-01

    The application of silver nanoparticles (AgNPs) in food contact materials has recently become a subject of dispute due to the possible migration of silver in nanoform into foods and beverages. Therefore, the analysis of the interaction of AgNPs with food components, especially proteins, is of high importance in order to increase our knowledge of the behavior of nanoparticles in food matrices. AgPURE™ W10 (20 nm), an industrially applied nanomaterial, was compared with AgNPs of similar size frequently investigated for scientific purposes differing in the surface capping agent (spherical AgNP coated with either PVP or citrate). The interactions of the AgNPs with whey proteins (BSA, α-lactalbumin and β-lactoglobulin) at different pH values (4.2, 7 or 7.4) were investigated using surface plasmon resonance, SDS-PAGE, and asymmetric flow field-flow fractionation. The data obtained by the three different methods correlated well. Besides the nature of the protein and the nanoparticle coating, the environment was shown to affect the interaction significantly. The strongest interaction was obtained with BSA and AgNPs in an acidic environment. Neutral and slightly alkaline conditions however, seemed to prevent the AgNP-protein interaction almost completely. Furthermore, the interaction of whey proteins with AgPURE™ W10 was found to be weaker compared to the interaction with the other two AgNPs under all conditions investigated.

  1. Enzyme directed formation of un-natural side-chains for covalent surface attachment of proteins.

    Science.gov (United States)

    Cho, Hwayoung; Jaworski, Justyn

    2014-10-01

    The covalent immobilization of proteins onto surfaces is an essential aspect of several fields of research, including proteomics, sensing, heterogeneous biocatalysis, and more broadly biotechnology. Site-specific, covalent attachment of proteins has been achieved in recent years by the use of expanded genetic codes to produce proteins with controlled placement of un-natural amino acids bearing bio-orthogonal functional groups. Unfortunately, the complexity of developing such systems is impractical for most laboratories; hence, a less complicated approach to generating un-natural amino acid side-chains has been employed. Utilizing a straightforward reaction with formylglycine generating enzyme, we use the site-specific modification of engineered proteins to yield un-natural amino acid side-chains for protein immobilization. Using this approach, we demonstrate the controlled immobilization of various enzymes onto a variety of amine coated surfaces. Our results reveal reusability of the immobilized enzymes via this strategy, and furthermore, we find the activity of the immobilized enzymes to remain even after a month of use indicating significant stability of the linkage.

  2. Serum antibody responses to Wolbachia surface protein in patients with human lymphatic filariasis.

    Science.gov (United States)

    Shiny, Chandanapurath; Krushna, Nagampalli S A; Archana, Bairavasundaram; Farzana, Begum; Narayanan, Rangarajan B

    2009-12-01

    Wolbachia surface protein (WSP), which is the most abundantly expressed protein of Wolbachia from the human filarial parasite Brugia malayi, was chosen for the present study. B-cell epitope prediction of the WSP protein sequence indicates a high antigenicity, surface probability and hydrophilicity by DNA STAR software analysis. ProPred analysis suggests the presence of HLA class II binding regions in the WSP protein that contribute to T-cell responses and isotype reactivity. In order to validate these findings, the gene coding for endosymbiont WSP was PCR-amplified from the genomic DNA of the human filarial parasite Brugia malayi and cloned in T-7 expression vector pRSET-A. Western blot and ELISA at the total IgG level with recombiant WSP indicated a significantly elevated reactivity in CP compared to MF, EN and NEN individuals. Isotype ELISA also suggested an elevated reactivity in CP patients at the IgG1 level. In contrast, WSP-specific IgG4 levels were found to be elevated in MF patients compared to CP and EN. Besides this, WSP-specific IgE levels indicated an elevated reactivity in CP and MF patients compared to normals. Observations from ELISA supported the in silico predictions that indicate the presence of B- and T-cell epitopes. Hence, a combinatorial approach of in silico predictions and wet-lab studies provides interesting insights into the role of Wolbachia proteins in filarial pathogenesis.

  3. Evaluation of the immunodiagnostic potential of a recombinant surface protein domain from Acanthamoeba castellanii.

    Science.gov (United States)

    Sánchez, Alemao G Carpinteyro; Virginio, Veridiana Gomes; Maschio, Vinicius José; Ferreira, Henrique Bunselmeyer; Rott, Marilise Brittes

    2016-10-01

    Acanthamoeba spp. are free-living protists widely distributed in environment, able to cause keratitis, encephalitis and skin lesions in humans and animals. Acanthamoeba spp. exist in two forms: an infective trophozoite and a dormant cyst. Several factors contribute to the pathogenesis of Acanthamoeba spp. The parasite adhesion to the host cell is the primary step for infection and is mediated by a mannose binding-protein, expressed in the surface and considered the main pathogenicity factor in Acanthamoeba spp. So far, there was no evidence of another surface protein of Acanthamoeba spp. relevant for host invasion or infection by these organisms. The aims of this study were to identify and characterize an Acanthamoeba castellanii surface protein and to evaluate its diagnostic potential. In silico predictions of surface proteins allowed to identify the A. castellanii calreticulin as a possible surface antigen. The coding sequence of a predicted extracellular domain of A. castellanii calreticulin was cloned by in vivo homologous recombination and the recombinant polypeptide (AcCRT29-130) was produced. Its immunodiagnostic potential was assessed in a recombinant antigen-based ELISA with sera from experimentally infected rats that developed keratitis and encephalitis, and sera from patients with encephalitis. The AcCRT29-130 was significantly more recognized by sera from encephalitis infected rats in comparison with the non-infected controls. Human sera from encephalitis patients, however presented no significant response. These results showed the AcCRT29-130 potential for A. castellanii infection immunodiagnosis in animals, with further studies being required for assessment of its use for human infections.

  4. Unfolding of a model protein on ion exchange and mixed mode chromatography surfaces.

    Science.gov (United States)

    Gospodarek, Adrian M; Hiser, Diana E; O'Connell, John P; Fernandez, Erik J

    2014-08-15

    Recent studies with proteins indicate that conformational changes and aggregation can occur during ion exchange chromatography (IEC). Such behavior is not usually expected, but could lead to decreased yield and product degradation from both IEC and multi mode chromatography (MMC) that has ligands of both hydrophobic and charged functionalities. In this study, we used hydrogen exchange mass spectrometry to investigate unfolding of the model protein BSA on IEC and MMC surfaces under different solution conditions at 25°C. Increased solvent exposure, indicating greater unfolding relative to that in solution, was found for protein adsorbed on cationic IEC and MMC surfaces in the pH range of 3.0 to 4.5, where BSA has decreased stability in solution. There was no effect of anionic surfaces at pH values in the range from 6.0 to 9.0. Differences of solvent exposure of whole molecules when adsorbed and in solution suggest that adsorbed BSA unfolds at lower pH values and may show aggregation, depending upon pH and the surface type. Measurements on digested peptides showed that classifications of stability can be made for various regions; these are generally retained as pH is changed. When salt was added to MMC systems, where electrostatic interactions would be minimized, less solvent exposure was seen, implying that it is the cationic moieties, rather than the hydrophobic ligands, which cause greater surface unfolding at low salt concentrations. These results suggest that proteins of lower stability may exhibit unfolding and aggregation during IEC and MMC separations, as they can with hydrophobic interaction chromatography.

  5. Conjugation of Hyaluronic Acid onto Surfaces via the Interfacial Polymerization of Dopamine to Prevent Protein Adsorption.

    Science.gov (United States)

    Huang, Renliang; Liu, Xia; Ye, Huijun; Su, Rongxin; Qi, Wei; Wang, Libing; He, Zhimin

    2015-11-10

    A versatile, convenient, and cost-effective method that can be used for grafting antifouling materials onto different surfaces is highly desirable in many applications. Here, we report the one-step fabrication of antifouling surfaces via the polymerization of dopamine and the simultaneous deposition of anionic hyaluronic acid (HA) on Au substrates. The water contact angle of the Au surfaces decreased from 84.9° to 24.8° after the attachment of a highly uniform polydopamine (PDA)/HA hybrid film. The results of surface plasmon resonance analysis showed that the Au-PDA/HA surfaces adsorbed proteins from solutions of bovine serum albumin, lysozyme, β-lactoglobulin, fibrinogen, and soybean milk in ultralow or low amounts (4.8-31.7 ng/cm(2)). The hydrophilicity and good antifouling performance of the PDA/HA surfaces is attributable to the HA chains that probably attached onto their upper surface via hydrogen bonding between PDA and HA. At the same time, the electrostatic repulsion between PDA and HA probably prevents the aggregation of PDA, resulting in the formation of a highly uniform PDA/HA hybrid film with the HA chains (with a stretched structure) on the upper surface. We also developed a simple method for removing this PDA/HA film and recycling the Au substrates by using an aqueous solution of NaOH as the hydrolyzing agent. The Au surface remained undamaged, and a PDA/HA film could be redeposited on the surface, with the surface exhibiting good antifouling performance even after 10 such cycles. Finally, it was found that this grafting method is applicable to other substrates, including epoxy resins, polystyrene, glass, and steel, owing to the strong adhesion of PDA with these substrates.

  6. Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

    OpenAIRE

    Hermann, M.; Jaconi, M E; Dahlgren, C; Waldvogel, F A; Stendahl, O; Lew, D P

    1990-01-01

    The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (m...

  7. Cytochrome C on a gold surface: investigating structural relaxations and their role in protein-surface electron transfer by molecular dynamics simulations.

    Science.gov (United States)

    Siwko, Magdalena E; Corni, Stefano

    2013-04-28

    Proteins immobilized on inorganic surfaces are important in technological fields such as biosensors, enzymatic biofuel cells and biomolecular electronics. In these frameworks, it has been demonstrated that some proteins are able to keep their functionality, although the latter may be somewhat modified by the interaction with the surface. Cytochrome C, an heme-based electron transfer protein, has been found to be able to exchange electrons with the gold surface on which it is immobilized, but some deviations from the expected electron transfer rates were evidenced [C. A. Bortolotti, et al., J. Phys. Chem. C 2007, 111, 12100-12105]. In this work we have used molecular dynamics simulations of (native and mutated) yeast cytochrome C supported on Au(111) to investigate the microscopic picture behind the experimental behavior of the molecule. In particular, we have focused on the structural re-arrangements due to the interactions with the surface. We found that, despite being secondary-structure preserving, they can profoundly affect protein-surface electronic coupling and, in turn, electron transfer rates, explaining experimental findings. The conformational flexibility of the protein in the region of the protein-surface bond is thus pivotal in determining the resulting ET functionality of the immobilized protein.

  8. Multicolor surface plasmon resonance imaging of ink jet-printed protein microarrays.

    Science.gov (United States)

    Singh, Bipin K; Hillier, Andrew C

    2007-07-15

    We report a technique that utilizes surface plasmon resonance dispersion as a mechanism to provide multicolor contrast for imaging thin molecular films. Illumination of gold surfaces with p-polarized white light in the Kretschmann configuration produces distinct reflected colors due to excitation of surface plasmons and the resulting absorption of specific wavelengths from the source light. In addition, these colors transform in response to the formation of thin molecular films. This process represents a simple detection method for distinguishing between films of varying thickness in sensor applications. As an example, we interrogated a protein microarray formed by a commercial drop-on-demand chemical ink jet printer. Submonolayer films of a test protein (bovine serum albumin) were readily detected by this method. Analysis of the dispersion relations and absorbance sensitivities illustrate the performance and characteristics of this system. Higher detection sensitivity was achieved at angles where red wavelengths coupled to surface plasmons. However, improved contrast and spatial resolution occurred when the angle of incidence was such that shorter wavelengths coupled to the surface plasmons. Simplified optics combined with the robust microarray printing platform are used to demonstrate the applicability of this technique as a rapid and versatile, high-throughput tool for label-free detection of adsorbed films and macromolecules.

  9. Contribution of Long Fibrils and Peptides to Surface and Foaming Behavior of Soy Protein Fibril System.

    Science.gov (United States)

    Wan, Zhili; Yang, Xiaoquan; Sagis, Leonard M C

    2016-08-16

    When soy glycinin (11S) is heated for a prolonged time at pH 2 (20 h at 85 °C), a mixture is formed consisting of long semiflexible 11S fibrils and small peptides. The surface and foaming properties of this mixture were investigated at different pHs, and compared to the behavior of pure fibrils and pure peptides, to determine the individual contributions of these two factions to the behavior of the mixture. The adsorption of these three systems at air-water interfaces and the resulting surface rheological properties were studied by combining drop shape analysis tensiometry, ellipsometry, and surface large amplitude oscillatory dilatational (LAOD) rheology. Lissajous plots of surface pressure versus deformation were used to analyze the surface rheological response in terms of interfacial microstructure. Our results show that the adsorption kinetics, dilatational rheological properties, and the foaming behavior of the mixture were mainly dominated by the small peptides in the fibril system. Compared to pH 2, the fibril mixture at pH 5 and 7 provides much better foam stability and appears to be a very promising protein material to make stable foams, even at low protein concentration (0.1 wt %). The presence of fibril clusters and peptide aggregates at pH 5 and 7 contributed to foam stability of the mixture. In contrast, pure fibril formed an interface with a highly pH-responsive adsorption and rheological behavior, and the foamability and foam stability of the pure fibrils were very poor.

  10. A transducing bacteriophage for Caulobacter crescentus uses the paracrystalline surface layer protein as a receptor.

    OpenAIRE

    P. Edwards; Smit, J

    1991-01-01

    The bacteriophage phi Cr30, a transducing phage for Caulobacter crescentus strains, required the paracrystalline surface (S) layer for infectivity. Wild-type strains were phage resistant when rsaA, the gene for the 130K S-layer protein, was interrupted with an antibiotic resistance cassette. Strains that had lost the S layer by mutation were phage resistant, as were mutants that produce an S layer but which do not attach the structure to the cell surface. Phage sensitivity was restored to 130...

  11. Scaling properties of the radius of gyration and surface area for EF-hand calcium binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Pitulice, L. [West University of Timisoara, Department of Chemistry, Pestalozzi 16, 300115 Timisoara (Romania); Isvoran, A. [West University of Timisoara, Department of Chemistry, Pestalozzi 16, 300115 Timisoara (Romania)], E-mail: aisvoran@cbg.uvt.ro; Craescu, C.T. [INSERM U759/Institute Curie-Recherche, Centre Universitaire Paris-Sud, Batiment 112, 91405 Orsay (France); Chiriac, A. [West University of Timisoara, Department of Chemistry, Pestalozzi 16, 300115 Timisoara (Romania)

    2009-04-30

    In this paper, we analyze the scaling properties of both the radius of gyration and the surface area for EF-hand calcium binding proteins. These properties are different for two conformational subfamilies: proteins with extended and compact structures, respectively. The radius of gyration is a measure of the shape of protein, whereas its surface fractal dimension is a measure of its interatomic packing. Different scaling properties for the radius of gyration underline that these two subfamilies present different shapes whilst different scaling properties for the surface area reveal different strengths of their intermolecular forces. All these data suggest different mechanisms responsible for the global folding of proteins belonging to these two subfamilies.

  12. 3D-SURFER 2.0: web platform for real-time search and characterization of protein surfaces.

    Science.gov (United States)

    Xiong, Yi; Esquivel-Rodriguez, Juan; Sael, Lee; Kihara, Daisuke

    2014-01-01

    The increasing number of uncharacterized protein structures necessitates the development of computational approaches for function annotation using the protein tertiary structures. Protein structure database search is the basis of any structure-based functional elucidation of proteins. 3D-SURFER is a web platform for real-time protein surface comparison of a given protein structure against the entire PDB using 3D Zernike descriptors. It can smoothly navigate the protein structure space in real-time from one query structure to another. A major new feature of Release 2.0 is the ability to compare the protein surface of a single chain, a single domain, or a single complex against databases of protein chains, domains, complexes, or a combination of all three in the latest PDB. Additionally, two types of protein structures can now be compared: all-atom-surface and backbone-atom-surface. The server can also accept a batch job for a large number of database searches. Pockets in protein surfaces can be identified by VisGrid and LIGSITE (csc) . The server is available at http://kiharalab.org/3d-surfer/.

  13. ErpC, a member of the complement regulator-acquiring family of surface proteins from Borrelia burgdorferi, possesses an architecture previously unseen in this protein family.

    Science.gov (United States)

    Caesar, Joseph J E; Johnson, Steven; Kraiczy, Peter; Lea, Susan M

    2013-06-01

    Borrelia burgdorferi is a spirochete responsible for Lyme disease, the most commonly occurring vector-borne disease in Europe and North America. The bacterium utilizes a set of proteins, termed complement regulator-acquiring surface proteins (CRASPs), to aid evasion of the human complement system by recruiting and presenting complement regulator factor H on its surface in a manner that mimics host cells. Presented here is the atomic resolution structure of a member of this protein family, ErpC. The structure provides new insights into the mechanism of recruitment of factor H and other factor H-related proteins by acting as a molecular mimic of host glycosaminoglycans. It also describes the architecture of other CRASP proteins belonging to the OspE/F-related paralogous protein family and suggests that they have evolved to bind specific complement proteins, aiding survival of the bacterium in different hosts.

  14. Probing protein orientation near charged surfaces with an implicit-solvent model and the PyGBe code

    CERN Document Server

    Cooper, Christopher D

    2015-01-01

    Protein-surface interactions are ubiquitous in biological processes and bioengineering, yet are not fully understood. In the field of biosensors, a key factor in biosensor performance is the orientation of biomolecules near charged surfaces. The aim of this work is developing and assessing a computational model to study proteins interacting with charged surfaces and obtain orientation data. After extending the implicit-solvent model used in the open-source code PyGBe and deriving an analytical solution for simple geometry, our careful grid-convergence analysis builds confidence on the correctness and value of our approach for probing protein orientation. Further computational experiments support it: they study preferred orientations for protein GB1 D4' and immunoglobulin G. Sampling the free energy for protein GB1 at a range of tilt and rotation angles with respect to the charged surface, we calculated the probability of the protein orientation and observed a dipolar behavior. This result is consistent with p...

  15. Influence of membrane surface charge on adsorption of complement proteins onto supported lipid bilayers.

    Science.gov (United States)

    Yorulmaz, Saziye; Jackman, Joshua A; Hunziker, Walter; Cho, Nam-Joon

    2016-12-01

    The complement system is an important part of the innate immune response, and there is great interest in understanding how complement proteins interact with lipid membrane interfaces, especially in the context of recognizing foreign particulates (e.g., liposomal nanomedicines). Herein, a supported lipid bilayer platform was employed in order to investigate the effect of membrane surface charge (positive, negative, or neutral) on the adsorption of three complement proteins. Quartz crystal microbalance-dissipation (QCM-D) experiments measured the real-time kinetics and total uptake of protein adsorption onto supported lipid bilayers. The results demonstrate that all three proteins exhibit preferential, mainly irreversible adsorption onto negatively charged lipid bilayers, yet there was also significant variation in total uptake and the relative degree of adsorption onto negatively charged bilayers versus neutral and positively charged bilayers. The total uptake was also observed to strongly depend on the bulk protein concentration. Taken together, our findings contribute to a broader understanding of the factors which influence adsorption of complement proteins onto lipid membranes and offer guidance towards the design of synthetic lipid bilayers with immunocompetent features.

  16. Facile Discovery of Cell-Surface Protein Targets of Cancer Cell Aptamers.

    Science.gov (United States)

    Bing, Tao; Shangguan, Dihua; Wang, Yinsheng

    2015-10-01

    Cancer biomarker discovery constitutes a frontier in cancer research. In recent years, cell-binding aptamers have become useful molecular probes for biomarker discovery. However, there are few successful examples, and the critical barrier resides in the identification of the cell-surface protein targets for the aptamers, where only a limited number of aptamer targets have been identified so far. Herein, we developed a universal SILAC-based quantitative proteomic method for target discovery of cell-binding aptamers. The method allowed for distinguishing specific aptamer-binding proteins from nonspecific proteins based on abundance ratios of proteins bound to aptamer-carrying bait and control bait. In addition, we employed fluorescently labeled aptamers for monitoring and optimizing the binding conditions. We were able to identify and validate selectin L and integrin α4 as the protein targets for two previously reported aptamers, Sgc-3b and Sgc-4e, respectively. This strategy should be generally applicable for the discovery of protein targets for other cell-binding aptamers, which will promote the applications of these aptamers.

  17. Aptamer-based surface plasmon resonance sensing of glycated human blood proteins

    Science.gov (United States)

    Reaver, Nathan G. F.; Zheng, Rui; Kim, Dong-Shik; Cameron, Brent D.

    2013-02-01

    The concentration ratio of glycated to non-glycated forms of various blood proteins can be used as a diagnostic measure in diabetes to determine a history of glycemic compliance. Depending on a protein's half-life in blood, compliance can be assessed from a few days to several months in the past, which can then be used to provide additional therapeutic guidance. Current glycated protein detection methods are limited in their ability to measure multiple proteins, and are susceptible to interference from other blood pathologies. In this study, we developed and characterized DNA aptamers for use in Surface Plasmon Resonance (SPR) sensors to assess the blood protein hemoglobin. The aptamers were developed by way of a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process which selects DNA sequences that have a high binding affinity to a specific protein. DNA products resulting from this process are sequenced and identified aptamers are then synthesized. The SELEX process was performed to produce aptamers for a glycated form of hemoglobin. Equilibrium dissociation constants for the binding of the identified aptamer to glycated hemoglobin, hemoglobin, and fibrinogen were calculated from fitted Langmuir isotherms obtained through SPR. These constants were determined to be 94 nM, 147 nM, and 244 nM respectively. This aptamer can potentially be used to create a SPR aptamer based biosensor for detection of glycated hemoglobin, a technology that has the potential to deliver low-cost and immediate glycemic compliance assessment in either a clinical or home setting.

  18. Predictive response surface model for heat-induced rheological changes and aggregation of whey protein concentrate.

    Science.gov (United States)

    Alvarez, Pedro A; Emond, Charles; Gomaa, Ahmed; Remondetto, Gabriel E; Subirade, Muriel

    2015-02-01

    Whey proteins are now far more than a by-product of cheese processing. In the last 2 decades, food manufacturers have developed them as ingredients, with the dairy industry remaining as a major user. For many applications, whey proteins are modified (denatured) to alter their structure and functional properties. The objective of this research was to study the influence of 85 to 100 °C, with protein concentration of 8% to 12%, and treatment times of 5 to 30 min, while measuring rheological properties (storage modulus, loss modulus, and complex viscosity) and aggregation (intermolecular beta-sheet formation) in dispersions of whey protein concentrate (WPC). A Box-Behnken Response Surface Methodology modeled the heat denaturation of liquid sweet WPC at 3 variables and 3 levels. The model revealed a very significant fit for viscoelastic properties, and a lesser fit for protein aggregation, at temperatures not previously studied. An exponential increase of rheological parameters was governed by protein concentration and temperature, while a modest linear relationship of aggregation was governed by temperature. Models such as these can serve as valuable guides to the ingredient and dairy industries to develop target products, as whey is a major ingredient in many functional foods.

  19. Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH.

    Science.gov (United States)

    Krishna Kumar, Kaavya; Jacques, David A; Pishchany, Gleb; Caradoc-Davies, Tom; Spirig, Thomas; Malmirchegini, G Reza; Langley, David B; Dickson, Claire F; Mackay, Joel P; Clubb, Robert T; Skaar, Eric P; Guss, J Mitchell; Gell, David A

    2011-11-01

    Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents.

  20. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Akiyoshi Uezumi

    2016-08-01

    Full Text Available Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases.

  1. Mammalian carboxylesterase (CES) releases GPI-anchored proteins from the cell surface upon lipid raft fluidization.

    Science.gov (United States)

    Orihashi, Kaoru; Tojo, Hiromasa; Okawa, Katsuya; Tashima, Yuko; Morita, Takashi; Kondoh, Gen

    2012-03-01

    Mammalian carboxylesterase (CES) is well known as a biotransformation enzyme for prodrugs and xenobiotics. Here, we purified CES as a GPI-anchored protein (GPI-AP)-releasing factor (GPIase) that releases such protein from the cell surface. All five isoforms of CES showed this activity to various degrees. When the serine residue of the catalytic triad for esterase was replaced by alanine, esterase activity was completely disrupted, while full GPIase activity remained, suggesting that these two activities are exhibited via different mechanisms. CES6, a new class of mammalian CES, exhibited the highest GPIase activity and released specific GPI-APs from the cell surface after lipid raft fluidization. The released product contained a GPI component, indicating that GPI-AP was released by cleavage in GPI. These results revealed for the first time that CES recognizes and catalyzes macromolecule GPI-AP as well as small molecules.

  2. Restricted mobility of side chains on concave surfaces of solenoid proteins may impart heightened potential for intermolecular interactions.

    Science.gov (United States)

    Ramya, L; Gautham, N; Chaloin, Laurent; Kajava, Andrey V

    2015-09-01

    Significant progress has been made in the determination of the protein structures with their number today passing over a hundred thousand structures. The next challenge is the understanding and prediction of protein-protein and protein-ligand interactions. In this work we address this problem by analyzing curved solenoid proteins. Many of these proteins are considered as "hub molecules" for their high potential to interact with many different molecules and to be a scaffold for multisubunit protein machineries. Our analysis of these structures through molecular dynamics simulations reveals that the mobility of the side-chains on the concave surfaces of the solenoids is lower than on the convex ones. This result provides an explanation to the observed preferential binding of the ligands, including small and flexible ligands, to the concave surface of the curved solenoid proteins. The relationship between the landscapes and dynamic properties of the protein surfaces can be further generalized to the other types of protein structures and eventually used in the computer algorithms, allowing prediction of protein-ligand interactions by analysis of protein surfaces.

  3. Streptococcal collagen-like surface protein 1 promotes adhesion to the respiratory epithelial cell

    Directory of Open Access Journals (Sweden)

    Chang Cherng-Shyang

    2010-12-01

    Full Text Available Abstract Background Collagen-like surface proteins Scl1 and Scl2 on Streptococcus pyogenes contain contiguous Gly-X-X triplet amino acid motifs, the characteristic structure of human collagen. Although the potential role of Scl1 in adhesion has been studied, the conclusions may be affected by the use of different S. pyogenes strains and their carriages of various adhesins. To explore the bona fide nature of Scl1 in adherence to human epithelial cells without the potential interference of other streptococcal surface factors, we constructed a scl1 isogenic mutant from the Scl2-defective S. pyogenes strain and a Scl1-expressed Escherichia coli. Results Loss of Scl1 in a Scl2-defective S. pyogenes strain dramatically decreased the adhesion of bacteria to HEp-2 human epithelial cells. Expression of Scl1 on the surface of the heterologous bacteria E. coli significantly increased adhesion to HEp-2. The increase in adhesion was nullified when Scl1-expressed E. coli was pre-incubated with proteases or antibodies against recombinant Scl1 (rScl1 protein. Treatment of HEp-2 cells with rScl protein or pronase drastically reduced the binding capability of Scl1-expressed E. coli. These findings suggest that the adhesion is mediated through Scl1 on bacterial surface and protein receptor(s on epithelial cells. Further blocking of potential integrins revealed significant contributions of α2 and β1 integrins in Scl1-mediated binding to epithelial cells. Conclusions Together, these results underscore the importance of Scl1 in the virulence of S. pyogenes and implicate Scl1 as an adhesin during pathogenesis of streptococcal infection.

  4. Microarray analysis of genes associated with cell surface NIS protein levels in breast cancer

    Directory of Open Access Journals (Sweden)

    Richardson Andrea L

    2011-10-01

    Full Text Available Abstract Background Na+/I- symporter (NIS-mediated iodide uptake allows radioiodine therapy for thyroid cancer. NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer. However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy. We aimed to identify biomarkers associated with NIS expression such that mechanisms underlying NIS modulation in human breast tumors may be elucidated. Methods Published oligonucleotide microarray data within the National Center for Biotechnology Information Gene Expression Omnibus database were analyzed to identify gene expression tightly correlated with NIS mRNA level among human breast tumors. NIS immunostaining was performed in a tissue microarray composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor such that gene expression associated with cell surface NIS protein level could be identified. Results and Discussion NIS mRNA levels do not vary among breast tumors or when compared to normal breast tissues when detected by Affymetrix oligonucleotide microarray platforms. Cell surface NIS protein levels are much more variable than their corresponding NIS mRNA levels. Despite a limited number of breast tumors examined, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein levels in the ER-positive breast cancer subtype. Conclusions Further investigation on genes associated with cell surface NIS protein levels within each breast cancer molecular subtype may lead to novel targets for selectively increasing NIS expression/function in a subset of breast cancers patients.

  5. [Vaccination of mice against murine coccidiosis by ingestion of surface proteins of Eimeria falciformis incorporated in liposomes].

    Science.gov (United States)

    Rhalem, A; Bekhti, K; Bourdieu, C; Luffau, G; Péry, P

    1989-01-01

    Proteins are released from the surface of sporozoites of Eimeria falciformis during their in vitro incubation in a detergent solution. Some of these proteins reacted with antibodies from infected mice and specifically stimulated the proliferation of mesenteric lymph node cells of these mice. Oral immunization of mice with liposome encapsulated sporozoite surface antigens protected mice against a challenge infection. Two proteins (M.W. 27 and 180 K) induced an antibody synthesis in these vaccinated mice.

  6. Saliva analysis combining membrane protein purification with surface-enhanced Raman spectroscopy for nasopharyngeal cancer detection

    Science.gov (United States)

    Feng, Shangyuan; Lin, Duo; Lin, Juqiang; Huang, Zufang; Chen, Guannan; Li, Yongzeng; Huang, Shaohua; Zhao, Jianhua; Chen, Rong; Zeng, Haishan

    2014-02-01

    A method for saliva analysis combining membrane protein purification with silver nanoparticle-based surface-enhanced Raman spectroscopy (SERS) for non-invasive nasopharyngeal cancer detection was present in this paper. In this method, cellulose acetate membrane was used to obtain purified whole proteins from human saliva while removing other native saliva constituents and exogenous substances. The purified proteins were mixed with silver nanoparticle for SERS analysis. A diagnostic accuracy of 90.2% can be achieved by principal components analysis combined with linear discriminate analysis, for saliva samples obtained from patients with nasopharyngeal cancer (n = 62) and healthy volunteers (n = 30). This exploratory study demonstrated the potential for developing non-invasive, rapid saliva SERS analysis for nasopharyngeal cancer detection.

  7. Baculovirus superinfection: a probable restriction factor on the surface display of proteins for library screening.

    Directory of Open Access Journals (Sweden)

    Xiaodong Xu

    Full Text Available In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed. The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells co-expressing the two fluorescent proteins at 48 hours post infection (hpi, and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the re-infection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple

  8. Surface Plasmon Resonance Investigations of Bioselective Element Based on the Recombinant Protein A for Immunoglobulin Detection

    Science.gov (United States)

    Bakhmachuk, A.; Gorbatiuk, O.; Rachkov, A.; Dons'koi, B.; Khristosenko, R.; Ushenin, I.; Peshkova, V.; Soldatkin, A.

    2017-02-01

    The developed surface plasmon resonance (SPR) biosensor based on the recombinant Staphylococcal protein A with an additional cysteine residue (SPA-Cys) used as a biorecognition component showed a good selectivity and sensitivity for the immunoglobulin detection. The developed biosensor with SPA-Cys-based bioselective element can also be used as a first step of immunosensor creation. The successful immobilization of SPA-Cys on the nanolayer gold sensor surface of the SPR spectrometer was performed. The efficiency of blocking nonspecific sorption sites on the sensor surface with milk proteins, gelatin, BSA, and HSA was studied, and a rather high efficiency of using gelatin was confirmed. The SPR biosensor selectively interacted with IgG and did not interact with the control proteins. The linear dependence of the sensor response on the IgG concentration in the range from 2 to 10 μg/ml was shown. Using the calibration curve, the IgG concentration was measured in the model samples. The determined concentrations are in good agreement ( r 2 = 0.97) with the given concentration of IgG.

  9. A simple strategy based on photobiotin irradiation for the photoelectrochemical immobilization of proteins on electrode surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Cosnier, Serge [Laboratoire d' Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Moleculaire de Grenoble, FR CNRS 2607, Universite Joseph Fourier, 38042 Grenoble cedex 9 (France)]. E-mail: Serge.Cosnier@ujf-grenoble.fr; Molins, Carmen [Departamento Quimica Analitica, Universitat de Valencia (Spain); Mousty, Christine [Laboratoire d' Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Moleculaire de Grenoble, FR CNRS 2607, Universite Joseph Fourier, 38042 Grenoble cedex 9 (France); Galland, Bruno [Laboratoire d' Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Moleculaire de Grenoble, FR CNRS 2607, Universite Joseph Fourier, 38042 Grenoble cedex 9 (France); Lepellec, Arielle [Laboratoire d' Electrochimie Organique et de Photochimie Redox, UMR CNRS 5630, Institut de Chimie Moleculaire de Grenoble, FR CNRS 2607, Universite Joseph Fourier, 38042 Grenoble cedex 9 (France)

    2006-03-15

    A photoactivable organic polymer was prepared first by electrogeneration of a conductive biotinylated polypyrrole film in acetonitrile electrolyte. The successive anchoring of avidin and photobiotin led to a multilayer configuration. The latter was illuminated with light (wavelength 370-400 nm) in the presence of proteins adsorbed onto its surface. The irradiation allowed the covalent linking of the proteins to the modified electrode. As a result of the photochemical reaction, a monolayer of enzyme (glucose oxidase, GOX or alkaline phosphatase, AP) was covalently bound to the photobiotin-modified surface with retention of their catalytic activities. The surfacic activities were 34 and 1.69 mU cm{sup -2} for GOX and AP photobiotin electrodes, respectively. These enzyme electrodes were compared to similar configurations obtained through the immobilization of biotinylated glucose oxidase or avidin-conjugated alkaline phosphatase on biotinylated polypyrrole film. Our results suggest that both procedures led to the immobilization of the same enzyme amount, namely a protein monolayer. This novel photo-immobilization methodology was also successfully applied to the anchoring of an anti-cholera toxin antibody which was then detected by a secondary antibody labelled with a peroxidase.

  10. Fixation and stabilization of Escherichia coli cells displaying genetically engineered cell surface proteins

    Energy Technology Data Exchange (ETDEWEB)

    Freeman, A.; Abramov, S. [Tel-Aviv Univ. (Israel); Georgiou, G. [Univ. of Texas, Austin, TX (United States). Dept. of Chemical Engineering

    1996-12-05

    A large biotechnological potential is inherent in the display of proteins. Applications such as immobilized whole-cell biocatalysts or cellular adsorbents require cell fixation to prevent disintegration, stabilization of the anchored protein from leakage, denaturation or proteolysis, and total loss of cell viability, preventing medium and potential product contamination with cells. In this article the authors describe the adaptation of a simple two-stage chemical crosslinking procedure based on bi-layer encagement for stabilizing Escherichia coli cells expressing an Lpp-OmpA-{beta}-lactamase fusion that displays {beta}-lactamase on the cell surface. Bilayer crosslinking and coating the bacteria with a polymeric matrix is accomplished by treating the cells first with either glutaraldehyde or polyglutaraldehyde, followed by secondary crosslinking with polyacrylamide hydrazide. These treatments resulted in a 5- to 25-fold reduction of the thermal inactivation rate constant at 55 C of surface anchored {beta}-lactamase and completely prevented the deterioration of the cells for at least a week of storage at 4 C. The stabilization procedure developed paves the way to scalable biotechnological applications of E. coli displaying surface anchored proteins as whole-cell biocatalysts and adsorbents.

  11. Analysis of Borrelia burgdorferi surface proteins as determinants in establishing host cell interactions

    Directory of Open Access Journals (Sweden)

    Virginia L Schmit

    2011-07-01

    Full Text Available Borrelia burgdorferi infection causes Lyme borreliosis in humans, a condition which can involve a systemic spread of the organism to colonize various tissues and organs. If the infection is left untreated by antimicrobials, it can lead to manifestations including, arthritis, carditis, and/or neurological problems. Identification and characterization of B. burgdorferi outer membrane proteins that facilitate cellular attachment and invasion to establish infection continue to be investigated. In this study, we sought to further define putative cell binding properties of surface-exposed B. burgdorferi proteins by observing whether cellular adherence could be blocked by antibodies. B. burgdorferi mixed separately with monoclonal antibodies against outer surface protein (Osp A, OspC, decorin-binding protein (Dbp A, BBA64, and RevA antigens were incubated with human umbilical vein endothelial cells (HUVEC and human neuroglial cells (H4. B. burgdorferi treated with anti-OspA, -DbpA, and –BBA64 monoclonal antibodies showed a significant decrease in cellular association compared to controls, whereas B. burgdorferi treated with anti-OspC and anti-RevA showed no reduction in cellular attachment. Additionally, temporal transcriptional analyses revealed upregulated expression of bba64, ospA, and dbpA during coincubation with cells. Together, the data provide evidence that OspA, DbpA, and BBA64 function in host cell adherence and infection mechanisms.

  12. Surface proteins in normal and transformed rat liver epithelial cells in culture.

    Science.gov (United States)

    Bannikov, G. A.; Saint Vincent, L.; Montesano, R.

    1980-01-01

    The pattern of surface proteins of different types of normal and transformed rat liver cells have been studied in culture by means of lactoperoxidase-catalysed iodination procedures, followed by SDS-gel electrophoresis. The cells examined were primary cultures of epithelial liver cells, long-term cultures of epithelial liver cells, in vitro transformed epithelial liver cell lines and liver tumour-cell lines; mesenchymal cells from liver and skin were also examined. The principal surface proteins of primary cultures of epithelial cells from adult or neonatal rats had components with mol. wts of 140,000-160,000, 100,000 and 40,000-70,000. A band that had the same position as fibronectin from mesenchymal cells was also present and this band, as well as other iodinated components, were less sensitive to trypsin than fibroblastic fibronectin. A similar pattern of iodinated proteins was seen in long-term cultures of epithelial liver cells, with a great reduction in the number and intensity of the bands in the mol. wt region below 100,000. Almost all the in vitro transformed and tumour epithelial cell lines contain a protein with a mol. wt 135,000 as one of the major iodinated bands, and in contrast to the observation in transformed fibroblasts, the fibronectin was retained by most of these transformed cell lines. Images Fig. 1 Fig. 2 Fig. 3 PMID:7053205

  13. Conformational dynamics and antigenicity in the disordered malaria antigen merozoite surface protein 2.

    Directory of Open Access Journals (Sweden)

    Christopher A MacRaild

    Full Text Available Merozoite surface protein 2 (MSP2 of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27 using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design.

  14. Surface coatings shape the protein corona of SPIONs with relevance to their application in vivo.

    Science.gov (United States)

    Jedlovszky-Hajdú, Angéla; Bombelli, Francesca Baldelli; Monopoli, Marco P; Tombácz, Etelka; Dawson, Kenneth A

    2012-10-23

    Superparamagnetic iron oxide nanoparticles (SPIONs) have proved their use in many biomedical applications, such as drug delivery, hyperthermia, and MRI (magnetic resonance imaging) contrast agents. Due to their instability in fluids, several surface coatings have been used to both stabilize and tune the properties of these nanoparticles (NPs) according to their applications. These coatings will strongly modify their surface properties and influence their interaction with the environment proteins in a relevant biological medium with a clear impact on their function. It is well-accepted that a protein corona is immediately formed when nanoparticles come in contact with a biological milieu, and the emergent bionano interface represents the biological identity of the particles. Here, we investigate how a different coating on the same magnetic core can influence the protein corona composition and structure with clear relevance to application of these NPs in medicine. In particular, we have studied the structure and composition of the protein corona-SPION complexes of magnetite nanoparticles stabilized with citric acid, poly(acrylic acid), or double layer oleic acid by a range of approaches, including dynamic light scattering, nanoparticle tracking analysis, differential centrifugal sedimentation, infrared spectroscopy, 1-D SDS gel electrophoresis, and mass spectroscopy.

  15. Conformational Dynamics and Antigenicity in the Disordered Malaria Antigen Merozoite Surface Protein 2

    Science.gov (United States)

    Andrew, Dean; Krishnarjuna, Bankala; Nováček, Jiří; Žídek, Lukáš; Sklenář, Vladimír; Richards, Jack S.; Beeson, James G.; Anders, Robin F.; Norton, Raymond S.

    2015-01-01

    Merozoite surface protein 2 (MSP2) of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27) using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design. PMID:25742002

  16. The role of basic residues in the adsorption of blood proteins onto the graphene surface

    Science.gov (United States)

    Gu, Zonglin; Yang, Zaixing; Wang, Lingle; Zhou, Hong; Jimenez-Cruz, Camilo A.; Zhou, Ruhong

    2015-06-01

    With its many unique properties, graphene has shown great potential in various biomedical applications, while its biocompatibility has also attracted growing concerns. Previous studies have shown that the formation of protein-graphene corona could effectively reduce its cytotoxicity; however, the underlying molecular mechanism remains not well-understood. Herein, we use extensive molecular dynamics simulations to demonstrate that blood proteins such as bovine fibrinogen (BFG) can absorb onto the graphene surface quickly and tightly to form a corona complex. Aromatic residues contributed significantly during this adsorption process due to the strong π-π stacking interactions between their aromatic rings and the graphene sp2-carbons. Somewhat surprisingly, basic residues like arginine, also played an equally or even stronger role during this process. The strong dispersion interactions between the sidechains of these solvent-exposed basic residues and the graphene surface provide the driving force for a tight binding of these basic residues. To the best of our knowledge, this is the first study with blood proteins to show that, in addition to the aromatic residues, the basic residues also play an important role in the formation of protein-graphene corona complexes.

  17. Cloning, and Molecular Characterization of Polymorphic Iranian Isolate Theileria annulata Surface Protein (Tasp

    Directory of Open Access Journals (Sweden)

    E Ebrahimzadeh

    2012-06-01

    Full Text Available Background: Because of the strong immunologic responses of surface protein TaSp in Theileria annu­lata infected host, we tried to characterize this protein in a T. annulata isolate from Iran.Methods: The RNA prepared from T. annulata infected cells was used to produce SMART-DS-cDNA. The Double strand cDNA was then amplified with primers derived from TaSp mRNA se­quences. The PCR product was cloned in pTZ57R/T vector, sequenced and registered under acces­sion no. JQ003240 in GenBank.Results: The sequence analysis showed 90%-94% nucleotide sequence identity and 68%-94% amino acid homology to the corresponding sequences of TaSp gene by T. annulata, T. sp. china I, T. sp. china and T. lestoquardi and three T. annulata reported from Iran respectively. Interestingly, the sequence analysis also showed small nucleotide sequence region near the 5` end in which the presented TaSp protein differed very strongly from the other known TaSp sequences. For the preparation of the recombi­nant protein, the cDNA was cloned in pQE-32 vector, the recombinant protein was pre­pared and assayed by Theileria infected bovine serum.Conclusion: The polymorphism in TaSp gene could be detected in intra- as well as inter species. The different characterized TaSp proteins had a common identic region, which may be helpful for develop­ment of broad band vaccine based on the recombinant proteins. The polymorphism in this gene, make this protein also interesting for the diagnostic purposes.

  18. Staphylococcus pseudintermedius expresses surface proteins that closely resemble those from Staphylococcus aureus.

    Science.gov (United States)

    Geoghegan, Joan A; Smith, Emma J; Speziale, Pietro; Foster, Timothy J

    2009-09-18

    Staphylococcus pseudintermedius is a commensal of dogs that is implicated in the pathogenesis of canine pyoderma. This study aimed to determine if S. pseudintermedius expresses surface proteins resembling those from Staphylococcus aureus and to characterise them. S. pseudintermedius strain 326 was shown to adhere strongly to purified fibrinogen, fibronectin and cytokeratin 10. It adhered to the alpha-chain of fibrinogen which, along with binding to cytokeratin 10, is the hallmark of clumping factor B of S. aureus, a surface protein that is in part responsible for colonisation of the human nares. Ligand-affinity blotting with cell-wall extracts demonstrated that S. pseudintermedius 326 expressed a cell-wall anchored fibronectin binding protein which recognised the N-terminal 29kDa fragment. The ability to bind fibronectin is an important attribute of pathogenic S. aureus and is associated with the ability of S. aureus to colonise skin of human atopic dermatitis patients. S. pseudintermedius genomic DNA was probed with labelled DNA amplified from the serine-aspartate repeat encoding region of clfA of S. aureus. This probe hybridised to a single SpeI fragment of S. pseudintermedius DNA. In the cell-wall extract of S. pseudintermedius 326, a 180kDa protein was discovered which bound to fibrinogen by ligand-affinity blotting and reacted in a Western blot with antibodies raised against the serine-aspartate repeat region of ClfA and the B-repeats of SdrD of S. aureus. It is proposed that this is an Sdr protein with B-repeats that has an A domain that binds to fibrinogen. Whether it is the same protein that binds cytokeratin 10 is not clear.

  19. Protein analysis in dissolved organic matter: What proteins from organic debris, soil leachate and surface water can tell us - a perspective

    Science.gov (United States)

    Schulze, W. X.

    Mass spectrometry based analysis of proteins is widely used to study cellular processes in model organisms. However, it has not yet routinely been applied in environmental research. Based on observations that protein can readily be detected as a component of dissolved organic matter (DOM), this article gives an example about the possible use of protein analysis in ecology and environmental sciences focusing on different terrestrial ecosystems. At this stage, there are two areas of interest: (1) the identification of phylogenetic groups contributing to the environmental protein pool, and (2) identification of the organismic origin of specific enzymes that are important for ecosystem processes. In this paper, mass spectrometric protein analysis was applied to identify proteins from decomposing plant material and DOM of soil leachates and surface water samples derived from different environments. It is concluded, that mass spectrometric protein analysis is capable of distinguishing phylogenetic origin of proteins from litter protein extracts, leachates of different soil horizons, and from various sources of terrestrial surface water. Current limitation is imposed by the limited knowledge of complete genomes of soil organisms. The protein analysis allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific active enzymes. Further applications, such as in pollution research are conceivable. In summary, the analysis of proteins opens a new area of research between the fields of microbiology and biogeochemistry.

  20. Protein analysis in dissolved organic matter: What proteins from organic debris, soil leachate and surface water can tell us - a perspective

    Directory of Open Access Journals (Sweden)

    W. X. Schulze

    2005-01-01

    Full Text Available Mass spectrometry based analysis of proteins is widely used to study cellular processes in model organisms. However, it has not yet routinely been applied in environmental research. Based on observations that protein can readily be detected as a component of dissolved organic matter (DOM, this article gives an example about the possible use of protein analysis in ecology and environmental sciences focusing on different terrestrial ecosystems. At this stage, there are two areas of interest: (1 the identification of phylogenetic groups contributing to the environmental protein pool, and (2 identification of the organismic origin of specific enzymes that are important for ecosystem processes. In this paper, mass spectrometric protein analysis was applied to identify proteins from decomposing plant material and DOM of soil leachates and surface water samples derived from different environments. It is concluded, that mass spectrometric protein analysis is capable of distinguishing phylogenetic origin of proteins from litter protein extracts, leachates of different soil horizons, and from various sources of terrestrial surface water. Current limitation is imposed by the limited knowledge of complete genomes of soil organisms. The protein analysis allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific active enzymes. Further applications, such as in pollution research are conceivable. In summary, the analysis of proteins opens a new area of research between the fields of microbiology and biogeochemistry.

  1. Bacterial surface-displayed GII.4 human norovirus capsid proteins bound to surface of Romaine lettuce through HBGA-like molecules

    Science.gov (United States)

    Human Noroviruses (HuNoVs) are the main cause of nonbacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein (INP) mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein (G...

  2. Protein imprinting and recognition via forming nanofilms on microbeads surfaces in aqueous media

    Energy Technology Data Exchange (ETDEWEB)

    Lu Yan, E-mail: yanlu2001@sohu.com [College of Chemistry and Environmental Science, Henan Normal University, Xinxiang 453007 (China); Yan Changling [College of Chemistry and Environmental Science, Henan Normal University, Xinxiang 453007 (China); Wang Xuejing [Chemistry and Chemical Engineer School, Henna Institute of Science and Technology, Xinxiang 453003 (China); Wang Gongke [College of Chemistry and Environmental Science, Henan Normal University, Xinxiang 453007 (China)

    2009-12-15

    In this paler, we present a technique of forming nanofilms of poly-3-aminophenylboronic acid (pAPBA) on the surfaces of polystyrene (PS) microbeads for proteins (papain and trypsin) in aqueous. Papain was chosen as a model to study the feasibility of the technique and trypsin as an extension. Obtained core-shell microbeads were characterized using scanning electron microscopy (SEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and BET methods. The results show that pAPBA formed nanofilms (60-100 nm in thickness) on the surfaces of PS microbeads. The specific surface area of the papain-imprinted beads was about 180 m{sup 2} g{sup -1} and its pore size was 31 nm. These imprinted microbeads exhibit high recognition specificity and fast mass transfer kinetics. The specificity of these imprinted beads mainly originates from the spatial effect of imprinted sites. Because the protein-imprinted sites were located at, or close to, the surface, the imprinted beads have good site accessibility toward the template molecules. The facility of the imprinting protocol and the high recognition properties of imprinted microbeads make the approach an attractive solution to problems in the field of biotechnology.

  3. Improvement in physical and biological properties of chitosan/soy protein films by surface grafted heparin.

    Science.gov (United States)

    Wang, Xiaomei; Hu, Ling; Li, Chen; Gan, Li; He, Meng; He, Xiaohua; Tian, Weiqun; Li, Mingming; Xu, Li; Li, Yinping; Chen, Yun

    2016-02-01

    A series of chitosan/soy protein isolate (SPI) composite films (CS-n, n=0, 10 and 30, corresponding to SPI content in the composites) were prepared. Heparin was grafted onto the surface of CS-n to fabricate a series of heparinized films (HCS-n). CS-n and HCS-n were characterized by ATR-Fourier transform infrared spectroscopy and water contact angle. The surface heparin density was measured by toluidine blue assay. The results showed that heparin has been successfully grafted onto the surface of CS-n. Heparin evenly distributed on the surface of the films and the heparin content increased with the increase of SPI content, and the hydrophilicity of the films was enhanced due to the grafted heparin. The cytocompatibility and hemocompatibility of CS-n and HCS-n were evaluated by cell culture (MTT assay, live/dead assay, cell morphology and cell density observation), platelet adhesion test, plasma recalcification time (PRT) measurement, hemolysis assay and thrombus formation test. HCS-n showed higher cell adhesion rate and improved cytocompatibility compared to the corresponding CS-n. HCS-n also exhibited lower platelet adhesion, longer PRT, higher blood anticoagulant indexes (BCI) and lower hemolysis rate than the corresponding CS-n. The improved cytocompatibility and hemocompatibility of HCS-n would shed light on the potential applications of chitosan/soy protein-based biomaterials that may come into contact with blood.

  4. A review of chemical surface modification of bioceramics: effects on protein adsorption and cellular response.

    Science.gov (United States)

    Lee, Wing-Hin; Loo, Ching-Yee; Rohanizadeh, Ramin

    2014-10-01

    Calcium phosphates (CaPs) are ideal biomaterials for bone repair because of the similarities between their chemical structure and the mineral phase of hard biological tissues (e.g., bones and teeth). Since CaP bone grafts exhibit superior biocompatibility and strong osseointegration properties, they have been widely investigated for use as an in situ carrier for delivery of anti-resorptive and osteogenic drugs. The surface properties of CaP govern the affinity and the binding mechanisms between biological macromolecules (e.g., proteins) and the CaP surface, which indirectly determines the interactions between bone cells and implanted CaP biomaterials. These surface properties ultimately play a pivotal role in determining the success of CaP as bone implants and/or drug carriers. This review provides an in-depth discussion of the current methodologies used to regulate the surface chemistry of CaP and their subsequent effects in regards to protein adsorption and delivery, as well as cell/materials interactions.

  5. Ice-surface adsorption enhanced colligative effect of antifreeze proteins in ice growth inhibition

    Science.gov (United States)

    Mao, Yougang; Ba, Yong

    2006-09-01

    This Communication describes a mechanism to explain antifreeze protein's function to inhibit the growth of ice crystals. We propose that the adsorption of antifreeze protein (AFP) molecules on an ice surface induces a dense AFP-water layer, which can significantly decrease the mole fraction of the interfacial water and, thus, lower the temperature for a seed ice crystal to grow in a super-cooled AFP solution. This mechanism can also explain the nearly unchanged melting point for the ice crystal due to the AFP's ice-surface adsorption. A mathematical model combining the Langmuir theory of adsorption and the colligative effect of thermodynamics has been proposed to find the equilibrium constants of the ice-surface adsorptions, and the interfacial concentrations of AFPs through fitting the theoretical curves to the experimental thermal hysteresis data. This model has been demonstrated by using the experimental data of serial size-mutated beetle Tenebrio molitor (Tm) AFPs. It was found that the AFP's ice-surface adsorptions could increase the interfacial AFP's concentrations by 3 to 4 orders compared with those in the bulk AFP solutions.

  6. Protein Compatible Polymer Brushes on Polymeric Substrates Prepared by Surface-Initiated Transfer Radica Polymerization

    DEFF Research Database (Denmark)

    Fristrup, Charlotte Juel; Eskimergen, Rüya; Burkrinsky, J.T.

    2008-01-01

    as coating materials. ATR FTIR, water contact angle measurements, Thermal Gravimetric Analysis (TGA), and X-ray Photoelectron Spectroscopy (XPS) confirmed that hydrophilic polymers have been grafted from the surface. The surface topography which was evaluated by Atomic Force Microscopy (AFM) did not change......Materials for insulin containers and delivery systems should comply with requirements like compatibility with proteins, sterilisability, 'good barrier properties towards preservatives, and no toxic leachables. The number of commercially available polymer materials which can be u sed is rather...... limited. Therefore, a polymer coating containing some of the required properties may expand the use ofpolymers in medical devices. 'The approach was to graft polymer brushes from initiator-functionalized substrates using Surface-Initiated Atom TnlJlsfer Radical Polymerization (SI ATRP). Initial studies...

  7. Adsorption of proteins at physiological concentrations on pegylated surfaces and the compatibilizing role of adsorbed albumin with respect to other proteins according to optical waveguide lightmode spectroscopy (OWLS).

    Science.gov (United States)

    Leclercq, Laurent; Modena, Enrico; Vert, Michel

    2013-01-01

    In literature, contacts between pegylated compounds and blood proteins are generally discussed in terms of excluded volume-related repulsions although adsorption and compatibility have been reported for some of these proteins occasionally. The major problem to investigate the behavior of blood in contact with pegylated surfaces is the complexity of the medium and especially the presence of albumin in large excess. In a model approach, optical waveguide lightmode spectroscopy (OWLS) was used to monitor the fate of albumin, fibrinogen, and γ-globulins at physiological concentrations in pH = 7.4 isotonic HEPES buffer after contact with SiTiO2 chips coated with diblock poly(DL-lactic acid)-block-poly(ethylene oxide)s and triblock poly(DL-lactic acid)-block-poly(ethylene oxide)-block-poly(DL-lactic acid) copolymers. Corresponding homopolymers were used as controls. The three protein systems were investigated separately, as a mixture and when added successively according to different orders of addition. OWLS gave access to the mass and the thickness of adhering protein layers that resist washing with HEPES buffer. Protein depositions were detected regardless of the presence of poly(ethylene glycol) segments on surfaces. Adsorption depended on the protein, on the surface and also on the presence of the other proteins. Unexpectedly any surface coated with a layer of adsorbed albumin prevented deposition of other proteins, including albumin itself. This outstanding finding suggests that it was the presence of albumin adsorbed on a surface, pegylated or not, that made that surface compatible with other proteins. As a consequence, dipping a device to be in contact with the blood of a patient in a solution of albumin could be a very simple means to avoid further protein deposition and maybe platelets adhesion after in vivo implantation.

  8. Interfacial design and structure of protein/polymer films on oxidized AlGaN surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Samit K; Casal, Patricia; Nicholson III, Theodore R; Lee, Stephen Craig [Department of Biomedical Engineering, Ohio State University, Columbus, OH 43210 (United States); Wu, Hao-Hsuan; Wen Xuejin; Anisha, R; Berger, Paul R; Lu, Wu; Brillson, Leonard J [Department of Electrical and Computer Engineering, Ohio State University, Columbus, OH 43210 (United States); Kwak, Kwang J; Bhushan, Bharat, E-mail: lee.1996@osu.edu [Department of Mechanical Engineering, Ohio State University, Columbus, OH 43210 (United States)

    2011-01-26

    Protein detection using biologically or immunologically modified field-effect transistors (bio/immunoFETs) depends on the nanoscale structure of the polymer/protein film at sensor interfaces (Bhushan 2010 Springer Handbook of Nanotechnology 3rd edn (Heidelberg: Springer); Gupta et al 2010 The effect of interface modification on bioFET sensitivity, submitted). AlGaN-based HFETs (heterojunction FETs) are attractive platforms for many protein sensing applications due to their electrical stability in high osmolarity aqueous environments and favourable current drive capabilities. However, interfacial polymer/protein films on AlGaN, though critical to HFET protein sensor function, have not yet been fully characterized. These interfacial films are typically comprised of protein-polymer films, in which analyte-specific receptors are tethered to the sensing surface with a heterobifunctional linker molecule (often a silane molecule). Here we provide insight into the structure and tribology of silane interfaces composed of one of two different silane monomers deposited on oxidized AlGaN, and other metal oxide surfaces. We demonstrate distinct morphologies and wear properties for the interfacial films, attributable to the specific chemistries of the silane monomers used in the films. For each specific silane monomer, film morphologies and wear are broadly consistent on multiple oxide surfaces. Differences in interfacial film morphology also drive improvements in sensitivity of the underlying HFET (coincident with, though not necessarily caused by, differences in interfacial film thickness). We present a testable model of the hypothetical differential interfacial depth distribution of protein analytes on FET sensor interfaces with distinct morphologies. Empirical validation of this model may rationalize the actual behaviour of planar immunoFETs, which has been shown to be contrary to expectations of bio/immunoFET behaviour prevalent in the literature for the last 20 years

  9. Protein adsorption onto Fe3O4 nanoparticles with opposite surface charge and its impact on cell uptake

    OpenAIRE

    Catalayud, M. P.; Sanz, B; Raffa, V.; Riggio, C.; Ibarra, M. R.; Goya, G. F.

    2014-01-01

    Nanoparticles (NPs) engineered for biomedical applications are meant to be in contact with protein-rich physiological fluids. These proteins are usually adsorbed onto the NP surface, forming a swaddling layer called protein corona that influences cell internalization. We present a study on protein adsorption onto different magnetic NPs (MNPs) when immersed in cell culture medium, and how these changes affect the cellular uptake. Two colloids with magnetite cores of 25 nm, same hydrodynamic si...

  10. Spore surface proteins of Brevibacillus laterosporus are involved in insect pathogenesis

    Science.gov (United States)

    Marche, Maria Giovanna; Mura, Maria Elena; Falchi, Giovanni; Ruiu, Luca

    2017-01-01

    Outer spore envelope proteins of pathogenic bacteria often present specific virulence factors and tools to evade the defence system of their hosts. Brevibacillus laterosporus, a pathogen of invertebrates and an antimicrobial-producing species, is characterised by a unique spore coat and canoe-shaped parasporal body (SC-CSPB) complex surrounding the core spore. In the present study, we identified and characterised major proteins of the SC-CSPB complex of B. laterosporus, and we investigated their entomopathogenic role. Employing a proteomic approach and a B. laterosporus-house fly study model, we found four highly conserved proteins (ExsC, CHRD, CpbA and CpbB) that function as insect virulence factors. CpbA was associated with a significantly higher mortality of flies and greater relative gene expression levels during sporulation, compared to the other SC-CSPB proteins. Taken together, we suggest that spore surface proteins are a part of a complex set of toxins and virulence factors that B. laterosporus employs in its pathogenicity against flies. PMID:28256631

  11. Class I major histocompatibility proteins as cell surface receptors for simian virus 40.

    Science.gov (United States)

    Atwood, W J; Norkin, L C

    1989-10-01

    Class I major histocompatibility complex proteins appear to be the major cell surface receptors for simian virus 40 (SV40), as implied by the following observations. Adsorption of SV40 to LLC-MK2 rhesus monkey kidney cells specifically inhibited binding of a monoclonal antibody (MAb) against class I human lymphocyte antigen (HLA) proteins. Conversely, pretreatment of LLC-MK2 cells with anti-HLA MAbs inhibited infection by SV40. The ability of anti-HLA to inhibit infection was greatly reduced when the order of addition of the anti-HLA and the virus was reversed. Infection was also inhibited by preincubating SV40 with purified soluble class I protein. Finally, human lymphoblastoid cells of the Daudi line, which do not express class I major histocompatibility complex proteins, were infected at relatively low levels with SV40 virions. In a control experiment, we found that pretreatment of cells with a MAb specific for the leukocytic-function-associated antigen LFA-3 actually enhanced infection. This finding may also support the premise that class I major histocompatibility complex proteins are receptors for SV40.

  12. [Mechanisms of human plasma proteins adsorption on the surface of perfluorocarbon emulsion stabilized with proxanol 268].

    Science.gov (United States)

    Zhalimov, V K; Sklifas, A N; Kukushkin, N I

    2012-01-01

    It has been shown that sorption of most proteins with the molecular weight lower than 200 kDa from human blood plasma on the surface of perfluorocarbon emulsion, stabilized with proxanol 268, is mainly based on hydrophobic interaction, whereas sorption of immunoglobulin G is mainly the result of electrostatic interaction. The removal of lipidic components from plasma leads to the increase of a total amount of adsorbed proteins by 35%. Particularly, when lipidic components are removed, sorption of apolipoprotein AI and immunoglobulin G is considerably bettered as well as sorption of other proteins with the molecular weight of about 50 and 60 kDa occurs. It has been out that apolipoprotein AI in the adsorbed condition loses its capability of tryptophan fluorescence, which might be probably determined by the quenching influence of the perfluorocarbon core of nanoparticle. We think that the findings obtained also indicates considerable conformational rearrangements of this protein during adsorption. It was shown, that the fluorescence of proteins with sorption on nanoparticles in emulsion based on the hydrophobic interaction, is completely or partially quenched.

  13. TGP, an extremely stable, non-aggregating fluorescent protein created by structure-guided surface engineering

    Science.gov (United States)

    Close, Devin W.; Don Paul, Craig; Langan, Patricia S.; Wilce, Matthew C.J.; Traore, Daouda A.K.; Halfmann, Randal; Rocha, Reginaldo C.; Waldo, Geoffery S.; Payne, Riley J.; Rucker, Joseph B.; Prescott, Mark; Bradbury, Andrew R.M.

    2014-01-01

    In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization. PMID:25287913

  14. An amino acid code to define a protein's tertiary packing surface.

    Science.gov (United States)

    Fraga, Keith J; Joo, Hyun; Tsai, Jerry

    2016-02-01

    One difficult aspect of the protein-folding problem is characterizing the nonspecific interactions that define packing in protein tertiary structure. To better understand tertiary structure, this work extends the knob-socket model by classifying the interactions of a single knob residue packed into a set of contiguous sockets, or a pocket made up of 4 or more residues. The knob-socket construct allows for a symbolic two-dimensional mapping of pockets. The two-dimensional mapping of pockets provides a simple method to investigate the variety of pocket shapes to understand the geometry of protein tertiary surfaces. The diversity of pocket geometries can be organized into groups of pockets that share a common core, which suggests that some interactions in pockets are ancillary to packing. Further analysis of pocket geometries displays a preferred configuration that is right-handed in α-helices and left-handed in β-sheets. The amino acid composition of pockets illustrates the importance of nonpolar amino acids in packing as well as position specificity. As expected, all pocket shapes prefer to pack with hydrophobic knobs; however, knobs are not selective for the pockets they pack. Investigating side-chain rotamer preferences for certain pocket shapes uncovers no strong correlations. These findings allow a simple vocabulary based on knobs and sockets to describe protein tertiary packing that supports improved analysis, design, and prediction of protein structure.

  15. Lactobacillus acidophilus CP23 with weak immunomodulatory activity lacks anchoring structure for surface layer protein.

    Science.gov (United States)

    Yanagihara, Sae; Kato, Shinji; Ashida, Nobuhisa; Yamamoto, Naoyuki

    2015-05-01

    To determine the reason for the low levels of Surface layer protein A (SlpA) on CP23 cells, which might play a crucial role in the immunomodulatory effect of Lactobacillus acidophilus, the DNA sequence of the slpA gene of CP23 and L-92 strains, including the upstream region, were analyzed. Unexpectedly, there was no significant difference in the predicted amino acid sequence of the C-terminus needed for cell anchoring, and only an additional Ala-Val-Ala sequence inserted in the N-terminal region of the mature CP23 protein. Therefore, anchoring of SlpA on the cell wall of CP23 and L-92 was evaluated by a reconstitution assay, which showed that SlpA released by LiCl treatment from both CP23 and L-92 was successfully anchored on LiCl-treated L-92 cells, but not on LiCl-treated CP23 cells. Moreover, quantitative analysis of SlpA protein in the culture medium of CP23 and L-92 by ELISA revealed higher levels of SlpA secretion in CP23 cells than in L-92 cells. Collectively, these results suggest that the lower levels of SlpA on the surface of CP23 cells might be caused by less cell wall capacity for SlpA anchoring, leading to an accumulation of SlpA in the culture medium of CP23 cells. The present study supports the importance of cell surface structure of L. acidophilus L-92 for SlpA anchoring on the cell surface needed for immunomodulatory effect.

  16. Protein-resistant polyurethane via surface-initiated atom transfer radical polymerization of oligo(ethylene glycol) methacrylate.

    Science.gov (United States)

    Jin, Zhilin; Feng, Wei; Zhu, Shiping; Sheardown, Heather; Brash, John L

    2009-12-15

    Protein-resistant polyurethane (PU) surfaces were prepared by surface-initiated simultaneous normal and reverse atom transfer radical polymerization (s-ATRP) of poly(oligo(ethylene glycol) methacrylate) (poly (OEGMA)). Oxygen plasma treatment was employed for initial activation of the PU surface. The grafted polymer chain length was adjusted by varying the molar ratio of monomer to sacrificial initiator in solution from 5:1 to 200:1. The modified PU surfaces were characterized by water contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Protein adsorption experiments from tris-buffered saline (TBS) and plasma were carried out to evaluate the protein-resistance of the surfaces. Adsorption from single and binary protein solutions as well as from plasma was significantly reduced after modification. Adsorption decreased with increasing poly(OEGMA) chain length. Fibrinogen (Fg) adsorption on the 200:1 monomer/initiator surface was in the range of 3-33 ng/cm(2) representing 96-99% reduction compared with the unmodified PU. Fg adsorption from 0.01-10% plasma was as low as 1-5 ng/cm(2). Moreover, binary protein adsorption experiments using Fg and lysozyme (Lys) showed that protein size is a factor in the protein resistance of these surfaces.

  17. Protein analysis in dissolved organic matter: what free proteins from soil leachate and surface water can tell us – a perspective

    Directory of Open Access Journals (Sweden)

    W. Schulze

    2004-12-01

    Full Text Available Mass spectrometry based analysis of proteins is widely used to study cellular processes in model organisms. However, it has not yet routinely been applied in environmental research. Based on observations that protein can readily be detected as a component of dissolved organic matter (DOM, this article gives an example about the possible use of protein analysis in ecology and environmental sciences focusing on different terrestrial ecosystems. At this stage, there are two areas of interest: (1 the identification of phylogenetic groups contributing to the DOM protein pool, and (2 identification of the organismic origin of specific enzymes that are important for ecosystem processes. In this paper, mass spectrometric protein analysis was applied to identify proteins from DOM and organism-free surface water samples derived from different environments. It is concluded, that mass spectrometric protein analysis is capable of distinguishing phylogenetic origin of proteins from leachates of different soil horizons, and from various sources of terrestrial surface water. Current limitation is imposed by the limited knowledge of complete genomes of soil organisms. The protein analysis allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific active enzymes. Further applications, such as in pollution research are conceivable. In summary, the analysis of proteins opens a new area of research between the fields of microbiology and biogeochemistry.

  18. Binding of dengue virus particles and dengue proteins onto solid surfaces.

    Science.gov (United States)

    Pereira, Edla M A; Dario, Aline F; França, Rafael F O; Fonseca, Benedito A L; Petri, Denise F S

    2010-09-01

    The interaction between dengue virus particles (DENV), sedimentation hemagglutinin particles (SHA), dengue virus envelope protein (Eprot), and solid surfaces was investigated by means of ellipsometry and atomic force microscopy (AFM). The surfaces chosen are bare Si/SiO2 wafers and Si/SiO2 wafers covered with concanavalin A (ConA), jacalin (Jac), polystyrene (PS), or poly(styrene sulfonate) (PSS) films. Adsorption experiments at pH 7.2 and pH 3 onto all surfaces revealed that (i) adsorption of DENV particles took place only onto ConA under pH 7.2, because of specific recognition between glycans on DENV surface and ConA binding site; (ii) DENV particles did not attach to any of the surfaces at pH 3, suggesting the presence of positive charges on DENV surface at this pH, which repel the positively charged lectin surfaces; (iii) SHA particles are positively charged at pH 7.2 and pH 3 because they adhered to negatively charged surfaces at pH 7.2 and repelled positively charged layers at pH 3; and (iv) SHA particles carry polar groups on the surface because they attached to silanol surfaces at pH 3 and avoided hydrophobic PS films at pH 3 and pH 7.2. The adsorption behavior of Eprot at pH 7.2 revealed affinity for ConA>Jac>PSS>PS≈bare Si/SiO2 layers. These findings indicate that selectivity of the Eprot adsorption is higher when it is part of virus structure than when it is free in solution. The correlation between surface energy values determined by means of contact angle measurements and DENV, SHA, or Eprot adsorption behavior was used to understand the intermolecular forces at the interfaces. A direct correlation was not found because the contributions from surface energy were probably surpassed by specific contributions.

  19. PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis

    Science.gov (United States)

    Mitra, Avishek; Speer, Alexander; Lin, Kan; Ehrt, Sabine

    2017-01-01

    ABSTRACT Iron is essential for replication of Mycobacterium tuberculosis, but iron is efficiently sequestered in the human host during infection. Heme constitutes the largest iron reservoir in the human body and is utilized by many bacterial pathogens as an iron source. While heme acquisition is well studied in other bacterial pathogens, little is known in M. tuberculosis. To identify proteins involved in heme utilization by M. tuberculosis, a transposon mutant library was screened for resistance to the toxic heme analog gallium(III)-porphyrin (Ga-PIX). Inactivation of the ppe36, ppe62, and rv0265c genes resulted in resistance to Ga-PIX. Growth experiments using isogenic M. tuberculosis deletion mutants showed that PPE36 is essential for heme utilization by M. tuberculosis, while the functions of PPE62 and Rv0265c are partially redundant. None of the genes restored growth of the heterologous M. tuberculosis mutants, indicating that the proteins encoded by the genes have separate functions. PPE36, PPE62, and Rv0265c bind heme as shown by surface plasmon resonance spectroscopy and are associated with membranes. Both PPE36 and PPE62 proteins are cell surface accessible, while the Rv0265c protein is probably located in the periplasm. PPE36 and PPE62 are, to our knowledge, the first proline-proline-glutamate (PPE) proteins of M. tuberculosis that bind small molecules and are involved in nutrient acquisition. The absence of a virulence defect of the ppe36 deletion mutant indicates that the different iron acquisition pathways of M. tuberculosis may substitute for each other during growth and persistence in mice. The emerging model of heme utilization by M. tuberculosis as derived from this study is substantially different from those of other bacteria. PMID:28119467

  20. Spontaneous surface self-assembly in protein-surfactant mixtures: interactions between hydrophobin and ethoxylated polysorbate surfactants.

    Science.gov (United States)

    Tucker, Ian M; Petkov, Jordan T; Penfold, Jeffrey; Thomas, Robert K; Li, Peixun; Cox, Andrew R; Hedges, Nick; Webster, John R P

    2014-05-01

    The synergistic interactions between certain ethoxylated polysorbate nonionic surfactants and the protein hydrophobin result in spontaneous self-assembly at the air-water interface to form layered surface structures. The surface structures are characterized using neutron reflectivity. The formation of the layered surface structures is promoted by the hydrophobic interaction between the polysorbate alkyl chain and the hydrophobic patch on the surface of the globular hydrophobin and the interaction between the ethoxylated sorbitan headgroup and hydrophilic regions of the protein. The range of the ethoxylated polysorbate concentrations over which the surface ordering occurs is a maximum for the more hydrophobic surfactant polyoxyethylene(8) sorbitan monostearate. The structures at the air-water interface are accompanied by a profound change in the wetting properties of the solution on hydrophobic substrates. In the absence of the polysorbate surfactant, hydrophobin wets a hydrophobic surface, whereas the hydrophobin/ethoxylated polysorbate mixtures where multilayer formation occurs result in a significant dewetting of hydrophobic surfaces. The spontaneous surface self-assembly for hydrophobin/ethoxylated polysorbate surfactant mixtures and the changes in surface wetting properties provide a different insight into protein-surfactant interactions and potential for manipulating surface and interfacial properties and protein surface behavior.

  1. The Biological Function of the Prion Protein: A Cell Surface Scaffold of Signaling Modules

    Science.gov (United States)

    Linden, Rafael

    2017-01-01

    The prion glycoprotein (PrPC) is mostly located at the cell surface, tethered to the plasma membrane through a glycosyl-phosphatydil inositol (GPI) anchor. Misfolding of PrPC is associated with the transmissible spongiform encephalopathies (TSEs), whereas its normal conformer serves as a receptor for oligomers of the β-amyloid peptide, which play a major role in the pathogenesis of Alzheimer’s Disease (AD). PrPC is highly expressed in both the nervous and immune systems, as well as in other organs, but its functions are controversial. Extensive experimental work disclosed multiple physiological roles of PrPC at the molecular, cellular and systemic levels, affecting the homeostasis of copper, neuroprotection, stem cell renewal and memory mechanisms, among others. Often each such process has been heralded as the bona fide function of PrPC, despite restricted attention paid to a selected phenotypic trait, associated with either modulation of gene expression or to the engagement of PrPC with a single ligand. In contrast, the GPI-anchored prion protein was shown to bind several extracellular and transmembrane ligands, which are required to endow that protein with the ability to play various roles in transmembrane signal transduction. In addition, differing sets of those ligands are available in cell type- and context-dependent scenarios. To account for such properties, we proposed that PrPC serves as a dynamic platform for the assembly of signaling modules at the cell surface, with widespread consequences for both physiology and behavior. The current review advances the hypothesis that the biological function of the prion protein is that of a cell surface scaffold protein, based on the striking similarities of its functional properties with those of scaffold proteins involved in the organization of intracellular signal transduction pathways. Those properties are: the ability to recruit spatially restricted sets of binding molecules involved in specific signaling

  2. Construction and in vitro Expression of Streptococcus Mutans Surface Protein Encoding DNA Vaccine

    Institute of Scientific and Technical Information of China (English)

    PENG; Zhixiang(

    2001-01-01

    [1]樊明文主编.口腔生物学.北京:人民卫生出版社 1996.132[2]Senpuku H Iizima T Yamaguchi Y et al.Immunogenicity of peptides coupled with multiple T-cell epitopes of a surface protein antigen of Streptococcus mutans.Immunology 1996 88:2275[3]Kato H Takeuchi H Oishi Y et al.The immunogenicity of various peptide antigens inducing cross-reacting antibodies to a cell surface protein antigen of Streptococcus mutans.Oral Microbiol Immunol 1999 14:213[4]Okahashi N Sasakawa C Yoshikawa M et al.Molecular characterization of a surface protein antigen gene from serotype c Streptococcus mutans implicated in dental caries.Mol Microbiol 1989 3:673[5]Okahashi N Takahashi I Nakai M et al.Identification of antigenic epitopes in an alanine-rich repeating region of a surface protein antigen of Streptococcus mutans.Infeet Immun 1993 61(4):1301[6]Brady L J Cvitkovitch D G Geric C M et al.Deletion of the central proline-rich repeat domain results in altered antigenicity and lack of surface expression of the Streptococcus mutans P1 adhesin molecule.Infect Immun 1998 66(9):4274[7]彭志翔 樊明文 边专.变形链球菌表面蛋白PAc结构基因克隆工程数据分析.口腔医学纵横杂志 2000 16(2):90[8]彭志翔 钟燕 樊明文等.含变链菌PAc蛋白编码基因保守区重组质粒pCIA-P的亚克隆构建.中华口腔医学杂志 2000 35(5):339[9]Peng Z X Zhong Y Fan M W et al.Design and preparation of cloned DNA fragment from pac gene of Streptococcus mutans.J Comprehensive Stomatology 2000 16(1):54

  3. Experimental verification of the identity of variant-specific surface proteins in Giardia lamblia trophozoites.

    Science.gov (United States)

    Li, Wei; Saraiya, Ashesh A; Wang, Ching C

    2013-05-21

    The cell membrane of a Giardia lamblia trophozoite is covered with a single species of variant-specific surface protein (VSP) that is replaced by another VSP every 6 to 13 generations of cell growth, possibly for an evasion of host immunity. Experimentally, only six VSP species have been verified to localize to the cell membrane thus far. By assuming that VSP contains multiple CXXC motifs, 219 vsp genes were annotated in GiardiaDB of the WB isolate. By further assuming that VSP possesses both CXXC motifs and a CRGKA tail at the C terminus, Adam et al. (BMC Genomics 11:424, 2010) identified a total of 303 potential vsp genes in Giardia WB. The discrepancies between these two assumed VSP identities have caused some confusion. Here, we used experimental approaches to further verify what is required of the structures of a VSP to localize to the surface of cell membrane. The data led to the following conclusions. (i) The C-terminal CRGKA sequence is not essential for localizing VSPs to the cell membrane. (ii) A "motif 1" of 45 residues, consisting of two CXXCs separated by 12 to 15 amino acid residues, located close to the C terminus and a hydrophobic "motif 2" of 38 residues at the C terminus are both essential and sufficient for localizing the protein to the cell membrane. (ii) An N-terminal sequence upstream from motif 1 is not required for targeting VSPs to the cell membrane. By these criteria, we are able to identify 73 open reading frames as the putative vsp genes in Giardia. IMPORTANCE The intestinal pathogen Giardia lamblia expresses only one variant-specific surface protein (VSP) on the cell membrane surface at a given time, but it changes spontaneously every 6 to 13 generations of growth, presumably for evading the host immunity. Only 6 VSPs have been empirically shown to localize to the cell membrane surface thus far. Here, we used mutations of VSPs and methods of identifying their locations in Giardia cells and found that a "motif 1" of 45 residues

  4. High performance workflow implementation for protein surface characterization using grid technology

    Directory of Open Access Journals (Sweden)

    Clematis Andrea

    2005-12-01

    Full Text Available Abstract Background This study concerns the development of a high performance workflow that, using grid technology, correlates different kinds of Bioinformatics data, starting from the base pairs of the nucleotide sequence to the exposed residues of the protein surface. The implementation of this workflow is based on the Italian Grid.it project infrastructure, that is a network of several computational resources and storage facilities distributed at different grid sites. Methods Workflows are very common in Bioinformatics because they allow to process large quantities of data by delegating the management of resources to the information streaming. Grid technology optimizes the computational load during the different workflow steps, dividing the more expensive tasks into a set of small jobs. Results Grid technology allows efficient database management, a crucial problem for obtaining good results in Bioinformatics applications. The proposed workflow is implemented to integrate huge amounts of data and the results themselves must be stored into a relational database, which results as the added value to the global knowledge. Conclusion A web interface has been developed to make this technology accessible to grid users. Once the workflow has started, by means of the simplified interface, it is possible to follow all the different steps throughout the data processing. Eventually, when the workflow has been terminated, the different features of the protein, like the amino acids exposed on the protein surface, can be compared with the data present in the output database.

  5. Design of cyclic peptides that bind protein surfaces with antibody-like affinity.

    Science.gov (United States)

    Millward, Steven W; Fiacco, Stephen; Austin, Ryan J; Roberts, Richard W

    2007-09-21

    There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein G alpha i1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic G alpha i binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity ( K i approximately 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the betagamma heterodimer, an endogenous G alpha i1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces.

  6. Proteome analysis and serological characterization of surface-exposed proteins of Rickettsia heilongjiangensis.

    Directory of Open Access Journals (Sweden)

    Yong Qi

    Full Text Available BACKGROUND: Rickettsia heilongjiangensis, the agent of Far-Eastern spotted fever (FESF, is an obligate intracellular bacterium. The surface-exposed proteins (SEPs of rickettsiae are involved in rickettsial adherence to and invasion of host cells, intracellular bacterial growth, and/or interaction with immune cells. They are also potential molecular candidates for the development of diagnostic reagents and vaccines against rickettsiosis. METHODS: R. heilongjiangensis SEPs were identified by biotin-streptavidin affinity purification and 2D electrophoreses coupled with ESI-MS/MS. Recombinant SEPs were probed with various sera to analyze their serological characteristics using a protein microarray and an enzyme-linked immune sorbent assay (ELISA. RESULTS: Twenty-five SEPs were identified, most of which were predicted to reside on the surface of R. heilongjiangensis cells. Bioinformatics analysis suggests that these proteins could be involved in bacterial pathogenesis. Eleven of the 25 SEPs were recognized as major seroreactive antigens by sera from R. heilongjiangensis-infected mice and FESF patients. Among the major seroreactive SEPs, microarray assays and/or ELISAs revealed that GroEL, OmpA-2, OmpB-3, PrsA, RplY, RpsB, SurA and YbgF had modest sensitivity and specificity for recognizing R. heilongjiangensis infection and/or spotted fever. CONCLUSIONS: Many of the SEPs identified herein have potentially important roles in R. heilongjiangensis pathogenicity. Some of them have potential as serodiagnostic antigens or as subunit vaccine antigens against the disease.

  7. Relevance of glycosylation of S-layer proteins for cell surface properties.

    Science.gov (United States)

    Schuster, Bernhard; Sleytr, Uwe B

    2015-06-01

    Elucidating the building principles and intrinsic features modulating certain water-associated processes (e.g., surface roughness in the nanometer scale, surface hydration and accompanied antifouling property, etc.) of surface structures from (micro)organisms is nowadays a highly challenging task in fields like microbiology, biomimetic engineering and (bio)material sciences. Here, we show for the first time the recrystallization of the wild-type S-layer glycoprotein wtSgsE from Geobacillus stearothermophilus NRS 2004/3a and its recombinantly produced non-glycosylated form, rSgsE, on gold sensor surfaces. Whereas the proteinaceous lattice of the S-layer proteins is forming a rigid layer on the sensor surface, the glycan chains are developing an overall soft, highly dissipative film. Interestingly, to the wtSgsE lattice almost twice the amount of water is bound and/or coupled in comparison with the non-glycosylated rSgsE with the preferred region being the extending glycan residues. The present results are discussed in terms of the effect of the glycan residues on the recrystallization, the adjoining hydration layer, and the nanoscale roughness and fluidic behavior. The latter features may turn out to be one of the most general ones among bacterial and archaeal S-layer lattices.

  8. Hydration behavior at the ice-binding surface of the Tenebrio molitor antifreeze protein.

    Science.gov (United States)

    Midya, Uday Sankar; Bandyopadhyay, Sanjoy

    2014-05-08

    Molecular dynamics (MD) simulations have been carried out at two different temperatures (300 and 220 K) to study the conformational rigidity of the hyperactive Tenebrio molitor antifreeze protein (TmAFP) in aqueous medium and the structural arrangements of water molecules hydrating its surface. It is found that irrespective of the temperature the ice-binding surface (IBS) of the protein is relatively more rigid than its nonice-binding surface (NIBS). The presence of a set of regularly arranged internally bound water molecules is found to play an important role in maintaining the flat rigid nature of the IBS. Importantly, the calculations reveal that the strategically located hydroxyl oxygens of the threonine (Thr) residues in the IBS influence the arrangements of five sets of ordered waters around it on two parallel planes that closely resemble the basal plane of ice. As a result, these waters can register well with the ice basal plane, thereby allowing the IBS to preferentially bind at the ice interface and inhibit its growth. This provides a possible molecular reason behind the ice-binding activity of TmAFP at the basal plane of ice.

  9. Photo-induced formation of nitrous acid (HONO) on protein surfaces

    Science.gov (United States)

    Meusel, Hannah; Elshorbany, Yasin; Bartels-Rausch, Thorsten; Selzle, Kathrin; Lelieveld, Jos; Ammann, Markus; Pöschl, Ulrich; Su, Hang; Cheng, Yafang

    2014-05-01

    The study of nitrous acid (HONO) is of great interest, as the photolysis of HONO leads to the OH radical, which is the most important oxidant in the troposphere. HONO is directly emitted by combustion of fossil fuel and from soil biogenic nitrite (Su et al., 2011), and can also be formed by gas phase reactions of NO and OH and heterogeneous reactions of NO2. Previous atmospheric measurements have shown unexpectedly high HONO concentrations during daytime. Measured mixing ratios were about one order of magnitude higher than model simulations (Kleffmann et al. 2005, Vogel et al. 2003). The additional daytime source of HONO might be attributed to the photolysis of adsorbed nitric acid or heterogeneous photochemistry of NO2 on organic substrates, such as humic acids or polyphenolic compounds (Stemmler et al., 2006), or indirectly through nitration of phenols and subsequent photolysis of nitrophenols (Sosedova et al., 2011, Bejan et al., 2006). An important reactive surface for the heterogeneous formation of HONO could involve proteins, which are ubiquitous in the environment. They are part of coarse biological aerosol particles like pollen grains, fine particles (fragments of pollen, microorganism, plant debris) and dissolved in rainwater, soil and road dust (Miguel et al. 1999). In this project a thin film of bovine serum albumin (BSA), a model protein with 67 kDa and 21 tyrosine residues per molecule, is irradiated and exposed to nitrogen dioxide in humidified nitrogen. The formation of HONO is measured with long path absorption photometry (LOPAP). The generated HONO is in the range of 100 to 1100 ppt depending on light intensity, NO2 concentration and film thickness. Light induced HONO formation on protein surfaces is stable over the 20-hours experiment of irradiation and exposure. On the other hand, light activated proteins reacting with NO2 form nitrated proteins, as detected by liquid chromatography (LC-DAD). Our experiments on tetranitromethane (TNM) nitrated

  10. In vitro affinity screening of protein and peptide binders by megavalent bead surface display.

    Science.gov (United States)

    Diamante, Letizia; Gatti-Lafranconi, Pietro; Schaerli, Yolanda; Hollfelder, Florian

    2013-10-01

    The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 10(3) and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead K(d) measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).

  11. A Gravity-Responsive Time-Keeping Protein of the Plant and Animal Cell Surface

    Science.gov (United States)

    Morre, D. James

    2003-01-01

    The hypothesis under investigation was that a ubiquinol (NADH) oxidase protein of the cell surface with protein disulfide-thiol interchange activity (= NOX protein) is a plant and animal time-keeping ultradian (period of less than 24 h) driver of both cell enlargement and the biological clock that responds to gravity. Despite considerable work in a large number of laboratories spanning several decades, this is, to my knowledge, our work is the first demonstration of a time-keeping biochemical reaction that is both gravity-responsive and growth-related and that has been shown to determine circadian periodicity. As such, the NOX protein may represent both the long-sought biological gravity receptor and the core oscillator of the cellular biological clock. Completed studies have resulted in 12 publications and two issued NASA-owned patents of the clock activity. The gravity response and autoentrainment were characterized in cultured mammalian cells and in two plant systems together with entrainment by light and small molecules (melatonin). The molecular basis of the oscillatory behavior was investigated using spectroscopic methods (Fourier transform infrared and circular dichroism) and high resolution electron microscopy. We have also applied these findings to an understanding of the response to hypergravity. Statistical methods for analysis of time series phenomena were developed (Foster et al., 2003).

  12. Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance

    Directory of Open Access Journals (Sweden)

    Markus Ritzefeld

    2012-01-01

    Full Text Available Several proteins, like transcription factors, bind to certain DNA sequences, thereby regulating biochemical pathways that determine the fate of the corresponding cell. Due to these key positions, it is indispensable to analyze protein-DNA interactions and to identify their mode of action. Surface plasmon resonance is a label-free method that facilitates the elucidation of real-time kinetics of biomolecular interactions. In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides. After a description of the physical phenomenon and the instrumental realization including fiber-optic-based SPR and SPR imaging, we will continue with a survey of immobilization methods. Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published. Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.

  13. Environmental proteomics what proteins from soil and surface water can tell us: a perspective

    Science.gov (United States)

    Schulze, W.

    2004-07-01

    Mass spectrometry based proteomics is widely used to study cellular processes in model organisms. However, it has not much been applied in environmental research because it was thought that free proteins would not be sufficiently stable in the environments. Based on recent observations that protein can readily be detected as a component of dissolve organic carbon, this article gives an overview about the possible use of proteomic methods in ecology and environmental sciences. At this stage, there are two areas of interest: (1) the identification of phylogenetic groups contributing to the DOC pool, and (2) identification of the origin of specific enzymes that are important for ecosystem processes. In this paper methods of mass spectrometry based proteomics were applied to identify proteins from DOC and water samples from different environments. It is demonstrated, that environmental proteomics is capable to distinguish the active set of organisms of different horizons of soils, and from various sources of surface water. Currently the limitation is given by the present knowledge of the genome of soil organisms. In addition, environmental proteomics allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific enzymes. Taking laccases as an example, it is shown that this enzyme is excreted into soils by a whole range of organisms from different phylogenetic groups. Further applications, such as in pollution reseach are conceivable. In summary, environmental proteomcis opens a new area of research between the fields of microbiology and biogeochemistry.

  14. The surface-anchored NanA protein promotes pneumococcal brain endothelial cell invasion.

    Science.gov (United States)

    Uchiyama, Satoshi; Carlin, Aaron F; Khosravi, Arya; Weiman, Shannon; Banerjee, Anirban; Quach, Darin; Hightower, George; Mitchell, Tim J; Doran, Kelly S; Nizet, Victor

    2009-08-31

    In humans, Streptococcus pneumoniae (SPN) is the leading cause of bacterial meningitis, a disease with high attributable mortality and frequent permanent neurological sequelae. The molecular mechanisms underlying the central nervous system tropism of SPN are incompletely understood, but include a primary interaction of the pathogen with the blood-brain barrier (BBB) endothelium. All SPN strains possess a gene encoding the surface-anchored sialidase (neuraminidase) NanA, which cleaves sialic acid on host cells and proteins. Here, we use an isogenic SPN NanA-deficient mutant and heterologous expression of the protein to show that NanA is both necessary and sufficient to promote SPN adherence to and invasion of human brain microvascular endothelial cells (hBMECs). NanA-mediated hBMEC invasion depends only partially on sialidase activity, whereas the N-terminal lectinlike domain of the protein plays a critical role. NanA promotes SPN-BBB interaction in a murine infection model, identifying the protein as proximal mediator of CNS entry by the pathogen.

  15. Structural and biological mimicry of protein surface recognition by [alpha/beta]-peptide foldamers

    Energy Technology Data Exchange (ETDEWEB)

    Horne, W. Seth; Johnson, Lisa M.; Ketas, Thomas J.; Klasse, Per Johan; Lu, Min; Moore, John P.; Gellman, Samuel H.; (Cornell); (UW)

    2009-10-05

    Unnatural oligomers that can mimic protein surfaces offer a potentially useful strategy for blocking biomedically important protein-protein interactions. Here we evaluate an approach based on combining {alpha}- and {beta}-amino acid residues in the context of a polypeptide sequence from the HIV protein gp41, which represents an excellent testbed because of the wealth of available structural and biological information. We show that {alpha}/{beta}-peptides can mimic structural and functional properties of a critical gp41 subunit. Physical studies in solution, crystallographic data, and results from cell-fusion and virus-infectivity assays collectively indicate that the gp41-mimetic {alpha}/{beta}-peptides effectively block HIV-cell fusion via a mechanism comparable to that of gp41-derived {alpha}-peptides. An optimized {alpha}/{beta}-peptide is far less susceptible to proteolytic degradation than is an analogous {alpha}-peptide. Our findings show how a two-stage design approach, in which sequence-based {alpha} {yields} {beta} replacements are followed by site-specific backbone rigidification, can lead to physical and biological mimicry of a natural biorecognition process.

  16. Environmental proteomics – what proteins from soil and surface water can tell us: a perspective

    Directory of Open Access Journals (Sweden)

    W. Schulze

    2004-07-01

    Full Text Available Mass spectrometry based proteomics is widely used to study cellular processes in model organisms. However, it has not much been applied in environmental research because it was thought that free proteins would not be sufficiently stable in the environments. Based on recent observations that protein can readily be detected as a component of dissolve organic carbon, this article gives an overview about the possible use of proteomic methods in ecology and environmental sciences. At this stage, there are two areas of interest: (1 the identification of phylogenetic groups contributing to the DOC pool, and (2 identification of the origin of specific enzymes that are important for ecosystem processes. In this paper methods of mass spectrometry based proteomics were applied to identify proteins from DOC and water samples from different environments. It is demonstrated, that environmental proteomics is capable to distinguish the active set of organisms of different horizons of soils, and from various sources of surface water. Currently the limitation is given by the present knowledge of the genome of soil organisms. In addition, environmental proteomics allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific enzymes. Taking laccases as an example, it is shown that this enzyme is excreted into soils by a whole range of organisms from different phylogenetic groups. Further applications, such as in pollution reseach are conceivable. In summary, environmental proteomcis opens a new area of research between the fields of microbiology and biogeochemistry.

  17. Site-selective adsorption of protein induced by a metal pattern on a poly(ethylene terephthalate) surface.

    Science.gov (United States)

    Cao, Jiali; Wu, Zhongkui; Li, Shaoying; Tang, Hongxiao; Mei, Qilin

    2013-11-01

    A novel technique for inducing site-selective adsorption of protein through constructing metal patterns on flexible poly(ethylene terephthalate) surfaces is presented. The substrates were first modified by vacuum ultraviolet (VUV) irradiation through a photomask to introduce regions with different functional groups. Then the designed metal patterns were constructed on the surfaces of VUV-treated substrates. The surface rearrangement was effectively prevented by constructing silver patterns on poly(ethylene terephthalate) surfaces, thus significantly improving the stability and selectivity of protein adsorption on the surfaces. Moreover, the protein-repulsive layer further reinforced the effect. Finally, protein patterns were successfully obtained. As confirmed by fluorescence microscope, field emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and static water contact angle measurement, the protein patterns possess both excellent selectivity and high fidelity. Feature size of the protein patterns surrounded by a protein-repulsive layer was exactly the same as that of the photomask. And the grain sizes of silver particles were approximately 50 nm. This work could potentially be used in various fields such as biomedicine, bioelectronic components, and tissue repair and replacement, where selective adsorption of protein is desired.

  18. Restoration of proper trafficking to the cell surface for membrane proteins harboring cysteine mutations.

    Directory of Open Access Journals (Sweden)

    Angelica Lopez-Rodriguez

    Full Text Available A common phenotype for many genetic diseases is that the cell is unable to deliver full-length membrane proteins to the cell surface. For some forms of autism, hereditary spherocytosis and color blindness, the culprits are single point mutations to cysteine. We have studied two inheritable cysteine mutants of cyclic nucleotide-gated channels that produce achromatopsia, a common form of severe color blindness. By taking advantage of the reactivity of cysteine's sulfhydryl group, we modified these mutants with chemical reagents that attach moieties with similar chemistries to the wild-type amino acids' side chains. We show that these modifications restored proper delivery to the cell membrane. Once there, the channels exhibited normal functional properties. This strategy might provide a unique opportunity to assess the chemical nature of membrane protein traffic problems.

  19. Protein separation and identification using magnetic beads encoded with surface-enhanced Raman spectroscopy.

    Science.gov (United States)

    Jun, Bong-Hyun; Noh, Mi Suk; Kim, Gunsung; Kang, Homan; Kim, Jong-Ho; Chung, Woo-Jae; Kim, Min-Soo; Kim, Yong-Kweon; Cho, Myung-Haing; Jeong, Dae Hong; Lee, Yoon-Sik

    2009-08-01

    This article presents a prototype of a surface-enhanced Raman spectroscopy (SERS)-encoded magnetic bead of 8mum diameter. The core part of the bead is composed of a magnetic nanoparticle (NP)-embedded sulfonated polystyrene bead. The outer part of the bead is embedded with Ag NPs on which labeling molecules generating specific SERS bands are adsorbed. A silica shell is fabricated for further bioconjugation and protection of SERS signaling. Benzenethiol, 4-mercaptotoluene, 2-naphthalenethiol, and 4-aminothiophenol are used as labeling molecules. The magnetic SERS beads are used as substrates for protein sensing and screening with easy handling. As a model application, streptavidin-bound magnetic SERS beads are used to illustrate selective separation in a flow cytometry system, and the screened beads are spectrally recognized by Raman spectroscopy. The proposed magnetic SERS beads are likely to be used as a versatile solid support for protein sensing and screening in multiple assay technology.

  20. Data on the role of accessible surface area on osmolytes-induced protein stabilization

    Directory of Open Access Journals (Sweden)

    Safikur Rahman

    2017-02-01

    Full Text Available This paper describes data related to the research article “Testing the dependence of stabilizing effect of osmolytes on the fractional increase in the accessible surface area on thermal and chemical denaturations of proteins” [1]. Heat- and guanidinium chloride (GdmCl-induced denaturation of three disulfide free proteins (bovine cytochrome c (b-cyt-c, myoglobin (Mb and barstar in the presence of different concentrations of methylamines (sarcosine, glycine-betaine (GB and trimethylamine-N-oxide (TMAO was monitored by [ϴ]222, the mean residue ellipticity at 222 nm at pH 7.0. Methylamines belong to a class of osmolytes known to protect proteins from deleterious effect of urea. This paper includes comprehensive thermodynamic data obtained from the heat- and GdmCl-induced denaturations of barstar, b-cyt-c and Mb.

  1. Surface adsorption of lattice HP proteins: Thermodynamics and structural transitions using Wang-Landau sampling

    Science.gov (United States)

    Li, Ying Wai; Wüst, Thomas; Landau, David P.

    2012-12-01

    Wang-Landau sampling has been applied to investigate the thermodynamics and structural properties of a lattice hydrophobic-polar heteropolymer (the HP protein model) interacting with an attractive substrate. For simplicity, we consider a short HP sequence consisting of only 36 monomers interacting with a substrate which attracts all monomers in the sequence. The conformational “phase transitions” have been identified by a canonical analysis of the specific heat and suitable structural observables. Three major “transitions”, namely, adsorption, hydrophobic core formation and “flattening” of adsorbed structures, are observed. Depending on the surface attractive strength relative to the intra-protein attraction among the H monomers, these processes take place in different sequences upon cooling.

  2. Tailoring odorant-binding protein coatings characteristics for surface acoustic wave biosensor development

    Energy Technology Data Exchange (ETDEWEB)

    Di Pietrantonio, F., E-mail: fabio.dp@idasc.cnr.it [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Benetti, M. [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Dinca, V. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125 Magurele (Romania); Cannatà, D. [Institute of Acoustics and Sensors “O. M. Corbino”, National Research Council of Italy, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Verona, E. [Institute for Photonics and Nanotechnologies, National Research Council of Italy, Via del Cineto Romano 42, 00156 Rome (Italy); D’Auria, S. [Institute of Protein Biochemistry, National Research Council of Italy, Via Pietro Castellino 111, 80131 Naples (Italy); Dinescu, M. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125 Magurele (Romania)

    2014-05-01

    In this study, wild type bovine odorant-binding proteins (wtbOBPs) were deposited by matrix-assisted pulsed laser evaporation (MAPLE) and utilized as active material on surface acoustic wave (SAW) biosensors. Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM) were used to determine the chemical, morphological characteristics of the protein thin films. The FTIR data demonstrates that the functional groups of wtbOBPs do not suffer significant changes in the MAPLE-deposited films when compared to the reference one. The topographical studies show that the homogeneity, density and the roughness of the coatings are related mainly to the laser parameters (fluence and number of pulses). SAW biosensor responses to different concentrations of R-(–)-1-octen-3-ol (octenol) and R-(–)-carvone (carvone) were evaluated. The obtained sensitivities, achieved through the optimization of deposition parameters, demonstrated that MAPLE is a promising deposition technique for SAW biosensor implementation.

  3. Tailoring odorant-binding protein coatings characteristics for surface acoustic wave biosensor development

    Science.gov (United States)

    Di Pietrantonio, F.; Benetti, M.; Dinca, V.; Cannatà, D.; Verona, E.; D'Auria, S.; Dinescu, M.

    2014-05-01

    In this study, wild type bovine odorant-binding proteins (wtbOBPs) were deposited by matrix-assisted pulsed laser evaporation (MAPLE) and utilized as active material on surface acoustic wave (SAW) biosensors. Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM) were used to determine the chemical, morphological characteristics of the protein thin films. The FTIR data demonstrates that the functional groups of wtbOBPs do not suffer significant changes in the MAPLE-deposited films when compared to the reference one. The topographical studies show that the homogeneity, density and the roughness of the coatings are related mainly to the laser parameters (fluence and number of pulses). SAW biosensor responses to different concentrations of R-(-)-1-octen-3-ol (octenol) and R-(-)-carvone (carvone) were evaluated. The obtained sensitivities, achieved through the optimization of deposition parameters, demonstrated that MAPLE is a promising deposition technique for SAW biosensor implementation.

  4. Cell cycle-dependent phosphorylation of Theileria annulata schizont surface proteins.

    Directory of Open Access Journals (Sweden)

    Olga Wiens

    Full Text Available The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1, are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr, serine (p-Ser and threonine-proline (p-Thr-Pro epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state.

  5. Evolution of hepatitis B virus surface gene and protein among Iranian chronic carriers from different provinces

    Directory of Open Access Journals (Sweden)

    Fatemeh Ramezani

    2015-11-01

    Full Text Available Background and Objectives:  Iranian chronic HBV carrier’s population has shown a unique pattern of genotype D distri- bution all around the country. The aim of this study was to explore more details of evolutionary history of carriers based on structural surface proteins from different provinces.Materials and Methods: Sera obtained from 360 isolates from 12 Different regions of country were used for amplificationand sequencing of surface proteins. A detailed mutational analysis was undertaken.Results: The total ratio for Missense/Silent nucleotide substitutions was 0.96. Sistan and Kermanshah showed the lowest rate of evolution between provinces (P = 0.055. On the other hand, Khorasan Razavi and Khoozestan contained the highest ratio (P = 0.055. The rest of regions were laid between these two extremes. Azarbayjan and Guilan showed the highest proportion of immune epitope distribution (91.3% and 96%, respectively. Conversely, Sistan and Tehran harbored the least percentage (66.6% and 68.8%, respectively. Kermanshah province contained only 5.2%, whereas Isfahan had 54.5% of B cell epitope distribution. In terms of T helper epitopes, all provinces showed a somehow homogeneity: 22.58% (Fars to 46.6% (Khuz- estan. On the other hand, distribution of substitutions within the CTL epitopes showed a wide range of variation between 6.6% (Khuzestan and 63% (Kermanshah.Conclusion: Further to low selection pressure found in Iranian population, the variations between different regions designate random genetic drift within the surface proteins. These finding would have some applications in terms of specific antiviral regimen, design of more efficient vaccine and public health issues.

  6. Mussel inspired protein-mediated surface modification to electrospun fibers and their potential biomedical applications.

    Science.gov (United States)

    Xie, Jingwei; Michael, Praveesuda Lorwattanapongsa; Zhong, Shaoping; Ma, Bing; MacEwan, Matthew R; Lim, Chwee Teck

    2012-04-01

    Mussel inspired proteins have been demonstrated to serve as a versatile biologic adhesive with numerous applications. The present study illustrates the use of such Mussel inspired proteins (polydopamine) in the fabrication of functionalized bio-inspired nanomaterials capable of both improving cell response and sustained delivery of model probes. X-ray photoelectron spectroscopy analysis confirmed the ability of dopamine to polymerize on the surface of plasma-treated, electrospun poly(ε-caprolactone) (PCL) fiber mats to form polydopamine coating. Transmission electron microscopy images demonstrated that self-polymerization of dopamine was induced by pH shift and that the thickness of polydopamine coating was readily modulated by adjusting the concentration of dopamine and reaction time. Polydopamine coatings were noted to affect the mechanical properties of underlying fiber mats, as mechanical testing demonstrated a decrease in elasticity and increase in stiffness of polydopamine-coated fiber mats. Polydopamine coatings were also utilized to effectively immobilize extracellular matrix proteins (i.e., fibronectin) on the surface of polydopamine-coated, electrospun fibers, resulting in enhancement of NIH3T3 cell attachment, spreading, and cytoskeletal development. Comparison of release rates of rhodamine 6G encapsulated in coated and uncoated PCL fibers also confirmed that polydopamine coatings modulate the release rate of loaded payloads. The authors further demonstrate the significant difference of rhodamine 6G adsorption kinetics in water between PCL fibers and polydopamine-coated PCL fibers. Taken together, polydopamine-mediated surface modification to electrospun fibers may be an effective means of fabricating a wide range of bio-inspired nanomaterials with unique properties for use in tissue engineering, drug delivery, and advanced biomedical applications.

  7. Selective cell-surface labeling of the molecular motor protein prestin

    Energy Technology Data Exchange (ETDEWEB)

    McGuire, Ryan M. [Department of Bioengineering, Rice University, Houston, TX 77251 (United States); Silberg, Jonathan J., E-mail: joff@rice.edu [Department of Bioengineering, Rice University, Houston, TX 77251 (United States); Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251 (United States); Pereira, Fred A. [Department of Bioengineering, Rice University, Houston, TX 77251 (United States); Huffington Center on Aging, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Raphael, Robert M., E-mail: rraphael@rice.edu [Department of Bioengineering, Rice University, Houston, TX 77251 (United States)

    2011-06-24

    Highlights: {yields} Trafficking to the plasma membrane is required for prestin function. {yields} Biotin acceptor peptide (BAP) was fused to prestin through a transmembrane domain. {yields} BAP-prestin can be metabolically labeled with biotin in HEK293 cells. {yields} Biotin-BAP-prestin allows for selective imaging of fully trafficked prestin. {yields} The biotin-BAP-prestin displays voltage-sensitive activity. -- Abstract: Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

  8. Influence of surface modification on protein retention in ion-exchange chromatography. Evaluation using different retention models.

    Science.gov (United States)

    Bruch, Thomas; Graalfs, Heiner; Jacob, Lothar; Frech, Christian

    2009-02-06

    A large number of different stationary phases for ion-exchange chromatography (IEC) from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (alpha-lactalbumin, beta-lactoglobulin A, bovine serum albumin and alcohol dehydrogenase) were determined for three anion-exchange adsorbents based on synthetic copolymer beads with differences in the functional group chemistry. Fractogel EMD DEAE and Fractoprep DEAE consist of functional groups bound to the surface via "tentacles", ToyopearlDEAE by a short linker. Three models which describe chromatographic retention were used to analyse the characteristic parameters of the protein/stationary-phase interactions. The number of electrostatic interaction between the stationary phase and the model proteins, the protein specific surface charge densities and the interacting surface of the proteins with the adsorptive layer of the chromatographic media depend on the surface modification as well as on the molecular mass of the model proteins. In general, protein retention of the model proteins on the weak anion exchangers was found to be greater if the stationary phase carries tentacles and protein mass is above 60 kDa.

  9. Protein attachment onto silica surfaces--a survey of molecular fundamentals, resulting effects and novel preventive strategies in CE.

    Science.gov (United States)

    Stutz, Hanno

    2009-06-01

    This review addresses the fundamentals governing the adsorption of individual protein molecules onto the surface of fused-silica capillaries, the protein aggregation to adsorbate clusters and their final accretion to monolayers with subsequent stratification to protein multilayers. The attention in CE protein separation has primarily been focused on (i) tuning the BGE including the buffer type, ionic strength, pH and additives, (ii) tailored post-rinse procedures to detach adhered protein residues and (iii) the optimization of capillary wall shielding in order to reduce protein attachment. Improvements in protein separation as well as related adverse effects are mainly discussed on the basis of parameters known to become deteriorated in case of protein adhesion, e.g. repeatability of the EOF and of migration times, peak width, theoretical plate numbers, resolution and asymmetry factor. However, knowledge of the molecular principles controlling protein adsorption onto silica surfaces is indispensable for separation optimization. Furthermore, it facilitates troubleshooting and the interpretation of undesired concomitant phenomena. This review comprehensively discusses protein adsorption models derived from surface chemistry primarily in terms of their relevance for CE, clearly showing that the adsorption process in its complexity is only partially revealed by models, which address single or binary protein solutions. In a further section theoretical concepts and surface models are related to surface phenomena encountered in CE. The final part of the review surveys recent concepts for prevention of protein adhesion, thereby addressing capillary treatment, favorable buffer types, dynamic and adhesive semi-permanent coating strategies covering the literature from 2000-2008.

  10. Specific Interactions of Neutral Side Chains of an Adsorbed Protein with the Surface of α-Quartz and Silica Gel.

    Science.gov (United States)

    Odinokov, Alexey V; Bagaturyants, Alexander A

    2015-07-16

    Many key features of the protein adsorption on the silica surfaces still remain unraveled. One of the open questions is the interaction of nonpolar side chains with siloxane cavities. Here, we use nonequilibrium molecular dynamics simulations for the detailed investigation of the binding of several hydrophobic and amphiphilic protein side chains with silica surface. These interactions were found to be a possible driving force for protein adsorption. The free energy gain was larger for the disordered surface of amorphous silica gel as compared to α-quartz, but the impact depended on the type of amino acid. The dependence was analyzed from the structural point of view. For every amino acid an enthalpy-entropy compensation behavior was observed. These results confirm a hypothesis of an essential role of hydrophobic interactions in protein unfolding and irreversible adsorption on the silica surface.

  11. The effect of amorphous silicon surface hydrogenation on morphology, wettability and its implication on the adsorption of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Filali, Larbi, E-mail: larbifilali5@gmail.com [Laboratoire de Physique des Couches Minces et Matériaux pour l' Electronique, Université d' Oran 1, Ahmed Ben Bella, BP 1524, El M' naouar 31100 Oran (Algeria); Brahmi, Yamina; Sib, Jamal Dine [Laboratoire de Physique des Couches Minces et Matériaux pour l' Electronique, Université d' Oran 1, Ahmed Ben Bella, BP 1524, El M' naouar 31100 Oran (Algeria); Bouhekka, Ahmed [Laboratoire de Physique des Couches Minces et Matériaux pour l' Electronique, Université d' Oran 1, Ahmed Ben Bella, BP 1524, El M' naouar 31100 Oran (Algeria); Département de Physique, Université Hassiba Ben Bouali, 02000 Chlef (Algeria); Benlakehal, Djamel; Bouizem, Yahya; Kebab, Aissa; Chahed, Larbi [Laboratoire de Physique des Couches Minces et Matériaux pour l' Electronique, Université d' Oran 1, Ahmed Ben Bella, BP 1524, El M' naouar 31100 Oran (Algeria)

    2016-10-30

    Highlights: • Hydrogenation of the surfaces had the effect of reducing the roughness by way of shadow etching. • Roughness was the driving factor affecting the wettability of the hydrogenated surfaces. • Bovine Serum Albumin proteins favored the surfaces with highest hydrogen content. • Surface modification induced secondary structure change of adsorbed proteins. - Abstract: We study the effect of amorphous silicon (a-Si) surface hydrogenation on Bovine Serum Albumin (BSA) adsorption. A set of (a-Si) films was prepared by radio frequency magnetron sputtering (RFMS) and after deposition; they were treated in molecular hydrogen ambient at different pressures (1–3 Pa). Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy and spectroscopic ellipsometry (SE) were used to study the hydrogenation effect and BSA adsorption. Atomic force microscopy (AFM) was used to evaluate morphological changes caused by hydrogenation. The wettability of the films was measured using contact angle measurement, and in the case of the hydrogenated surfaces, it was found to be driven by surface roughness. FTIR-ATR spectroscopy and SE measurements show that proteins had the strongest affinity toward the surfaces with the highest hydrogen content and their secondary structure was affected by a significant decrease of the α-helix component (-27%) compared with the proteins adsorbed on the un-treated surface, which had a predominantly α-helix (45%) structure. The adsorbed protein layer was found to be densely packed with a large thickness (30.9 nm) on the hydrogen-rich surfaces. The most important result is that the surface hydrogen content was the dominant factor, compared to wettability and morphology, for protein adsorption.

  12. Crystal Structure of Neurotropism-Associated Variable Surface Protein 1 (VSP1) of Borrelia Turicatae

    Energy Technology Data Exchange (ETDEWEB)

    Lawson,C.; Yung, B.; Barbour, A.; Zuckert, W.

    2006-01-01

    Vsp surface lipoproteins are serotype-defining antigens of relapsing fever spirochetes that undergo multiphasic antigenic variation to allow bacterial persistence in spite of an immune response. Two isogenic serotypes of Borrelia turicatae strain Oz1 differ in their Vsp sequences and in disease manifestations in infected mice: Vsp1 is associated with the selection of a neurological niche, while Vsp2 is associated with blood and skin infection. We report here crystal structures of the Vsp1 dimer at 2.7 and 2.2 Angstroms. The structures confirm that relapsing fever Vsp proteins share a common helical fold with OspCs of Lyme disease-causing Borrelia. The fold features an inner stem formed by highly conserved N and C termini and an outer 'dome' formed by the variable central residues. Both Vsp1 and OspC structures possess small water-filled cavities, or pockets, that are lined largely by variable residues and are thus highly variable in shape. These features appear to signify tolerance of the Vsp-OspC fold for imperfect packing of residues at its antigenic surface. Structural comparison of Vsp1 with a homology model for Vsp2 suggests that observed differences in disease manifestation may arise in part from distinct differences in electrostatic surface properties; additional predicted positively charged surface patches on Vsp2 compared to Vsp1 may be sufficient to explain the relative propensity of Vsp2 to bind to acidic glycosaminoglycans.

  13. Protein-nanoparticle interaction in bioconjugated silver nanoparticles: A transmission electron microscopy and surface enhanced Raman spectroscopy study

    Science.gov (United States)

    Reymond-Laruinaz, Sébastien; Saviot, Lucien; Potin, Valérie; Marco de Lucas, María del Carmen

    2016-12-01

    Understanding the mechanisms of interaction between proteins and noble metal nanoparticles (NPs) is crucial to extend the use of NPs in biological applications and nanomedicine. We report the synthesis of Ag-NPs:protein bioconjugates synthesized in total absence of citrates or other stabilizing agents in order to study the NP-protein interaction. Four common proteins (lysozyme, bovine serum albumin, cytochrome-C and hemoglobin) were used in this work. Transmission electron microscopy (TEM) and surface enhanced Raman spectroscopy (SERS) were mainly used to study these bioconjugated NPs. TEM images showed Ag NPs with sizes in the 5-40 nm range. The presence of a protein layer surrounding the Ag NPs was also observed by TEM. Moreover, the composition at different points of single bioconjugated NPs was probed by electron energy loss spectroscopy (EELS). The thickness of the protein layer varies in the 3-15 nm range and the Ag NPs are a few nanometers away. This allowed to obtain an enhancement of the Raman signal of the proteins in the analysis of water suspensions of bioconjugates. SERS results showed a broadening of the Raman bands of the proteins which we attribute to the contribution of different configurations of the proteins adsorbed on the Ag NPs surface. Moreover, the assignment of an intense and sharp peak in the low-frequency range to Ag-N vibrations points to the chemisorption of the proteins on the Ag-NPs surface.

  14. Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

    Science.gov (United States)

    Hermann, M; Jaconi, M E; Dahlgren, C; Waldvogel, F A; Stendahl, O; Lew, D P

    1990-09-01

    The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (mean survival: 34.2% +/- 9.0% of albumin, P less than 0.0001) despite similar efficient ingestion of extracellular bacteria. Enhancement of killing was observed when surfaces were coated with purified constituents of extracellular matrix, i.e., fibronectin, fibrinogen, laminin, vitronectin, or type IV collagen. In addition to matrix proteins, the tetrapeptide RGDS (the sequence recognized by integrins) crosslinked to surface bound albumin was also active (survival: 74.5% +/- 5.5% of albumin, P less than 0.02), and fibronectin-increased killing was inhibited by soluble RGDS. Chemiluminescence measurements and experiments with CGD neutrophils revealed that both oxygen-dependent and -independent bactericidal mechanisms are involved. In conclusion, matrix proteins enhance intracellular bactericidal activity of adherent neutrophils, presumably by integrin recognition of RGDS-containing ligands. These results indicate a role for extracellular matrix proteins in the enhancement of the host defense against pyogenic infections.

  15. Molecularly imprinted polymer grafted on polysaccharide microsphere surface by the sol-gel process for protein recognition.

    Science.gov (United States)

    Li, Feng; Li, Jing; Zhang, Shusheng

    2008-02-15

    An interfacial organic-inorganic hybridization concept was applied to the preparation of a new spherical imprinted material for protein recognition. The functional biopolymer chitosan (CS), shaped as microsphere and high-density cross-linked, constituted of the polysaccharide core for surface imprinting. After the model template protein, bovine serum albumin, was covalently immobilized by forming imine bonds with the functional amine groups of CS, two kinds of organic siloxane (3-aminopropyltrimethoxysiloxane: APTMS, and tetraethoxysiloxane: TEOS) assembled and polymerized on the polysaccharide-protein surface via sol-gel process in aqueous solution at room temperature. After template removal, the protein-imprinted sol-gel surface exhibited a prevalent preference for the template protein in adsorption experiments, as compared with four contrastive proteins. Bioinformatics methods were also employed to investigate the imprinting process and the recognition effect. The influence of siloxane type, pH, siloxane/water ratio on template removal and recognition selectivity was assessed. Under optimized imprinting conditions, a large quantity of well-distributed pores was observed on the immobilized-template imprinted surface. The surface-imprinted adsorbent offered a fast kinetics for template re-adsorption and could be reused. Compared with the imprinted material prepared with free-template, material prepared with immobilized-template possessed higher adsorption capacity towards template protein. Easy preparation of the described imprinted material, high affinity and good reusability make this approach attractive and broadly applicable in biotechnology for down-stream processing and biosensor.

  16. Modulation of Protein Fouling and Interfacial Properties at Carbon Surfaces via Immobilization of Glycans Using Aryldiazonium Chemistry

    Science.gov (United States)

    Zen, Federico; Angione, M. Daniela; Behan, James A.; Cullen, Ronan J.; Duff, Thomas; Vasconcelos, Joana M.; Scanlan, Eoin M.; Colavita, Paula E.

    2016-01-01

    Carbon materials and nanomaterials are of great interest for biological applications such as implantable devices and nanoparticle vectors, however, to realize their potential it is critical to control formation and composition of the protein corona in biological media. In this work, protein adsorption studies were carried out at carbon surfaces functionalized with aryldiazonium layers bearing mono- and di-saccharide glycosides. Surface IR reflectance absorption spectroscopy and quartz crystal microbalance were used to study adsorption of albumin, lysozyme and fibrinogen. Protein adsorption was found to decrease by 30–90% with respect to bare carbon surfaces; notably, enhanced rejection was observed in the case of the tested di-saccharide vs. simple mono-saccharides for near-physiological protein concentration values. ζ-potential measurements revealed that aryldiazonium chemistry results in the immobilization of phenylglycosides without a change in surface charge density, which is known to be important for protein adsorption. Multisolvent contact angle measurements were used to calculate surface free energy and acid-base polar components of bare and modified surfaces based on the van Oss-Chaudhury-Good model: results indicate that protein resistance in these phenylglycoside layers correlates positively with wetting behavior and Lewis basicity. PMID:27108562

  17. Compression/expansion rheology of oil/water interfaces with adsorbed proteins. Comparison with the air/water surface

    NARCIS (Netherlands)

    Benjamins, J.; Lyklema, J.; Lucassen-Reynders, E.H.

    2006-01-01

    Dynamic interfacial tensions and surface dilational moduli were measured for four proteins at three fluid interfaces, as a function of time and concentration. The proteins-ß-casein, ß-lactoglobulin, bovine serum albumin, and ovalbumin - were adsorbed from aqueous solution against air, n-tetradecane,

  18. The role of Listeria monocytogenes cell wall surface anchor protein LapB in virulence, adherence, and intracellular replication

    Science.gov (United States)

    Lmof2365_2117 is a Listeria monocytogenes putative cell wall surface anchor protein with a conserved domain found in collagen binding proteins. We constructed a deletion mutation in lmof2365_2117 in serotype 4b strain F2365, evaluated its virulence, and determined its ability to adhere and invade co...

  19. A novel Pfs38 protein complex on the surface of Plasmodium falciparum blood-stage merozoites

    DEFF Research Database (Denmark)

    Paul, Gourab; Deshmukh, Arunaditya; Kaur, Inderjeet

    2017-01-01

    and glycerol density gradient fractionation were carried out to confirm the respective interactions. Furthermore, erythrocyte binding assay with 6-cys proteins were undertaken to find out their possible role in host-parasite infection and seropositivity was assessed using Indian and Liberian sera. RESULTS...... the development of a multi-sub-unit malaria vaccine based on some of these protein complexes on merozoite surface....

  20. Hypothetical protein Avin_16040 as the S-layer protein of Azotobacter vinelandii and its involvement in plant root surface attachment.

    Science.gov (United States)

    Liew, Pauline Woan Ying; Jong, Bor Chyan; Najimudin, Nazalan

    2015-11-01

    A proteomic analysis of a soil-dwelling, plant growth-promoting Azotobacter vinelandii strain showed the presence of a protein encoded by the hypothetical Avin_16040 gene when the bacterial cells were attached to the Oryza sativa root surface. An Avin_16040 deletion mutant demonstrated reduced cellular adherence to the root surface, surface hydrophobicity, and biofilm formation compared to those of the wild type. By atomic force microscopy (AFM) analysis of the cell surface topography, the deletion mutant displayed a cell surface architectural pattern that was different from that of the wild type. Escherichia coli transformed with the wild-type Avin_16040 gene displayed on its cell surface organized motifs which looked like the S-layer monomers of A. vinelandii. The recombinant E. coli also demonstrated enhanced adhesion to the root surface.

  1. Degradable polyester scaffolds with controlled surface chemistry combining minimal protein adsorption with specific bioactivation

    Science.gov (United States)

    Grafahrend, Dirk; Heffels, Karl-Heinz; Beer, Meike V.; Gasteier, Peter; Möller, Martin; Boehm, Gabriele; Dalton, Paul D.; Groll, Jürgen

    2011-01-01

    Advanced biomaterials and scaffolds for tissue engineering place high demands on materials and exceed the passive biocompatibility requirements previously considered acceptable for biomedical implants. Together with degradability, the activation of specific cell-material interactions and a three-dimensional environment that mimics the extracellular matrix are core challenges and prerequisites for the organization of living cells to functional tissue. Moreover, although bioactive signalling combined with minimization of non-specific protein adsorption is an advanced modification technique for flat surfaces, it is usually not accomplished for three-dimensional fibrous scaffolds used in tissue engineering. Here, we present a one-step preparation of fully synthetic, bioactive and degradable extracellular matrix-mimetic scaffolds by electrospinning, using poly(D,L-lactide-co-glycolide) as the matrix polymer. Addition of a functional, amphiphilic macromolecule based on star-shaped poly(ethylene oxide) transforms current biomedically used degradable polyesters into hydrophilic fibres, which causes the suppression of non-specific protein adsorption on the fibres’ surface. The subsequent covalent attachment of cell-adhesion-mediating peptides to the hydrophilic fibres promotes specific bioactivation and enables adhesion of cells through exclusive recognition of the immobilized binding motifs. This approach permits synthetic materials to directly control cell behaviour, for example, resembling the binding of cells to fibronectin immobilized on collagen fibres in the extracellular matrix of connective tissue.

  2. Probing DNA-Protein Interactions on Surfaces Using Spectral Self-interference Fluorescence Microscopy

    Science.gov (United States)

    Dogan, Mehmet; Droge, Peter; Swan, Anna K.; Unlu, Selim; Goldberg, Bennett B.

    2007-03-01

    We are probing the interactions between double-stranded DNA and integration host factor (IHF) proteins [1] on surfaces using Spectral Self-interference Fluorescence Microscopy (SSFM) [2].The probing technique utilizes the spectral fringes produced by interference of direct and reflected emission from fluorescent molecules. The modified spectrum provides a unique signature of the axial position of the fluorophores. Using the SSFM technique, we probe the average location of the fluorescent markers attached to the DNA molecules to study the conformational changes in double-stranded DNA tethered to SiO2 surfaces. In the presence of IHF, a DNA bending protein, we observe reduction in the vertical position of fluorescent molecules suggesting the formation of IHF-DNA complex and IHF-induced DNA bending. We also discuss the results with different IHF strains and different binding conditions. [1] Q. Bao et. al., Gene, Vol.343 pp.99-106 (2004) [2] L.A. Moiseev et. al., Journal of Applied Physics, Vol.96, pp. 5311-5315 (2004)

  3. Proinflammatory Cytokine Gene Expression by Murine Macrophages in Response to Brugia malayi Wolbachia Surface Protein

    Directory of Open Access Journals (Sweden)

    Chantima Porksakorn

    2007-01-01

    Full Text Available Wolbachia, an endosymbiotic bacterium found in most species of filarial parasites, is thought to play a significant role in inducing innate inflammatory responses in lymphatic filariasis patients. However, the Wolbachia-derived molecules that are recognized by the innate immune system have not yet been identified. In this study, we exposed the murine macrophage cell line RAW 264.7 to a recombinant form of the major Wolbachia surface protein (rWSP to determine if WSP is capable of innately inducing cytokine transcription. Interleukin (IL-1β, IL-6, and tumor necrosis factor (TNF mRNAs were all upregulated by the rWSP stimulation in a dose-dependant manner. TNF transcription peaked at 3 hours, whereas IL-1β and IL-6 transcription peaked at 6 hours post-rWSP exposure. The levels of innate cytokine expression induced by a high-dose (9.0 μg/mL rWSP in the RAW 264.7 cells were comparable to the levels induced by 0.1 μg/mL E. coli-derived lipopolysaccharides. Pretreatment of the rWSP with proteinase-K drastically reduced IL-1β, IL-6, and TNF transcription. However, the proinflammatory response was not inhibited by polymyxin B treatment. These results strongly suggest that the major Wolbachia surface protein molecule WSP is an important inducer of innate immune responses during filarial infections.

  4. Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d

    Directory of Open Access Journals (Sweden)

    Moffatt Barbara A

    2010-08-01

    Full Text Available Abstract Background Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB for coplanar aromatic motifs similar to those found in known glycan-binding proteins. Results The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192 in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Conclusions Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

  5. Nonstructural protein 1 characteristic peak from NS1-saliva mixture with Surface-Enhanced Raman spectroscopy.

    Science.gov (United States)

    Radzol, A R M; Lee, Khuan Y; Mansor, W

    2013-01-01

    Surface Enhanced Raman spectroscopy (SERS) is an enhanced technique of Raman spectroscopy, which amplifies the intensity of Raman scattering to a practical range with adsorption of analyte onto nano-size plasmonic material such as gold, silver or copper. This feature of SERS has given it a niche in tracing molecular structure, especially useful for marking diseases specific biomarker. NS1 protein has been clinically accepted as an alternative biomarker for diseases caused by flavivirus. Detection of Nonstructural Protein 1 (NS1) will allow early diagnosis of the diseases. Its presence in the blood serum has been reported as early as first day of infection. With gold substrate, our work here intends to explore if SERS is suitable to detect NS1 from saliva, with saliva becoming the most favored alternative to blood as diagnostic fluid due to its advantages in sample collection. Our experimental results find both gold coated slide (GS) and saliva being Raman inactive, but the molecular fingerprint of NS1 protein at Raman shift 1012 cm(-1), which has never been reported before. The distinct peak is discovered to be attributed by breathing vibration of the benzene ring structure of NS1 side chain molecule. The characteristic peak is also found to vary in direct proportion to concentration of the NS1-saliva mixture, with a correlation coefficient of +0.96118 and a standard error estimation of 0.11382.

  6. Optimization of the Preparation of Fish Protein Anti-Obesity Hydrolysates Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Jinju Wang

    2013-02-01

    Full Text Available The enzymatic condition for producing the anti-obesity hydrolysates from fish water-soluble protein was optimized with the aid of response surface methodology, which also derived a statistical model for experimental validation. Compared with neutral protease, papain and protamex, the porcine pancreas lipase inhibitory rate of hydrolysates from fish water-soluble protein was higher with alkaline protease. Results showed that the model terms were significant, the terms of lack of fit were not significant, and the optimal conditions for the hydrolysis by alkaline protease were initial pH 11, temperature 39 °C, enzyme dosage 122 U/mL and 10 h of hydrolysis time. Under these conditions, the porcine pancreas lipase and the α-amylase inhibitory rate could reach 53.04% ± 1.32% and 20.03 ± 0.89%, while predicted value were 54.63% ± 1.75%, 21.22% ± 0.70%, respectively. In addition, Lineweaver-Burk plots showed noncompetitive inhibition. The Ki value calculated was 84.13 mg/mL. These results demonstrated that fish water-soluble protein could be used for obtaining anti-obesity hydrolysates.

  7. Electronic Wiring of a Multi-Redox Site Membrane Protein in a Biomimetic Surface Architecture

    Science.gov (United States)

    Friedrich, Marcel G.; Robertson, Joseph W. F.; Walz, Dieter; Knoll, Wolfgang; Naumann, Renate L. C.

    2008-01-01

    Bioelectronic coupling of multi-redox-site membrane proteins was accomplished with cytochrome c oxidase (CcO) as an example. A biomimetic membrane system was used for the oriented immobilization of the CcO oxidase on a metal electrode. When the protein is immobilized with the CcO binding side directed toward the electrode and reconstituted in situ into a lipid bilayer, it is addressable by direct electron transfer to the redox centers. Electron transfer to the enzyme via the spacer, referred to as electronic wiring, shows an exceptionally high rate constant. This allows a kinetic analysis of all four consecutive electron transfer steps within the enzyme to be carried out. Electron transfer followed by rapid scan cyclic voltametry in combination with surface-enhanced resonance Raman spectroscopy provides mechanistic and structural information about the heme centers. Probing the enzyme under turnover conditions showed mechanistic insights into proton translocation coupled to electron transfer. This bioelectronic approach opens a new field of activity to investigate complex processes in a wide variety of membrane proteins. PMID:18222995

  8. The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition.

    Science.gov (United States)

    Wienk, Hans; Slootweg, Jack C; Speerstra, Sietske; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2013-07-01

    To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition.

  9. Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

    Directory of Open Access Journals (Sweden)

    Fabio Cattaneo

    2014-10-01

    Full Text Available G protein-coupled receptors (GPCRs are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors

  10. A novel typing method for Streptococcus pneumoniae using selected surface proteins

    Directory of Open Access Journals (Sweden)

    Arnau eDomenech

    2016-03-01

    Full Text Available The diverse pneumococcal diseases are associated with different pneumococcal lineages, or clonal complexes. Nevertheless, intra-clonal genomic variability, which influences pathogenicity, has been reported for surface virulence factors. These factors constitute the communication interface between the pathogen and its host and their corresponding genes are subjected to strong selective pressures affecting functionality and immunogenicity. First, the presence and allelic dispersion of 97 outer protein families were screened in 19 complete pneumococcal genomes. Seventeen families were deemed variable and were then examined in 216 draft genomes. This procedure allowed the generation of binary vectors with 17 positions and the classification of strains into surfotypes. They represent the outer protein subsets with the highest inter-strain discriminative power. A total of 116 non-redundant surfotypes were identified. Those sharing a critical number of common protein features were hierarchically clustered into 18 surfogroups. Most clonal complexes with comparable epidemiological characteristics belonged to the same or similar surfogroups. However, the very large CC156 clonal complex was dispersed over several surfogroups. In order to establish a relationship between surfogroup and pathogenicity, the surfotypes of 95 clinical isolates with different serogroup/serotype combinations were analysed. We found a significant correlation between surfogroup and type of pathogenic behaviour (primary invasive, opportunistic invasive and non-invasive. We conclude that the virulent behaviour of S. pneumoniae is related to the activity of collections of, rather than individual, surface virulence factors. Since surfotypes evolve faster than MLSTs and directly reflect virulence potential, this novel typing protocol is appropriate for the identification of emerging clones.

  11. Francisella tularensis LVS surface and membrane proteins as targets of effective post-exposure immunization for tularemia.

    Science.gov (United States)

    Chandler, Jeffrey C; Sutherland, Marjorie D; Harton, Marisa R; Molins, Claudia R; Anderson, Rebecca V; Heaslip, Darragh G; Bosio, Catharine M; Belisle, John T

    2015-02-01

    Francisella tularensis causes disease (tularemia) in a large number of mammals, including man. We previously demonstrated enhanced efficacy of conventional antibiotic therapy for tularemia by postexposure passive transfer of immune sera developed against a F. tularensis LVS membrane protein fraction (MPF). However, the protein composition of this immunogenic fraction was not defined. Proteomic approaches were applied to define the protein composition and identify the immunogens of MPF. MPF consisted of at least 299 proteins and 2-D Western blot analyses using sera from MPF-immunized and F. tularensis LVS-vaccinated mice coupled to liquid chromatography-tandem mass spectrometry identified 24 immunoreactive protein spots containing 45 proteins. A reverse vaccinology approach that applied labeling of F. tularensis LVS surface proteins and bioinformatics was used to reduce the complexity of potential target immunogens. Bioinformatics analyses of the immunoreactive proteins reduced the number of immunogen targets to 32. Direct surface labeling of F. tularensis LVS resulted in the identification of 31 surface proteins. However, only 13 of these were reactive with MPF and/or F. tularensis LVS immune sera. Collectively, this use of orthogonal proteomic approaches reduced the complexity of potential immunogens in MPF by 96% and allowed for prioritization of target immunogens for antibody-based immunotherapies against tularemia.

  12. Transfer of Fas (CD95 protein from the cell surface to the surface of polystyrene beads coated with anti-Fas antibody clone CH-11

    Directory of Open Access Journals (Sweden)

    H. Sawai

    2010-02-01

    Full Text Available Mouse monoclonal anti-Fas (CD95 antibody clone CH-11 has been widely used in research on apoptosis. CH-11 has the ability to bind to Fas protein on cell surface and induce apoptosis. Here, we used polystyrene beads coated with CH-11 to investigate the role of lipid rafts in Fas-mediated apoptosis in SKW6.4 cells. Unexpectedly, by treatment of the cells with CH-11-coated beads Fas protein was detached from cell surface and transferred to the surface of CH-11-coated beads. Western blot analysis showed that Fas protein containing both extracellular and intracellular domains was attached to the beads. Fas protein was not transferred from the cells to the surface of the beads coated with other anti-Fas antibodies or Fas ligand. Similar phenomenon was observed in Jurkat T cells. Furthermore, CH-11-induced apoptosis was suppressed by pretreatment with CH-11-coated beads in Jurkat cells. These results suggest that CH-11 might possess distinct properties on Fas protein compared with other anti-Fas antibodies or Fas ligand, and also suggest that caution should be needed to use polystyrene beads coated with antibodies such as CH-11.

  13. Identification of immunodominant epitopes in Trypanosoma cruzi trypomastigote surface antigen-1 protein that mask protective epitopes.

    Science.gov (United States)

    Wrightsman, R A; Dawson, B D; Fouts, D L; Manning, J E

    1994-10-01

    The gene that encodes trypomastigote surface Ag-1 (TSA-1), a major surface Ag of the bloodstream trypomastigote stage of Trypanosoma cruzi, was expressed in a baculovirus expression system. To determine the epitope(s) in TSA-1 that was recognized during T. cruzi infection and after immunization with TSA-1, subregions of the TSA-1 gene were expressed in a bacterial expression system. As seen by Western blotting, both mice and rabbits immunized with recombinant TSA-1 protein, as well as T. cruzi-infected mice, developed strong immune responses to the carboxyl-proximal region of TSA-1, but show no reaction to the amino-proximal portion of TSA-1. When mice were immunized with either recombinant TSA-1 protein or the carboxyl-proximal region of TSA-1, they did not survive challenge with 10(3) bloodstream trypomastigotes. However, 70% of the mice immunized with the amino-proximal portion of TSA-1 survived challenge with 10(3) bloodstream trypomastigotes. Thus, the immune responses elicited by recombinant TSA-1 or the carboxyl-proximal portion of TSA-1 are nonprotective during T. cruzi infection. In contrast, vaccination with the amino proximal region of TSA-1 elicits a protective immune response. These results suggest that responses to immunodominant epitope(s) within the carboxyl-proximal portion of TSA-1 mask epitopes within the amino-proximal portion that are capable of stimulating host-protective immune responses. It is suggested that immunodominant regions in surface molecules such as TSA-1 may provide a mechanism for the parasite to evade the host immune response by directing the response away from epitopes that have the potential to elicit a reaction that is damaging to the parasite.

  14. Stress-strain curves of adsorbed protein layers at the air/water interface measured with surface shear rheology

    NARCIS (Netherlands)

    Martin, A.; Bos, M.; Stuart, M.C.; Vliet, T. van

    2002-01-01

    Interfacial shear properties of adsorbed protein layers at the air/water interface were determined using a Couette-type surface shear rheometer. Such experiments are often used to determine a steady-state ratio between stress and rate of strain, which is then denoted as "surface shear viscosity". Ho

  15. ProFASTA: a pipeline web server for fungal protein scanning with integration of cell surface prediction software

    NARCIS (Netherlands)

    de Groot, P.W.J.; Brandt, B.W.

    2012-01-01

    Surface proteins, such as those located in the cell wall of fungi, play an important role in the interaction with the surrounding environment. For instance, they mediate primary host-pathogen interactions and are crucial to the establishment of biofilms and fungal infections. Surface localization of

  16. Binding characteristics of thrombin-activatable fibrinolysis inhibitor to streptococcal surface collagen-like proteins A and B

    NARCIS (Netherlands)

    Seron, Mercedes Valls; Plug, Tom; Marquart, J. Arnoud; Marx, Pauline F.; Herwald, Heiko; de Groot, Philip G.; Meijers, Joost C. M.

    2011-01-01

    Streptococcus pyogenes is the causative agent in a wide range of diseases in humans. Thrombin-activatable fibrinolysis inhibitor (TAFI) binds to collagen-like proteins ScIA and ScIB at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a transient to chronic s

  17. Hierarchical self-assembly of designed 2x2-alpha-helix bundle proteins on Au(111) surfaces

    DEFF Research Database (Denmark)

    Wackerbarth, Hainer; Tofteng, A.P.; Jensen, K.J.

    2006-01-01

    Self-assembled monolayers of biomolecules on atomically planar surfaces offer the prospect of complex combinations of controlled properties, e. g., for bioelectronics. We have prepared a novel hemi-4-alpha-helix bundle protein by attaching two alpha-helical peptides to a cyclo-dithiothreitol (cyclo...... proteins retained. The surface properties of the DTT and 2 x 2- R-helix bundle protein adlayer were characterized by interfacial capacitance and impedance techniques. Reductive desorption was used to determine the coverage of the adlayers, giving values of 65 and 16 mu C cm(-2) for DTT and 2 x 2-helix...

  18. Influence of enterococcal surface protein (esp) on the transport of Enterococcus faecium within saturated quartz sands.

    Science.gov (United States)

    Johanson, Jennifer J; Feriancikova, Lucia; Xu, Shangping

    2012-02-07

    Enterococcus was selected by US EPA as a Gram-positive indicator microorganism for groundwater fecal contamination. It was recently reported that enterococcal surface protein (esp) was more prevalent in Enterococcus from human sources than in Enterococcus from nonhuman sources and esp could potentially be used as a source tracking tool for fecal contamination (Scott et al., 2005). In this research, we performed laboratory column transport experiments to investigate the transport of Enterococcus faecium within saturated quartz sands. Particularly, we used a wild type strain (E1162) and a mutant (E1162Δesp) to examine the influence of esp on the transport behavior of E. faecium. Our results showed that esp could significantly enhance the attachment of E. faecium cells onto the surface of silica sands and thus lower the mobility of E. faecium within sand packs. Cell surface properties (e.g., zeta potential) were determined and the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory was applied to explain the effects of esp on the retention of E. faecium. Overall, our results suggested that E. faecium strains with esp could display lower mobility within saturated sand packs than E. faecium strains without esp. The disparity in the transport behavior of E. faecium with and without esp could limit the effectiveness of esp as a source tracking tool within the groundwater system.

  19. Tight attachment of chitin-binding-domain-tagged proteins to surfaces coated with acetylated chitosan.

    Science.gov (United States)

    Bernard, Michael P; Cao, Donghui; Myers, Rebecca V; Moyle, William R

    2004-04-15

    Several excellent procedures for trapping tagged proteins have been devised, but many of these are expensive, cannot be used outside a limited pH range, fail to work in the presence of chaotropic agents, or are difficult to use. The chitin binding domain (CBD) of Bacillus circulans chitinase, which binds to chitin matrices prepared from inexpensive reagents isolated from crab shells, is an alternative tag that can be used under a variety of pH and denaturing conditions. Kits based on the interaction between the CBD and the chitin beads are available commercially. Here, we show that simultaneous treatment of microtiter plates with chitosan, a deacetylated form of chitin, and acetic anhydride produces a surface-bound film of chitin that also interacts tightly with the CBD. Chitin-coated microtiter well plates captured a CBD-tagged heterodimeric human glycoprotein hormone analog directly from mammalian cell culture media, even when present in trace amounts. Binding to the surface was stable in sodium dodecylsulfate and reversed only partially at low pH or in 8M urea at 37 degrees C. This technique appears well suited to surface attachment and permits biochemical or other analyses of molecules that can be tagged with a CBD.

  20. Honeycomb-patterned films of polystyrene/poly(ethylene glycol): Preparation, surface aggregation and protein adsorption

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Highly ordered honeycomb-patterned polystyrene (PS)/poly(ethylene glycol) (PEG) films were prepared by a water-assisted method using an improved setup, which facilitated the formation of films with higher regularity, better reproducibility, and larger area of honeycomb structures. Surface aggregation of hydrophilic PEG and adsorption of bovine serum albumin (BSA) on the honeycomb-patterned films were investigated. Field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) were used to observe the surface morphologies of the films before and after being rinsed with water. As confirmed by the FESEM images and the AFM phase images, PEG was enriched in the pores and could be gradually removed by water. The adsorption of fluorescence-labeled BSA on the films was studied in visual form using laser scanning confocal microscopy. Results clearly demonstrated that the protein-resistant PEG was selectively enriched in the pores. This water-assisted method may be a latent tool to prepare honeycomb-patterned biofunctional surfaces.

  1. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    Science.gov (United States)

    Yuan, Huihui; Qian, Bin; Zhang, Wei; Lan, Minbo

    2016-02-01

    An anti-fouling surface of polyurethane (PU) film grafted with Poly(N-vinylpyrrolidone) (PVP) was prepared through surface-initiated atom transfer radical polymerization (SI-ATRP). And the polymerization time was investigated to obtain PU films with PVP brushes of different lengths. The surface properties and protein adsorption of modified PU films were evaluated. The results showed that the hydrophilicity of PU-PVP films were improved with the increase of polymerization time, which was not positive correlation with the surface roughness due to the brush structure. Additionally, the protein resistance performance was promoted when prolonging the polymerization time. The best antifouling PU-PVP (6.0 h) film reduced the adsoption level of bovine serum albumin (BSA), lysozyme (LYS), and brovin serum fibrinogen (BFG) by 93.4%, 68.3%, 85.6%, respectively, compared to the unmodified PU film. The competitive adsorption of three proteins indicated that LYS preferentially adsorbed on the modified PU film, while BFG had the lowest adsorption selectivity. And the amount of BFG on PU-PVP (6.0 h) film reduced greatly to 0.08 μg/cm2, which was almost one-tenth of its adsorption from the single-protein system. Presented results suggested that both hydrophilicity and surface roughness might be the important factors in all cases of protein adsorption, and the competitive or selective adsorption might be related to the size of the proteins, especially on the non-charged films.

  2. Surface functionalization of liposomes with proteins and carbohydrates for use in anti-cancer applications

    Science.gov (United States)

    Platt, Virginia M.

    Liposomes can be used to exploit the altered biology of cancer thereby increasing delivery of liposome-associated anti-cancer drugs. In this dissertation, I explore methods that utilize the unique cancer expression of the polymeric glycosaminoglycan hyaluronan (HA) and the HA receptor CD44 to target liposomes to tumors, using liposomes functionalized with proteins or oligosaccharides on their surface. To make it easier to prepare protein-functionalized liposomes, a non-covalent protein/liposome association method based upon metal chelation/his 6 interaction was devised and characterized. I evaluated non-covalent attachment of the prodrug converting enzyme yeast cytosine deaminase, the far-red fluorescent protein mKate, two antigens ovalbumin and the membrane proximal region of an HIV GAG and hyaluronidase, a HA-degrading enzyme. In Chapter 2, I describe the synthesis of hyaluronan-oligosaccharide (HA-O) lipid conjugates and their incorporation into liposomes to target CD44-overexpressing cancer cells. HA-O ligands of defined-length, up to 10 monosaccharides, were attached to lipids via various linkers by reductive amination. The HA-lipids were easily incorporated into liposomes but did not mediate binding of liposomes to CD44 overexpressing cells. In Chapter 3, I evaluate the capacity of tris-NTA-Ni-lipids incorporated within a liposome bilayer to associate with his6-tagged proteins. Tris-NTA-lipids of differing structures and avidities were used to associate yeast cytosine deaminase and mKate to the surface of liposomes. Two tris-NTA-lipids and a mono-NTA lipid associated his-tagged proteins to a 1:1 molar ratio in solution. The proteins remained active while associated with the liposome surface. When challenged in vitro with fetal calf serum, tris-NTA-containing liposomes retained his-tagged proteins longer than mono-NTA. However, the tris-NTA/his6 interaction was found to be in a dynamic state; free yeast cytosine deaminase rapidly competed with pre-bound m

  3. Surface layer protein characterization by small angle x-ray scattering and a fractal mean force concept: From protein structure to nanodisk assemblies

    Science.gov (United States)

    Horejs, Christine; Pum, Dietmar; Sleytr, Uwe B.; Peterlik, Herwig; Jungbauer, Alois; Tscheliessnig, Rupert

    2010-11-01

    Surface layers (S-layers) are the most commonly observed cell surface structure of prokaryotic organisms. They are made up of proteins that spontaneously self-assemble into functional crystalline lattices in solution, on various solid surfaces, and interfaces. While classical experimental techniques failed to recover a complete structural model of an unmodified S-layer protein, small angle x-ray scattering (SAXS) provides an opportunity to study the structure of S-layer monomers in solution and of self-assembled two-dimensional sheets. For the protein under investigation we recently suggested an atomistic structural model by the use of molecular dynamics simulations. This structural model is now refined on the basis of SAXS data together with a fractal assembly approach. Here we show that a nondiluted critical system of proteins, which crystallize into monomolecular structures, might be analyzed by SAXS if protein-protein interactions are taken into account by relating a fractal local density distribution to a fractal local mean potential, which has to fulfill the Poisson equation. The present work demonstrates an important step into the elucidation of the structure of S-layers and offers a tool to analyze the structure of self-assembling systems in solution by means of SAXS and computer simulations.

  4. Evaluation of the effects of Streptococcus mutans chaperones and protein secretion machinery components on cell surface protein biogenesis, competence, and mutacin production.

    Science.gov (United States)

    Crowley, P J; Brady, L J

    2016-02-01

    The respective contributions of components of the protein translocation/maturation machinery to cell surface biogenesis in Streptococcus mutans are not fully understood. Here we used a genetic approach to characterize the effects of deletion of genes encoding the ribosome-associated chaperone RopA (Trigger Factor), the surface-localized foldase PrsA, and the membrane-localized chaperone insertases YidC1 and YidC2, both singly and in combination, on bacterial growth, chain length, self-aggregation, cell surface hydrophobicity, autolysis, and antigenicity of surface proteins P1 (AgI/II, PAc), WapA, GbpC, and GtfD. The single and double deletion mutants, as well as additional mutant strains lacking components of the signal recognition particle pathway, were also evaluated for their effects on mutacin production and genetic competence.

  5. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Huihui; Qian, Bin; Zhang, Wei [Shanghai Key Laboratory of Functional Materials Chemistry and Research Center of Analysis and Test, East China University of Science and Technology, Shanghai 200237 (China); Lan, Minbo, E-mail: minbolan@ecust.edu.cn [Shanghai Key Laboratory of Functional Materials Chemistry and Research Center of Analysis and Test, East China University of Science and Technology, Shanghai 200237 (China); State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China)

    2016-02-15

    Highlights: • Antifouling PVP brushes were successfully grafted on PU films by SI-ATRP. • The effect of polymerization time on surface property and topography was studied. • Hydrophilicity and protein fouling resistance of PVP–PU films were greatly promoted. • Competitive adsorption of three proteins on PVP–PU films was evaluated. - Abstract: An anti-fouling surface of polyurethane (PU) film grafted with Poly(N-vinylpyrrolidone) (PVP) was prepared through surface-initiated atom transfer radical polymerization (SI-ATRP). And the polymerization time was investigated to obtain PU films with PVP brushes of different lengths. The surface properties and protein adsorption of modified PU films were evaluated. The results showed that the hydrophilicity of PU–PVP films were improved with the increase of polymerization time, which was not positive correlation with the surface roughness due to the brush structure. Additionally, the protein resistance performance was promoted when prolonging the polymerization time. The best antifouling PU–PVP (6.0 h) film reduced the adsoption level of bovine serum albumin (BSA), lysozyme (LYS), and brovin serum fibrinogen (BFG) by 93.4%, 68.3%, 85.6%, respectively, compared to the unmodified PU film. The competitive adsorption of three proteins indicated that LYS preferentially adsorbed on the modified PU film, while BFG had the lowest adsorption selectivity. And the amount of BFG on PU–PVP (6.0 h) film reduced greatly to 0.08 μg/cm{sup 2}, which was almost one-tenth of its adsorption from the single-protein system. Presented results suggested that both hydrophilicity and surface roughness might be the important factors in all cases of protein adsorption, and the competitive or selective adsorption might be related to the size of the proteins, especially on the non-charged films.

  6. Analysis of novel iron-regulated, surface-anchored hemin-binding proteins in Corynebacterium diphtheriae.

    Science.gov (United States)

    Allen, Courtni E; Burgos, Jonathan M; Schmitt, Michael P

    2013-06-01

    Corynebacterium diphtheriae utilizes hemin and hemoglobin (Hb) as iron sources during growth in iron-depleted environments, and recent studies have shown that the surface-exposed HtaA protein binds both hemin and Hb and also contributes to the utilization of hemin iron. Conserved (CR) domains within HtaA and in the associated hemin-binding protein, HtaB, are required for the ability to bind hemin and Hb. In this study, we identified and characterized two novel genetic loci in C. diphtheriae that encode factors that bind hemin and Hb. Both genetic systems contain two-gene operons that are transcriptionally regulated by DtxR and iron. The gene products of these operons are ChtA-ChtB and ChtC-CirA (previously DIP0522-DIP0523). The chtA and chtB genes are carried on a putative composite transposon associated with C. diphtheriae isolates that dominated the diphtheria outbreak in the former Soviet Union in the 1990s. ChtA and ChtC each contain a single N-terminal CR domain and exhibit significant sequence similarity to each other but only limited similarity with HtaA. The chtB and htaB gene products exhibited a high level of sequence similarity throughout their sequences, and both proteins contain a single CR domain. Whole-cell binding studies as well as protease analysis indicated that all four of the proteins encoded by these two operons are surface exposed, which is consistent with the presence of a transmembrane segment in their C-terminal regions. ChtA, ChtB, and ChtC are able to bind hemin and Hb, with ChtA showing the highest affinity. Site-directed mutagenesis showed that specific tyrosine residues within the ChtA CR domain were critical for hemin and Hb binding. Hemin iron utilization assays using various C. diphtheriae mutants indicate that deletion of the chtA-chtB region and the chtC gene has no affect on the ability of C. diphtheriae to use hemin or Hb as iron sources; however, a chtB htaB double mutant exhibits a significant decrease in hemin iron use

  7. A Surface Biotinylation Strategy for Reproducible Plasma Membrane Protein Purification and Tracking of Genetic and Drug-Induced Alterations.

    Science.gov (United States)

    Hörmann, Katrin; Stukalov, Alexey; Müller, André C; Heinz, Leonhard X; Superti-Furga, Giulio; Colinge, Jacques; Bennett, Keiryn L

    2016-02-01

    Plasma membrane (PM) proteins contribute to the identity of a cell, mediate contact and communication, and account for more than two-thirds of known drug targets.1-8 In the past years, several protocols for the proteomic profiling of PM proteins have been described. Nevertheless, comparative analyses have mainly focused on different variations of one approach.9-11 We compared sulfo-NHS-SS-biotinylation, aminooxy-biotinylation, and surface coating with silica beads to isolate PM proteins for subsequent analysis by one-dimensional gel-free liquid chromatography mass spectrometry. Absolute and relative numbers of PM proteins and reproducibility parameters on a qualitative and quantitative level were assessed. Sulfo-NHS-SS-biotinylation outperformed aminooxy-biotinylation and surface coating using silica beads for most of the monitored criteria. We further simplified this procedure by a competitive biotin elution strategy achieving an average PM annotated protein fraction of 54% (347 proteins). Computational analysis using additional databases and prediction tools revealed that in total over 90% of the purified proteins were associated with the PM, mostly as interactors. The modified sulfo-NHS-SS-biotinylation protocol was validated by tracking changes in the plasma membrane proteome composition induced by genetic alteration and drug treatment. Glycosylphosphatidylinositol (GPI)-anchored proteins were depleted in PM purifications from cells deficient in the GPI transamidase component PIGS, and treatment of cells with tunicamycin significantly reduced the abundance of N-glycoproteins in surface purifications.

  8. The effect of physiological conditions on the surface structure of proteins: Setting the scene for human digestion of emulsions

    Science.gov (United States)

    Maldonado-Valderrama, J.; Gunning, A. P.; Ridout, M. J.; Wilde, P. J.; Morris, V. J.

    2009-10-01

    Understanding and manipulating the interfacial mechanisms that control human digestion of food emulsions is a crucial step towards improved control of dietary intake. This article reports initial studies on the effects of the physiological conditions within the stomach on the properties of the film formed by the milk protein ( β -lactoglobulin) at the air-water interface. Atomic force microscopy (AFM), surface tension and surface rheology techniques were used to visualize and examine the effect of gastric conditions on the network structure. The effects of changes in temperature, pH and ionic strength on a pre-formed interfacial structure were characterized in order to simulate the actual digestion process. Changes in ionic strength had little effect on the surface properties. In isolation, acidification reduced both the dilatational and the surface shear modulus, mainly due to strong repulsive electrostatic interactions within the surface layer and raising the temperature to body temperature accelerated the rearrangements within the surface layer, resulting in a decrease of the dilatational response and an increase of surface pressure. Together pH and temperature display an unexpected synergism, independent of the ionic strength. Thus, exposure of a pre-formed interfacial β -lactoglobulin film to simulated gastric conditions reduced the surface dilatational modulus and surface shear moduli. This is attributed to a weakening of the surface network in which the surface rearrangements of the protein prior to exposure to gastric conditions might play a crucial role.

  9. Fructose 1,6-Bisphosphate Aldolase, a Novel Immunogenic Surface Protein on Listeria Species

    Science.gov (United States)

    Conceição, Fabricio Rochedo; Hust, Michael; Mendonça, Karla Sequeira; Moreira, Ângela Nunes; França, Rodrigo Correa; da Silva, Wladimir Padilha; Aleixo, José Antonio G.

    2016-01-01

    Listeria monocytogenes is a ubiquitous food-borne pathogen, and its presence in food or production facilities highlights the importance of surveillance. Increased understanding of the surface exposed antigens on Listeria would provide potential diagnostic and therapeutic targets. In the present work, using mass spectrometry and genetic cloning, we show that fructose-1,6-bisphosphate aldolase (FBA) class II in Listeria species is the antigen target of the previously described mAb-3F8. Western and dot blot assays confirmed that the mAb-3F8 could distinguish all tested Listeria species from close-related bacteria. Localization studies indicated that FBA is present in every fraction of Listeria cells, including supernatant and the cell wall, setting Listeria spp. as one of the few bacteria described to have this protein on their cell surface. Epitope mapping using ORFeome display and a peptide membrane revealed a 14-amino acid peptide as the potential mAb-3F8 epitope. The target epitope in FBA allowed distinguishing Listeria spp. from closely-related bacteria, and was identified as part of the active site in the dimeric enzyme. However, its function in cell surface seems not to be host cell adhesion-related. Western and dot blot assays further demonstrated that mAb-3F8 together with anti-InlA mAb-2D12 could differentiate pathogenic from non-pathogenic Listeria isolated from artificially contaminated cheese. In summary, we report FBA as a novel immunogenic surface target useful for the detection of Listeria genus. PMID:27489951

  10. The effect of amorphous silicon surface hydrogenation on morphology, wettability and its implication on the adsorption of proteins

    Science.gov (United States)

    Filali, Larbi; Brahmi, Yamina; Sib, Jamal Dine; Bouhekka, Ahmed; Benlakehal, Djamel; Bouizem, Yahya; Kebab, Aissa; Chahed, Larbi

    2016-10-01

    We study the effect of amorphous silicon (a-Si) surface hydrogenation on Bovine Serum Albumin (BSA) adsorption. A set of (a-Si) films was prepared by radio frequency magnetron sputtering (RFMS) and after deposition; they were treated in molecular hydrogen ambient at different pressures (1-3 Pa). Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy and spectroscopic ellipsometry (SE) were used to study the hydrogenation effect and BSA adsorption. Atomic force microscopy (AFM) was used to evaluate morphological changes caused by hydrogenation. The wettability of the films was measured using contact angle measurement, and in the case of the hydrogenated surfaces, it was found to be driven by surface roughness. FTIR-ATR spectroscopy and SE measurements show that proteins had the strongest affinity toward the surfaces with the highest hydrogen content and their secondary structure was affected by a significant decrease of the α-helix component (-27%) compared with the proteins adsorbed on the un-treated surface, which had a predominantly α-helix (45%) structure. The adsorbed protein layer was found to be densely packed with a large thickness (30.9 nm) on the hydrogen-rich surfaces. The most important result is that the surface hydrogen content was the dominant factor, compared to wettability and morphology, for protein adsorption.

  11. Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria.

    Science.gov (United States)

    Hooda, Yogesh; Lai, Christine Chieh-Lin; Judd, Andrew; Buckwalter, Carolyn M; Shin, Hyejin Esther; Gray-Owen, Scott D; Moraes, Trevor F

    2016-02-29

    Lipoproteins decorate the surface of many Gram-negative bacterial pathogens, playing essential roles in immune evasion and nutrient acquisition. In Neisseria spp., the causative agents of gonorrhoea and meningococcal meningitis, surface lipoproteins (SLPs) are required for virulence and have been extensively studied as prime candidates for vaccine development. However, the machinery and mechanism that allow for the surface display of SLPs are not known. Here, we describe a transposon (Tn5)-based search for the proteins required to deliver SLPs to the surface of Neisseria meningitidis, revealing a family of proteins that we have named the surface lipoprotein assembly modulator (Slam). N. meningitidis contains two Slam proteins, each exhibiting distinct substrate preferences. The Slam proteins are sufficient to reconstitute SLP transport in laboratory strains of Escherichia coli, which are otherwise unable to efficiently display these lipoproteins on their cell surface. Immunoprecipitation and domain probing experiments suggest that the SLP, TbpB, interacts with Slam during the transit process; furthermore, the membrane domain of Slam is sufficient for selectivity and proper surface display of SLPs. Rather than being a Neisseria-specific factor, our bioinformatic analysis shows that Slam can be found throughout proteobacterial genomes, indicating a conserved but until now unrecognized virulence mechanism.

  12. Hydrogel Layers on the Surface of Polyester-Based Materials for Improvement of Their Biointeractions and Controlled Release of Proteins

    Directory of Open Access Journals (Sweden)

    Viktor Korzhikov-Vlakh

    2016-12-01

    Full Text Available The modification of bioresorbable polyester surfaces in order to alter their biointeractions presents an important problem in biomedical polymer science. In this study, the covalent modification of the surface of poly(lactic acid-based (PLA-based films with poly(acryl amide and sodium alginate hydrogels was performed to change the non-specific polyester interaction with proteins and cells, as well as to make possible the covalent attachment of low-molecular weight ligands and to control protein release. The effect of such modification on the film surface properties was studied. Parameters such as swelling, water contact angle, surface area, and binding capacity of low-molecular weight substances were evaluated and compared. The comparative study of adsorption of model protein (BSA on the surface of non-modified and modified films was investigated and the protein release was evaluated. Cell viability on the surface of hydrogel-coated films was also tested. The developed approach could be applied for the modification of PLA-based scaffolds for tissue engineering and will be further studied for molecular-imprinting of biomolecules on the surface of polyester-based materials for control of biointeractions.

  13. Protein adsorption resistant surface on polymer composite based on 2D- and 3D-controlled grafting of phospholipid moieties

    Energy Technology Data Exchange (ETDEWEB)

    Hoshi, Toru [Department of Materials Engineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Matsuno, Ryosuke [Department of Materials Engineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Center for NanoBio Integration, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Sawaguchi, Takashi [Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda-surugadai, Chiyoda-ku, Tokyo 101-8308 (Japan); Konno, Tomohiro; Takai, Madoka [Department of Materials Engineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Center for NanoBio Integration, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Ishihara, Kazuhiko [Department of Materials Engineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Department of Bioengineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan); Center for NanoBio Integration, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan)], E-mail: ishihara@mpc.t.u-tokyo.ac.jp

    2008-11-15

    To prepare the biocompatible surface, a phosphorylcholine (PC) group was introduced on this hydroxyl group generated by surface hydrolysis on the polymer composite composed of polyethylene (PE) and poly (vinyl acetate) (PVAc) prepared by supercritical carbon dioxide. Two different procedures such as two-dimensional (2D) modification and three-dimensional (3D) modification were applied to obtain the steady biocompatible surface. 2D modification was that PC groups were directly anchored on the surface of the polymer composite. 3D modification was that phospholipid polymer was grafted from the surface of the polymer composite by surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-methacryloyloxyethyl phosphorylcholine (MPC). The surfaces were characterized by X-ray photoelectron spectroscopy, dynamic water contact angle measurements, and atomic force microscope. The effects of the poly(MPC) chain length on the protein adsorption resistivity were investigated. The protein adsorption on the polymer composite surface with PC groups modified by 2D or 3D modification was significantly reduced as compared with that on the unmodified PE. Further, the amount of protein adsorbed on the 3D modified surface that is poly(MPC)-grafted surface decreased with an increase in the chain length of the poly(MPC). The surface with an arbitrary structure and the characteristic can be constructed by using 2D and 3D modification. We conclude that the polymer composites of PE/PVAc with PC groups on the surface are useful for fabricating biomedical devices due to their good mechanical and surface properties.

  14. Screening of hepatocyte proteins binding with the middle surface protein of the hepatitis B virus by the yeast two-hybrid system.

    Science.gov (United States)

    Li, Zhiqun; Linghu, Enqiang; Cheng, Jun

    2014-06-01

    The effect of the middle hepatitis B virus surface protein (MHBs) remains to be elucidated. To investigate the biological function of the MHBs protein, the present study performed yeast two-hybrid screening to search for proteins that interact with the MHBs protein in hepatocytes. The bait plasmid expressing the MHBs protein was constructed by cloning the gene of the MHBs protein into pGBKT7, then the recombinant plasmid DNA was transformed into AH109 yeast (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing the liver cDNA library plasmid in 2X yeast peptone dextrose adenine (YPDA) medium. The mated diploid yeast was plated on quadruple dropout medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. Following extracting and sequencing of the plasmids from positive (blue) colonies, the sequence analysis was conducted and analyzed by bioinformatics methods. Two colonies were selected and sequenced. Among them, one was the human DNA sequence from the clone RP11-490D19 on chromosome 9 and the other was homo sapiens 12 BAC RP11-180M15 (Roswell Park Cancer Institute Human BAC Library). The yeast two-hybrid system is an effective method for identifying hepatocyte proteins that interact with MHBs. The MHBs protein binds with different proteins suggesting that it has multiple functions in vivo.

  15. Murein hydrolase activity of surface layer proteins from Lactobacillus acidophilus against Escherichia coli.

    Science.gov (United States)

    Meng, Jun; Gao, Shu-Ming; Zhang, Qiu-Xiang; Lu, Rong-Rong

    2015-08-01

    The aim of this study was to investigate the murein hydrolase activities of the surface layer proteins (SLPs) from two strains of Lactobacillus acidophilus using zymography. The influence of these hydrolase activities on Escherichia coli ATCC 43893 was also evaluated by analysing their growth curve, cell morphology and physiological state. After the incubation of E. coli with SLPs, growth was inhibited, the number of viable cells was significantly reduced, examination by transmission electron microscopy showed that the cell wall was damaged and flow cytometry results indicated that the majority of the cells were sublethally injured. All of these results suggested that the SLPs of both L. acidophilus strains possessed murein hydrolase activities that were sublethal to E. coli cells.

  16. Contribution of the enterococcal surface protein Esp to pathogenesis of Enterococcus faecium endocarditis.

    Science.gov (United States)

    Heikens, Esther; Singh, Kavindra V; Jacques-Palaz, Karen D; van Luit-Asbroek, Miranda; Oostdijk, Evelien A N; Bonten, Marc J M; Murray, Barbara E; Willems, Rob J L

    2011-12-01

    The enterococcal surface protein Esp, specifically linked to nosocomial Enterococcus faecium, is involved in biofilm formation. To assess the role of Esp in endocarditis, a biofilm-associated infection, an Esp-expressing E. faecium strain (E1162) or its Esp-deficient mutant (E1162Δesp) were inoculated through a catheter into the left ventricle of rats. After 24 h, less E1162Δesp than E1162 were recovered from heart valve vegetations. In addition, anti-Esp antibodies were detected in Esp-positive E. faecium bacteremia and endocarditis patient sera. In conclusion, Esp contributes to colonization of E. faecium at the heart valves. Furthermore, systemic infection elicits an Esp-specific antibody response in humans.

  17. Magnetic molecularly imprinted polydopamine nanolayer on multiwalled carbon nanotubes surface for protein capture.

    Science.gov (United States)

    Yin, Yuli; Yan, Liang; Zhang, Zhaohui; Wang, Jing

    2015-11-01

    A novel, facile and low cost process for imprinting protein on the surface of magnetic multiwalled carbon nanotubes (MMWNTs) was developed using human serum albumin (HSA) as the template and dopamine as the functional monomer. The magnetic imprinted polymers were characterized with transmission electron microscope (TEM), scanning electron microscope (SEM), Fourier-transform infrared spectrometry (FT-IR), vibrating sample magnetometer (VSM) and thermogravimetric analysis (TGA) in detail. The maximum adsorption capacity of the magnetic imprinted polymers toward HSA was 66.23 mg g(-1) and it took 20 min to achieve the adsorption equilibrium. The magnetic imprinted polymers exhibited the specific selective adsorption toward HSA. Coupled with high performance liquid chromatography (HPLC) analysis, the magnetic imprinted polymers were used to solid-phase extract and detect HSA in urine samples successfully with the recoveries of 91.95-97.8%.

  18. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography Using Surface Acoustic Waves.

    Science.gov (United States)

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela H; French, Jarrod B; Huang, Tony Jun

    2015-06-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming, a surface acoustic wave-based method for manipulating and patterning crystals is developed. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and submicrometer-sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but will also make it possible to collect data on samples that were previously intractable.

  19. Mussel Coating Protein-Derived Complex Coacervates Mitigate Frictional Surface Damage.

    Science.gov (United States)

    Miller, Dusty Rose; Das, Saurabh; Huang, Kuo-Ying; Han, Songi; Israelachvili, Jacob N; Waite, J Herbert

    2015-11-09

    The role of friction in the functional performance of biomaterial interfaces is widely reckoned to be critical and complicated but poorly understood. To better understand friction forces, we investigated the natural adaptation of the holdfast or byssus of mussels that live in high-energy surf habitats. As the outermost covering of the byssus, the cuticle deserves particular attention for its adaptations to frictional wear under shear. In this study, we coacervated one of three variants of a key cuticular component, mussel foot protein 1, mfp-1 [(1) Mytilus californianus mcfp-1, (2) rmfp-1, and (3) rmfp-1-Dopa], with hyaluronic acid (HA) and investigated the wear protection capabilities of these coacervates to surfaces (mica) during shear. Native mcfp-1/HA coacervates had an intermediate coefficient of friction (μ ∼0.3) but conferred excellent wear protection to mica with no damage from applied loads, F⊥, as high as 300 mN (pressure, P, > 2 MPa). Recombinant rmfp-1/HA coacervates exhibited a comparable coefficient of friction (μ ∼0.3); however, wear protection was significantly inferior (damage at F⊥ > 60 mN) compared with that of native protein coacervates. Wear protection of rmfp-1/HA coacervates increased 5-fold upon addition of the surface adhesive group 3,4-dihydroxyphenylalanine, (Dopa). We propose a Dopa-dependent wear protection mechanism to explain the differences in wear protection between coacervates. Our results reveal a significant untapped potential for coacervates in applications that require adhesion, lubrication, and wear protection. These applications include artificial joints, contact lenses, dental sealants, and hair and skin conditioners.

  20. Campylobacter fetus surface layer proteins are transported by a type I secretion system.

    Science.gov (United States)

    Thompson, S A; Shedd, O L; Ray, K C; Beins, M H; Jorgensen, J P; Blaser, M J

    1998-12-01

    The virulence of Campylobacter fetus, a bacterial pathogen of ungulates and humans, is mediated in part by the presence of a paracrystalline surface layer (S-layer) that confers serum resistance. The subunits of the S-layer are S-layer proteins (SLPs) that are secreted in the absence of an N-terminal signal sequence and attach to either type A or B C. fetus lipopolysaccharide in a serospecific manner. Antigenic variation of multiple SLPs (encoded by sapA homologs) of type A strain 23D occurs by inversion of a promoter-containing DNA element flanked by two sapA homologs. Cloning and sequencing of the entire 6.2-kb invertible region from C. fetus 23D revealed a probable 5.6-kb operon of four overlapping genes (sapCDEF, with sizes of 1,035, 1,752, 1,284, and 1,302 bp, respectively) transcribed in the opposite direction from sapA. The four genes also were present in the invertible region of type B strain 84-107 and were virtually identical to their counterparts in the type A strain. Although SapC had no database homologies, SapD, SapE, and SapF had predicted amino acid homologies with type I protein secretion systems (typified by Escherichia coli HlyBD/TolC or Erwinia chrysanthemi PrtDEF) that utilize C-terminal secretion signals to mediate the secretion of hemolysins, leukotoxins, or proteases from other bacterial species. Analysis of the C termini of four C. fetus SLPs revealed conserved structures that are potential secretion signals. A C. fetus sapD mutant neither produced nor secreted SLPs. E. coli expressing C. fetus sapA and sapCDEF secreted SapA, indicating that the sapCDEF genes are sufficient for SLP secretion. C. fetus SLPs therefore are transported to the cell surface by a type I secretion system.

  1. Mussel Coating Protein-Derived Complex Coacervates Mitigate Frictional Surface Damage

    Science.gov (United States)

    2015-01-01

    The role of friction in the functional performance of biomaterial interfaces is widely reckoned to be critical and complicated but poorly understood. To better understand friction forces, we investigated the natural adaptation of the holdfast or byssus of mussels that live in high-energy surf habitats. As the outermost covering of the byssus, the cuticle deserves particular attention for its adaptations to frictional wear under shear. In this study, we coacervated one of three variants of a key cuticular component, mussel foot protein 1, mfp-1 [(1) Mytilus californianus mcfp-1, (2) rmfp-1, and (3) rmfp-1-Dopa], with hyaluronic acid (HA) and investigated the wear protection capabilities of these coacervates to surfaces (mica) during shear. Native mcfp-1/HA coacervates had an intermediate coefficient of friction (μ ∼0.3) but conferred excellent wear protection to mica with no damage from applied loads, F⊥, as high as 300 mN (pressure, P, > 2 MPa). Recombinant rmfp-1/HA coacervates exhibited a comparable coefficient of friction (μ ∼0.3); however, wear protection was significantly inferior (damage at F⊥ > 60 mN) compared with that of native protein coacervates. Wear protection of rmfp-1/HA coacervates increased 5-fold upon addition of the surface adhesive group 3,4-dihydroxyphenylalanine, (Dopa). We propose a Dopa-dependent wear protection mechanism to explain the differences in wear protection between coacervates. Our results reveal a significant untapped potential for coacervates in applications that require adhesion, lubrication, and wear protection. These applications include artificial joints, contact lenses, dental sealants, and hair and skin conditioners. PMID:26618194

  2. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Haizhen; Brown, Roslyn N.; Qian, Weijun; Monroe, Matthew E.; Purvine, Samuel O.; Moore, Ronald J.; Gritsenko, Marina A.; Shi, Liang; Romine, Margaret F.; Fredrickson, Jim K.; Pasa-Tolic, Ljiljana; Smith, Richard D.; Lipton, Mary S.

    2010-05-03

    We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope 18O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and environmental electron receptors. LC/MS/MS analysis resulted in the identification of about 79% membrane proteins among all proteins identified from the enriched sample. To illustrate the quantification of membrane proteome changes, enriched membrane protein samples from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) were further labeled with 16O and 18O at the peptide level prior to LC-MS analysis. A chemical-probe-labeled pure protein has also been used as an internal standard for normalization purpose. The quantitative data revealed reduced abundances of many outer membrane proteins such as OmcA and MtrC in ΔgspD mutant cells, which agreed well with previously published studies.

  3. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling

    Science.gov (United States)

    Zhang, Haizhen; Brown, Roslyn N.; Qian, Wei-Jun; Monroe, Matthew E.; Purvine, Samuel O.; Moore, Ronald J.; Gritsenko, Marina A.; Shi, Liang; Romine, Margaret F; Fredrickson, James K.; Paša-Tolić, Ljiljana; Smith, Richard D.; Lipton, Mary S.

    2010-01-01

    We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope 18O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level 16O and 18O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in ΔgspD mutant cells of many outer membrane proteins including the outer membrane c-cype cytochromes OmcA and MtrC, in agreement with previously investigation demonstrating that these proteins are substrates of the type II secretion system. PMID:20380418

  4. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling.

    Science.gov (United States)

    Zhang, Haizhen; Brown, Roslyn N; Qian, Wei-Jun; Monroe, Matthew E; Purvine, Samuel O; Moore, Ronald J; Gritsenko, Marina A; Shi, Liang; Romine, Margaret F; Fredrickson, James K; Pasa-Tolić, Ljiljana; Smith, Richard D; Lipton, Mary S

    2010-05-07

    We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope (18)O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a Gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level (16)O and (18)O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in Delta gspD mutant cells of many outer membrane proteins including the outer membrane c-type cytochromes OmcA and MtrC, in agreement with a previous report that these proteins are substrates of the type II secretion system.

  5. Adsorption of bovine serum albumin on CoCrMo surface: effect of temperature and protein concentration.

    Science.gov (United States)

    Valero Vidal, C; Olmo Juan, A; Igual Muñoz, A

    2010-10-01

    The adsorption of bovine serum albumin (BSA) onto CoCrMo surface has been studied as a function of concentration of BSA and temperature by electrochemical techniques. The electrochemical impedance spectroscopy (EIS) technique was used to investigate the interfacial behaviour of BSA at open circuit potential (OCP). The charge transfer resistance was very sensitive to the amount of adsorbed protein, indicating that the adsorption process was accompanied by the transfer of charge and influenced the mechanism and kinetics of the corrosion reaction. At all the temperatures studied, adsorption of BSA onto the CoCrMo surface was successfully described with a Langmuir adsorption isotherm. EIS study was also carried out for determine the surface charge density, resulting from protein adsorption, and it was shown to be directly proportional to the amount of adsorbed protein (surface concentration). Thermodynamic data of adsorption was obtained for analyzing the adsorption of BSA onto CoCrMo surface. Gibbs free energy of adsorption, DeltaG(ADS) values, for BSA in the investigated temperature range (-51kJmol(-1)) showed that the molecules have a strong affinity for the CoCrMo surface. Enthalpy (DeltaH(ADS)) and entropy (DeltaS(ADS)) of adsorption suggested that the adsorption process of BSA onto the CoCrMo surface is an endothermic process and the molecule suffers structural changes when adsorbing on the metallic surface.

  6. Biopolymer stochastic moments. I. Modeling human rhinovirus cellular recognition with protein surface electrostatic moments.

    Science.gov (United States)

    González-Díaz, Humberto; Uriarte, Eugenio

    2005-04-05

    Stochastic moments may be applied as molecular descriptors in quantitative structure-activity relationship (QSAR) studies for small molecules (H. González-Dìaz et al., Journal of Molecular Modeling, 2002, Vol. 8, pp. 237-245; 2003, Vol. 9, pp. 395-407). However, applications in the field of biopolymers are less known. Recently, the MARCH-INSIDE approach has been generalized to encode structural features of proteins and other biopolymers (H. González-Dáaz et al., Bioinformatics, 2003, Vol. 19, pp. 2079-2087; Bioorganic & Medicinal Chemistry Letters, 2004, Vol. 14, pp. 4691-4695; Polymers, 2004, Vol. 45, pp. 3845-3853; Bioorganic & Medicinal Chemistry, 2005, Vol. 13, pp. 323-331). The present article attempts to extend this research by introducing for the first time stochastic moments for a surface road map of viral proteins. These moments are afterward used to seek a model that predicts the cellular receptor for human rhinoviruses. The model correctly classified 100% of 10 viruses binding to low-density lipoprotein receptor (LDLR) and 88.9% of 9 viruses binding to the intracellular adhesion molecule (ICAM) receptors in training. The same results have been obtained in four cross-validation experiments using a resubstitution technique. The present model favorably compares, in terms of complexity, with other previously reported based on entropy considerations, and offers a quantitative basis for the visual rule previously reported by Vlasak et al.

  7. Staphylococcus saprophyticus surface-associated protein (Ssp) is associated with lifespan reduction in Caenorhabditis elegans.

    Science.gov (United States)

    Szabados, Florian; Mohner, Amelie; Kleine, Britta; Gatermann, Sören G

    2013-10-01

    Staphylococcal lipases have been proposed as pathogenicity factors. In Staphylococcus saprophyticus the surface-associated protein (Ssp) has been previously characterized as a cell wall-associated true lipase. A S. saprophyticus Δssp::ermB mutant has been described as less virulent in an in vivo model of urinary tract infection compared with its wild-type. This is the first report showing that S. saprophyticus induced a lifespan reduction in Caenorhabditis elegans similar to that of S. aureus RN4220. In two S. saprophyticus Δssp::ermB mutants lifespan reduction in C. elegans was partly abolished. In order to attribute virulence to the lipase activity itself and distinguish this phenomenon from the presence of the Ssp-protein, the conserved active site of the lipase was modified by site-directed ligase-independent mutagenesis and lipase activity-deficient mutants were constructed. These results indicate that the Ssp is associated with pathogenicity in C. elegans and one could speculate that the lipase activity itself is responsible for this virulence.

  8. Intentional formation of a protein corona on nanoparticles: Serum concentration affects protein corona mass, surface charge, and nanoparticle-cell interaction.

    Science.gov (United States)

    Gräfe, Christine; Weidner, Andreas; Lühe, Moritz V D; Bergemann, Christian; Schacher, Felix H; Clement, Joachim H; Dutz, Silvio

    2016-06-01

    The protein corona, which immediately is formed after contact of nanoparticles and biological systems, plays a crucial role for the biological fate of nanoparticles. In the here presented study we describe a strategy to control the amount of corona proteins which bind on particle surface and the impact of such a protein corona on particle-cell interactions. For corona formation, polyethyleneimine (PEI) coated magnetic nanoparticles (MNP) were incubated in a medium consisting of fetal calf serum (FCS) and cell culture medium. To modulate the amount of proteins bind to particles, the composition of the incubation medium was varied with regard to the FCS content. The protein corona mass was estimated and the size distribution of the participating proteins was determined by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, the zeta potential of incubated particles was measured. Human blood-brain barrier-representing cell line HBMEC was used for in vitro incubation experiments. To investigate the consequences of the FCS dependent protein corona formation on the interaction of MNP and cells flow cytometry and laser scanning microscopy were used. Zeta potential as well as SDS-PAGE clearly reveal an increase in the amount of corona proteins on MNP with increasing amount of FCS in incubation medium. For MNP incubated with lower FCS concentrations especially medium-sized proteins of molecular weights between 30kDa and 100kDa could be found within the protein corona, whereas for MNP incubated within higher FCS concentrations the fraction of corona proteins of 30kDa and less increased. The presence of the protein corona reduces the interaction of PEI-coated MNP with HBMEC cells within a 30min-incubation.

  9. Species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of Porphyromonas gingivalis HmuY

    Directory of Open Access Journals (Sweden)

    Ciuraszkiewicz Justyna

    2010-05-01

    Full Text Available Abstract Background Porphyromonas gingivalis is a major etiological agent of chronic periodontitis. The aim of this study was to examine the species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of the P. gingivalis heme-binding protein HmuY. Results HmuY is a unique protein of P. gingivalis since only low amino-acid sequence homology has been found to proteins encoded in other species. It is exposed on the cell surface and highly abundant in the outer membrane of the cell, in outer-membrane vesicles, and is released into culture medium in a soluble form. The protein is produced constitutively at low levels in bacteria grown under high-iron/heme conditions and at higher levels in bacteria growing under the low-iron/heme conditions typical of dental plaque. HmuY is immunogenic and elicits high IgG antibody titers in rabbits. It is also engaged in homotypic biofilm formation by P. gingivalis. Anti-HmuY antibodies exhibit inhibitory activity against P. gingivalis growth and biofilm formation. Conclusions Here it is demonstrated that HmuY may play a significant role not only in heme acquisition, but also in biofilm accumulation on abiotic surfaces. The data also suggest that HmuY, as a surface-exposed protein, would be available for recognition by the immune response during chronic periodontitis and the production of anti-HmuY antibodies may inhibit biofilm formation.

  10. Cobalt(III)-Mediated Permanent and Stable Immobilization of Histidine-Tagged Proteins on NTA-Functionalized Surfaces.

    Science.gov (United States)

    Wegner, Seraphine V; Schenk, Franziska C; Spatz, Joachim P

    2016-02-24

    We present the cobalt(III)-mediated interaction between polyhistidine (His)-tagged proteins and nitrilotriacetic acid (NTA)-modified surfaces as a general approach for a permanent, oriented, and specific protein immobilization. In this approach, we first form the well-established Co(2+) -mediated interaction between NTA and His-tagged proteins and subsequently oxidize the Co(2+) center in the complex to Co(3+) . Unlike conventionally used Ni(2+) - or Co(2+) -mediated immobilization, the resulting Co(3+) -mediated immobilization is resistant toward strong ligands, such as imidazole and ethylenediaminetetraacetic acid (EDTA), and washing off over time because of the high thermodynamic and kinetic stability of the Co(3+) complex. This immobilization method is compatible with a wide variety of surface coatings, including silane self-assembled monolayers (SAMs) on glass, thiol SAMs on gold surfaces, and supported lipid bilayers. Furthermore, once the cobalt center has been oxidized, it becomes inert toward reducing agents, specific and unspecific interactions, so that it can be used to orthogonally functionalize surfaces with multiple proteins. Overall, the large number of available His-tagged proteins and materials with NTA groups make the Co(3+) -mediated interaction an attractive and widely applicable platform for protein immobilization.

  11. Reversible Interactions of Proteins with Mixed Shell Polymeric Micelles: Tuning the Surface Hydrophobic/Hydrophilic Balance toward Efficient Artificial Chaperones.

    Science.gov (United States)

    Wang, Jianzu; Song, Yiqing; Sun, Pingchuan; An, Yingli; Zhang, Zhenkun; Shi, Linqi

    2016-03-22

    Molecular chaperones can elegantly fine-tune its hydrophobic/hydrophilic balance to assist a broad spectrum of nascent polypeptide chains to fold properly. Such precious property is difficult to be achieved by chaperone mimicking materials due to limited control of their surface characteristics that dictate interactions with unfolded protein intermediates. Mixed shell polymeric micelles (MSPMs), which consist of two kinds of dissimilar polymeric chains in the micellar shell, offer a convenient way to fine-tune surface properties of polymeric nanoparticles. In the current work, we have fabricated ca. 30 kinds of MSPMs with finely tunable hydrophilic/hydrophobic surface properties. We investigated the respective roles of thermosensitive and hydrophilic polymeric chains in the thermodenaturation protection of proteins down to the molecular structure. Although the three kinds of thermosensitive polymers investigated herein can form collapsed hydrophobic domains on the micellar surface, we found distinct capability to capture and release unfolded protein intermediates, due to their respective affinity for proteins. Meanwhile, in terms of the hydrophilic polymeric chains in the micellar shell, poly(ethylene glycol) (PEG) excels in assisting unfolded protein intermediates to refold properly via interacting with the refolding intermediates, resulting in enhanced chaperone efficiency. However, another hydrophilic polymer-poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) severely deteriorates the chaperone efficiency of MSPMs, due to its protein-resistant properties. Judicious combination of thermosensitive and hydrophilic chains in the micellar shell lead to MSPM-based artificial chaperones with optimal efficacy.

  12. High-performance liquid chromatography as a technique to determine protein adsorption onto hydrophilic/hydrophobic surfaces.

    Science.gov (United States)

    Huang, Tongtong; Anselme, Karine; Sarrailh, Segolene; Ponche, Arnaud

    2016-01-30

    The purpose of this study is to evaluate the potential of simple high performance liquid chromatography (HPLC) setup for quantification of adsorbed proteins on various type of plane substrates with limited area (hydrophobic (polydimethylsiloxane: PDMS) surfaces, kinetics of adsorption were determined and amounts of adsorbed bovine serum albumin, myoglobin and lysozyme were obtained: as expected for each protein, the amount adsorbed at the plateau on glass (between 0.15 μg/cm(2) and 0.4 μg/cm(2)) is lower than for hydrophobic PDMS surfaces (between 0.45 μg/cm(2) and 0.8 μg/cm(2)). These results were consistent with bicinchoninic acid protein determination. According to ICH guidelines, both Reversed Phase and Size Exclusion HPLC can be validated for quantification of adsorbed protein. However, we consider the size exclusion approach more interesting in this field because additional informations can be obtained for aggregative proteins. Indeed, monomer, dimer and oligomer of bovine serum albumin (BSA) were observed in the chromatogram. On increasing the temperature, we found a decrease of peak intensity of bovine serum albumin as well as the fraction of dimer and oligomer after contact with PDMS and glass surface. As the surface can act as a denaturation parameter, these informations can have a huge impact on the elucidation of the interfacial behavior of protein and in particular for aggregation processes in pharmaceutical applications.

  13. Corynebacterium diphtheriae invasion-associated protein (DIP1281 is involved in cell surface organization, adhesion and internalization in epithelial cells

    Directory of Open Access Journals (Sweden)

    Rheinlaender Johannes

    2010-01-01

    Full Text Available Abstract Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein. Results Microscopic inspection of DIP1281 mutant strains revealed an increased size of the single cells in combination with an altered less club-like shape and formation of chains of cells rather than the typical V-like division forms or palisades of growing C. diphtheriae cells. Cell viability was not impaired. Immuno-fluorescence microscopy, SDS-PAGE and 2-D PAGE of surface proteins revealed clear differences of wild-type and mutant protein patterns, which were verified by atomic force microscopy. DIP1281 mutant cells were not only altered in shape and surface structure but completely lack the ability to adhere to host cells and consequently invade these. Conclusions Our data indicate that DIP1281 is predominantly involved in the organization of the outer surface protein layer rather than in the separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface.

  14. Staphylococcus aureus surface protein SdrE binds complement regulator factor H as an immune evasion tactic.

    Directory of Open Access Journals (Sweden)

    Julia A Sharp

    Full Text Available Similar to other highly successful invasive bacterial pathogens, Staphylococcus aureus recruits the complement regulatory protein factor H (fH to its surface to inhibit the alternative pathway of complement. Here, we report the identification of the surface-associated protein SdrE as a fH-binding protein using purified fH overlay of S. aureus fractionated cell wall proteins and fH cross-linking to S. aureus followed by mass spectrometry. Studies using recombinant SdrE revealed that rSdrE bound significant fH whether from serum or as a purified form, in both a time- and dose-dependent manner. Furthermore, rSdrE-bound fH exhibited cofactor functionality for factor I (fI-mediated cleavage of C3b to iC3b which correlated positively with increasing amounts of fH. Expression of SdrE on the surface of the surrogate bacterium Lactococcus lactis enhanced recruitment of fH which resulted in increased iC3b generation. Moreover, surface expression of SdrE led to a reduction in C3-fragment deposition, less C5a generation, and reduced killing by polymorphonuclear cells. Thus, we report the first identification of a S. aureus protein associated with the staphylococcal surface that binds factor H as an immune evasion mechanism.

  15. Protein surface softness is the origin of enzyme cold-adaptation of trypsin.

    Directory of Open Access Journals (Sweden)

    Geir Villy Isaksen

    2014-08-01

    Full Text Available Life has effectively colonized most of our planet and extremophilic organisms require specialized enzymes to survive under harsh conditions. Cold-loving organisms (psychrophiles express heat-labile enzymes that possess a high specific activity and catalytic efficiency at low temperatures. A remarkable universal characteristic of cold-active enzymes is that they show a reduction both in activation enthalpy and entropy, compared to mesophilic orthologs, which makes their reaction rates less sensitive to falling temperature. Despite significant efforts since the early 1970s, the important question of the origin of this effect still largely remains unanswered. Here we use cold- and warm-active trypsins as model systems to investigate the temperature dependence of the reaction rates with extensive molecular dynamics free energy simulations. The calculations quantitatively reproduce the catalytic rates of the two enzymes and further yield high-precision Arrhenius plots, which show the characteristic trends in activation enthalpy and entropy. Detailed structural analysis indicates that the relationship between these parameters and the 3D structure is reflected by significantly different internal protein energy changes during the reaction. The origin of this effect is not localized to the active site, but is found in the outer regions of the protein, where the cold-active enzyme has a higher degree of softness. Several structural mechanisms for softening the protein surface are identified, together with key mutations responsible for this effect. Our simulations further show that single point-mutations can significantly affect the thermodynamic activation parameters, indicating how these can be optimized by evolution.

  16. Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162.

    Science.gov (United States)

    Heikens, Esther; Bonten, Marc J M; Willems, Rob J L

    2007-11-01

    Enterococci have emerged as important nosocomial pathogens with resistance to multiple antibiotics. Adhesion to abiotic materials and biofilm formation on medical devices are considered important virulence properties. A single clonal lineage of Enterococcus faecium, complex 17 (CC17), appears to be a successful nosocomial pathogen, and most CC17 isolates harbor the enterococcal surface protein gene, esp. In this study, we constructed an esp insertion-deletion mutant in a clinical E. faecium CC17 isolate. In addition, initial adherence and biofilm assays were performed. Compared to the wild-type strain, the esp insertion-deletion mutant no longer produced Esp on the cell surface and had significantly lower initial adherence to polystyrene and significantly less biofilm formation, resulting in levels of biofilm comparable to those of an esp-negative isolate. Capacities for initial adherence and biofilm formation were restored in the insertion-deletion mutant by in trans complementation with esp. These results identify Esp as the first documented determinant in E. faecium CC17 with an important role in biofilm formation, which is an essential factor in infection pathogenesis.

  17. Developmentally regulated epitopes of cell surface arabinogalactan proteins and their relation to root tissue pattern formation.

    Science.gov (United States)

    Knox, J P; Linstead, P J; Peart, J; Cooper, C; Roberts, K

    1991-11-01

    Two polymorphic forms of an extracellular arabinogalactan protein (AGP1 and AGP2), obtained from the conditioned media of two carrot suspension-cultured cell lines, have been identified in terms of binding of the anti-plasma membrane antibodies JIM4 and MAC207. AGP1 and AGP2 have been used as immunogens to generate further anti-AGP monoclonal antibodies. JIM14 identified an epitope carried by AGP2 and also by glycoproteins of low molecular weight localized to the plant cell wall. In addition, further antibodies (JIM13 and JIM15) identified carbohydrate epitopes of the AGPs that also occur on plasma membrane glycoproteins and are expressed by patterns of cells that reflect cell position at the carrot root apex. Indirect immunofluorescence microscopy indicated that JIM13 recognized the surface of cells forming the epidermis and cells marking the region and axis of the future xylem. JIM15 recognized a pattern of cells directly complementary to the JIM13 pattern. The panel of anti-AGP monoclonal antibodies now available indicates groups of cells within the root meristem that may reflect an early pre-pattern of the tissues of the mature root structure and suggests extensive modulation of cell surface AGPs during cell development and the positioning of cells within the apex.

  18. Structural insights into alginate binding by bacterial cell-surface protein.

    Science.gov (United States)

    Temtrirath, Kanate; Murata, Kousaku; Hashimoto, Wataru

    2015-03-02

    A gram-negative Sphingomonas sp. strain A1 inducibly forms a mouth-like pit on the cell surface in the presence of alginate and directly incorporates polymers into the cytoplasm via the pit and ABC transporter. Among the bacterial proteins involved in import of alginate, a cell-surface EfeO-like Algp7 shows an ability to bind alginate, suggesting its contribution to accumulate alginate in the pit. Here, we show identification of its positively charged cluster involved in alginate binding using X-ray crystallography, docking simulation, and site-directed mutagenesis. The tertiary structure of Algp7 was determined at a high resolution (1.99Å) by molecular replacement, although no alginates were included in the structure. Thus, an in silico model of Algp7/oligoalginate was constructed by docking simulation using atomic coordinates of Algp7 and alginate oligosaccharides, where some charged residues were found to be potential candidates for alginate binding. Site-directed mutagenesis was conducted and five purified mutants K68A, K69A, E194A, N221A, and K68A/K69A were subjected to a binding assay. UV absorption difference spectroscopy along with differential scanning fluorimetry analysis indicated that K68A/K69A exhibited a significant reduction in binding affinity with alginate than wild-type Algp7. Based on these data, Lys68/Lys69 residues of Algp7 probably play an important role in binding alginate.

  19. Using in situ X-ray reflectivity to study protein adsorption on hydrophilic and hydrophobic surfaces: benefits and limitations.

    Science.gov (United States)

    Richter, Andrew G; Kuzmenko, Ivan

    2013-04-30

    We have employed in situ X-ray reflectivity (IXRR) to study the adsorption of a variety of proteins (lysozyme, cytochrome c, myoglobin, hemoglobin, serum albumin, and immunoglobulin G) on model hydrophilic (silicon oxide) and hydrophobic surfaces (octadecyltrichlorosilane self-assembled monolayers), evaluating this recently developed technique for its applicability in the area of biomolecular studies. We report herein the highest resolution depiction of adsorbed protein films, greatly improving on the precision of previous neutron reflectivity (NR) results and previous IXRR studies. We were able to perform complete scans in 5 min or less with the maximum momentum transfer of at least 0.52 Å(-1), allowing for some time-resolved information about the evolution of the protein film structure. The three smallest proteins (lysozyme, cytochrome c, and myoglobin) were seen to deposit as fully hydrated, nondenatured molecules onto hydrophilic surfaces, with indications of particular preferential orientations. Time evolution was observed for both lysozyme and myoglobin films. The larger proteins were not observed to deposit on the hydrophilic substrates, perhaps because of contrast limitations. On hydrophobic surfaces, all proteins were seen to denature extensively in a qualitatively similar way but with a rough trend that the larger proteins resulted in lower coverage. We have generated high-resolution electron density profiles of these denatured films, including capturing the growth of a lysozyme film. Because the solution interface of these denatured films is diffuse, IXRR cannot unambiguously determine the film extent and coverage, a drawback compared to NR. X-ray radiation damage was systematically evaluated, including the controlled exposure of protein films to high-intensity X-rays and exposure of the hydrophobic surface to X-rays before adsorption. Our analysis showed that standard measuring procedures used for XRR studies may lead to altered protein films

  20. Size and surface functionalization of iron oxide nanoparticles influence the composition and dynamic nature of their protein corona.

    Science.gov (United States)

    Ashby, Jonathan; Pan, Songqin; Zhong, Wenwan

    2014-09-10

    Nanoparticles (NPs) adsorb proteins when in the biological matrix, and the resulted protein corona could affect NP-cell interactions. The corona has a dynamic nature with the adsorbed proteins constantly exchanging with the free proteins in the matrix at various rates. The rapidly exchanging proteins compose the soft corona, which responds more dynamically to environment changes than the hard corona established by the ones with slow exchange rates. In the present study, the corona formed on the superparamagnetic iron oxide NPs (SPIONs) in human serum was studied by flow field-flow fractionation and ultracentrifugation, which rapidly differentiated the corona proteins based on their exchange rates. By varying the surface hydrophobicity of the SPIONs with a core size around 10 nm, we found out that, the more hydrophobic surface ligand attracted proteins with higher surface hydrophobicity and formed a more dynamic corona with a larger portion of the involved proteins with fast exchange rates. Increasing the core diameter of the SPIONs but keeping the surface ligand the same could also result in a more dynamic corona. A brief investigation of the effect on the cellular uptake of SPIONs using one selected corona protein, transferrin, was conducted. The result showed that, only the stably bound transferrin could significantly enhance cellular uptake, while transferrin bound in a dynamic nature had negligible impact. Our study has led to a better understanding of the relationship between the particle properties and the dynamic nature of the corona, which can help with design of nanomaterials with higher biocompatibility and higher efficacy in biosystems for biomedical applications.

  1. Multi-parametric surface plasmon resonance platform for studying liposome-serum interactions and protein corona formation.

    Science.gov (United States)

    Kari, Otto K; Rojalin, Tatu; Salmaso, Stefano; Barattin, Michela; Jarva, Hanna; Meri, Seppo; Yliperttula, Marjo; Viitala, Tapani; Urtti, Arto

    2017-04-01

    When nanocarriers are administered into the blood circulation, a complex biomolecular layer known as the "protein corona" associates with their surface. Although the drivers of corona formation are not known, it is widely accepted that this layer mediates biological interactions of the nanocarrier with its surroundings. Label-free optical methods can be used to study protein corona formation without interfering with its dynamics. We demonstrate the proof-of-concept for a multi-parametric surface plasmon resonance (MP-SPR) technique in monitoring the formation of a protein corona on surface-immobilized liposomes subjected to flowing 100 % human serum. We observed the formation of formulation-dependent "hard" and "soft" coronas with distinct refractive indices, layer thicknesses, and surface mass densities. MP-SPR was also employed to determine the affinity (K D ) of a complement system molecule (C3b) with cationic liposomes with and without polyethylene glycol. Tendency to create a thick corona correlated with a higher affinity of opsonin C3b for the surface. The label-free platform provides a fast and robust preclinical tool for tuning nanocarrier surface architecture and composition to control protein corona formation.

  2. Protein

    Science.gov (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...

  3. Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium.

    Science.gov (United States)

    Knobloch, Denise; Ostermann, Kai; Rödel, Gerhard

    2012-01-01

    Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium, indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

  4. Heat shock protein 70 and glycoprotein 96 are differentially expressed on the surface of malignant and nonmalignant breast cells.

    Science.gov (United States)

    Melendez, Karla; Wallen, Erik S; Edwards, Bruce S; Mobarak, Charlotte D; Bear, David G; Moseley, Pope L

    2006-01-01

    Heat shock proteins (HSPs), which are important for a number of different intracellular functions, are occasionally found on the surface of cells. The function of heat shock protein on the cell surface is not understood, although it has been shown to be greater in some tumor cells and some virally infected cells. Surface expression of both glycoprotein 96 (gp96) and Hsp70 occurs on tumor cells, and this expression correlates with natural killer cell killing of the cells. We examined the surface expression of gp96 and Hsp70 on human breast cell lines MCF7, MCF10A, AU565, and HS578, and in primary human mammary epithelial cells by immunofluorescence microscopy and flow cytometry. The nonmalignant cell lines HS578, MCF10A, and HMEC showed no surface expression of gp96, whereas malignant cell lines MCF7 and AU565 were positive for gp96 surface expression. All of the breast cell lines examined showed Hsp70 surface expression. These results also confirm previous studies, demonstrating that Hsp70 is on the plasma membrane of tumor cell lines. Given the involvement of heat shock proteins, gp96 and Hsp70, in innate and adaptive immunity, these observations may be important in the immune response to tumor cells.

  5. Are the interactions between recombinant prion proteins and polymeric surfaces related to the hydrophilic/hydrophobic balance?

    Science.gov (United States)

    Vrlinic, Tjasa; Debarnot, Dominique; Legeay, Gilbert; Coudreuse, Arnaud; El Moualij, Benaissa; Zorzi, Willy; Perret-Liaudet, Armand; Quadrio, Isabelle; Mozetic, Miran; Poncin-Epaillard, Fabienne

    2012-06-01

    New non-fouling tubes are developed and their influence on the adhesion of neuroproteins is studied. Recombinant prion proteins are considered as a single component representative of hydrophobic proteins. Samples are stored for 24 h at 4 °C in tubes coated with two different coatings: poly(N-isopropylacrylamide) as a hydrophilic surface and a plasma-fluorinated coating as a hydrophobic one. The protein adhesion is monitored by ELISA tests, XPS and confocal microscopy. It appears that the highest recovery of recombinant prion protein in the liquid phase is obtained with the hydrophilic surface while the hydrophobic character of the storage tube induces an important amount of biological loss. However, the recovery is not complete even for tubes coated with poly(N-isopropylacrylamide).

  6. Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

    Science.gov (United States)

    Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

    2011-04-01

    Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

  7. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

    Directory of Open Access Journals (Sweden)

    McClafferty Heather

    2005-01-01

    Full Text Available Abstract Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP

  8. A bioactive elastin-like recombinamer reduces unspecific protein adsorption and enhances cell response on titanium surfaces.

    Science.gov (United States)

    Salvagni, Emiliano; Berguig, Geoffrey; Engel, Elisabeth; Rodriguez-Cabello, J Carlos; Coullerez, Geraldine; Textor, Marcus; Planell, Josep A; Gil, F Javier; Aparicio, Conrado

    2014-02-01

    We present the immobilization on synthetic substrates of elastin-like recombinamers (ELR) that combine a bioactive motif for cell adhesion with protein antifouling properties. Physical adsorption of the recombinamers and covalent-grafting through organosilane chemistry were investigated. The biochemically-modified surfaces were thoroughly characterized and tested for protein absorption in serum by fluorescence-labelling, XPS, Ellipsometry, and OWLS. The ELR were successfully grafted and stable, even upon mechanical stresses; being the covalent bonding favourable over physical adsorption. The coated metal surfaces exhibited excellent reduction of serum protein adsorption (9 ng/cm(2)) compared to the bare metal surface (310 ng/cm(2)). Non-specific protein adsorption may mask the introduced bioactive motifs; therefore, the bioactivated surfaces should display serum-protein antifouling properties. Finally, improved hMSCs response was assessed on the bioactivated substrates. In summary, the coatings simultaneously displayed anti-fouling and bioactive properties. These studies investigated key factors to enhance tissue material interactions fundamental for the design of bioactive devices and future biomedical applications.

  9. Scanning the cell surface proteome of cancer cells and identification of metastasis-associated proteins using a subtractive immunization strategy

    DEFF Research Database (Denmark)

    Rasmussen, Nicolaj; Ditzel, Henrik J

    2009-01-01

    characterization of the identified proteins. The strategy is based on subtractive immunization of mice, and we used the two isogenic cell lines, NM-2C5 and M-4A4, derived from the MDA-MB-435 cancer cell line, as a model system. Although the two cell lines are equally tumorigenic, only M-4A4 has metastatic...... of the immune system itself to scan the cell surface proteome for differentially expressed proteins. The subtractive immunization strategy should be broadly applicable as a quantitative and comparative proteomic approach for screening the cell surface and also allow generation of mAbs to study these cell...

  10. Influence of surface chemistry on the structural organization of monomolecular protein layers adsorbed to functionalized aqueous interfaces

    DEFF Research Database (Denmark)

    Lösche, M.; Piepenstock, M.; Diederich, A.;

    1993-01-01

    The molecular organization of streptavidin (SA) bound to aqueous surface monolayers of biotin-functionalized lipids and binary lipid mixtures has been investigated with neutron reflectivity and electron and fluorescence microscopy. The substitution of deuterons (2H) for protons (1H), both...... dependence of the structural properties of such self-assembled SA monolayers on the surface chemistry was observed: the lateral protein density depends on the length of the spacer connecting the biotin moiety and its hydrophobic anchor. The hydration of the lipid head groups in the protein-bound state...

  11. Modeling Condensation, Hydro- and Pepto-affinity of Surfaces in Medical Implant Devices and Surgical Lenses: Effect of Blood Proteins

    Science.gov (United States)

    Bennett-Kennett, Ross; Herbots, Nicole; Murphy, Ashlee; Sell, David; Kutz, Tyler; Benitez, Sophia; Acharya, Ajjya; Hughes, Brett; Watson, Clarizza; Culbertson, Eric; Sell, Clive; Kwong, H.

    2012-10-01

    Surgical lenses in laparoscopes and arthroscopes ``fog'' during surgery. Fogging increases by up to 40% surgery duration, infection rates, and scarring due to exposure from repeated scopes withdrawal for cleaning. Modeling nucleation on surfaces shows that 2-D layer-by-layer condensation maintains transparency while 3-D droplets refract at gas/fluid interfaces leading to opacity or ``fogging.'' This ProteinKnoxmodel for lenses made from bio-compatible polymers, and silica led us to a nano-scale molecular mesh applied as a bio-identical emulsion. ProteinKnox[1-5] meets a 100% success rate in eliminating fogging for up to 240 minutes over 300 experiments. Twenty surgical trials in the OR yield a success rate of 90%, with loss of vision due to the presence of blood or blood proteins, not fogging. We studied the common blood protein, heparin, which prevents coagulation, with the ProteinKnoxmodel. Heparin behaves like H2O on hydrophobic surfaces. It does not prevent fogging nor interferes with 2-D condensatio. Next, we investigated fibrinogen as agonist agent because it causes coagulation. Fibrinogen applied to various surfaces in emulsions prepared in accordance with the ProteinKnoxmodel can prevent not only

  12. Exploring the diameter and surface dependent conformational changes in carbon nanotube-protein corona and the related cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xingchen; Lu, Dawei; Hao, Fang [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Liu, Rutao, E-mail: rutaoliu@sdu.edu.cn [Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, China–America CRC for Environment & Health, Jinan 250100 (China)

    2015-07-15

    Highlights: • CNT diameter and surface area govern the stability of adsorbed proteins. • More BSA was loaded and destabilized on smaller CNTs. • Protein corona reduces the cytotoxicity of CNTs - Abstract: In this work, we investigated and compared carbon nanotubes (CNTs) of different diameters regarding their interaction with bovine serum albumin (BSA) and their ability to alter protein structure. BSA was exposed to CNT solutions, and the effects were assessed by utilizing fluorescence spectroscopy, UV–vis absorption spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), bichinchoninic acid (BCA) and zeta-potential measurement assays. We demonstrate that CNT diameter and surface area play key roles in influencing the stability of adsorbed proteins. Results showed that the secondary and tertiary structural stability of BSA decreased upon adsorption onto CNTs, with greater decrease on smaller-diametered nanotubes. Besides, more protein was loaded onto CNTs with small diameter, reducing the cytotoxicity. This study, therefore, provides fundamental information for the influence of CNT diameter and surface on protein behavior, which may be helpful to understand toxic effects of CNTs and prove beneficial for developing novel biomedical devices and safe use of nanomaterials.

  13. Another turn of the screw in shaving Gram-positive bacteria: Optimization of proteomics surface protein identification in Streptococcus pneumoniae.

    Science.gov (United States)

    Olaya-Abril, Alfonso; Gómez-Gascón, Lidia; Jiménez-Munguía, Irene; Obando, Ignacio; Rodríguez-Ortega, Manuel J

    2012-06-27

    Bacterial surface proteins are of outmost importance as they play critical roles in the interaction between cells and their environment. In addition, they can be targets of either vaccines or antibodies. Proteomic analysis through "shaving" live cells with proteases has become a successful approach for a fast and reliable identification of surface proteins. However, this protocol has not been able to reach the goal of excluding cytoplasmic contamination, as cell lysis is an inherent process during culture and experimental manipulation. In this work, we carried out the optimization of the "shaving" strategy for the Gram-positive human pathogen Streptococcus pneumoniae, a bacterium highly susceptible to autolysis, and set up the conditions for maximizing the identification of surface proteins containing sorting or exporting signals, and for minimizing cytoplasmic contamination. We also demonstrate that cell lysis is an inherent process during culture and experimental manipulation, and that a low level of lysis is enough to contaminate a "surfome" preparation with peptides derived from cytoplasmic proteins. When the optimized conditions were applied to several clinical isolates, we found the majority of the proteins described to induce protection against pneumococcal infection. In addition, we found other proteins whose protection capacity has not been yet tested. In addition, we show the utility of this approach for providing antigens that can be used in serological tests for the diagnosis of pneumococcal disease.

  14. Proteomic and transcriptomic profiling of Staphylococcus aureus surface LPXTG-proteins: correlation with agr genotypes and adherence phenotypes.

    Science.gov (United States)

    Ythier, Mathilde; Resch, Grégory; Waridel, Patrice; Panchaud, Alexandre; Gfeller, Aurélie; Majcherczyk, Paul; Quadroni, Manfredo; Moreillon, Philippe

    2012-11-01

    Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence

  15. Glioblastoma Inhibition by Cell Surface Immunoglobulin Protein EWI-2, In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Tatiana V. Kolesnikova

    2009-01-01

    Full Text Available EWI-2, a cell surface IgSF protein, is highly expressed in normal human brain but is considerably diminished in glioblastoma tumors and cell lines. Moreover, loss of EWI-2 expression correlated with a shorter survival time in human glioma patients, suggesting that EWI-2 might be a natural inhibitor of glioblastoma. In support of this idea, EWI-2 expression significantly impaired both ectopic and orthotopic tumor growth in nude mice in vivo. In vitro assays provided clues regarding EWI-2 functions. Expression of EWI-2 in T98G and/or U87-MG malignant glioblastoma cell lines failed to alter two-dimensional cell proliferation but inhibited glioblastoma colony formation in soft agar and caused diminished cell motility and invasion. At the biochemical level, EWI-2 markedly affects the organization of four molecules (tetraspanin proteins CD9 and CD81 and matrix metalloproteinases MMP-2 and MT1-MMP, which play key roles in the biology of astrocytes and gliomas. EWI-2 causes CD9 and CD81 to become more associated with each other, whereas CD81 and other tetraspanins become less associated with MMP-2 and MT1-MMP. We propose that EWI-2 inhibition of glioblastoma growth in vivo is at least partly explained by the capability of EWI-2 to inhibit growth and/or invasion in vitro. Underlying these functional effects, EWI-2 causes a substantial molecular reorganization of multiple molecules (CD81, CD9, MMP-2, and MT1-MMP known to affect proliferation and/or invasion of astrocytes and/or glioblastomas.

  16. Zwitterionic sulfobetaine-grafted poly(vinylidene fluoride) membrane surface with stably anti-protein-fouling performance via a two-step surface polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Li Qian; Bi Qiuyan; Zhou Bo [Membrane Technology and Engineering Research Center, Department of Chemical Engineering, Tsinghua University, Beijing 100084 (China); Wang Xiaolin, E-mail: xl-wang@tsinghua.edu.cn [Membrane Technology and Engineering Research Center, Department of Chemical Engineering, Tsinghua University, Beijing 100084 (China)

    2012-03-01

    A zwitterionic polymer, poly(3-(methacryloylamino) propyl-dimethyl-(3-sulfopropyl) ammonium hydroxide) (poly(MPDSAH)) was successfully grafted in high density from the surface of poly(vinylidene fluoride) (PVDF) hollow fiber membrane via a two-step polymerization. Poly(2-hydroxyethyl methacrylate) (poly(HEMA)) chains were firstly grafted from outside surface of PVDF membrane through atom transfer radical polymerization (ATRP) to provide the initiation sites for subsequent cerium (Ce (IV))-induced graft copolymerization of polyMPDSAH in the presence of N,N Prime -ethylene bisacrylamide (EBAA) as a cross-linking agent. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS) confirmed that the EBAA could stimulate zwitterionic polymers grafting onto the membrane surface. The dense poly(MPDSAH) layers on the PVDF membrane surface were revealed by the scanning electron microscope (SEM). The mechanical property of PVDF membrane was improved by the zwitterionic surface layers. The gravimetry results indicated the grafting amount increased to 520 {mu}g/cm{sup 2} for a copolymerization time of more than 3 h. Static and dynamic water contact angle measurements showed that the surface hydrophilicity of the PVDF membranes was significantly enhanced. As the grafting amount reached 513 {mu}g cm{sup -2}, the value of contact angle dropped to 22.1 Degree-Sign and the amount of protein adsorption decreased to zero. The cyclic experiments for BSA solution filtration demonstrated that the extent of protein fouling was significantly reduced and most of the fouling was reversible. The grafted polymer layer on the PVDF membrane showed a good stability during the membrane cleaning process. The experimental results concluded a good prospect in obtaining the sulfobetaine-modified PVDF membranes with high mechanical strength, good anti-protein-fouling performance, and long-term stability via the two-step polymerization.

  17. Off surface matrix based on-chip electrochemical biosensor platform for protein biomarker detection in undiluted serum.

    Science.gov (United States)

    Arya, Sunil K; Kongsuphol, Patthara; Park, Mi Kyoung

    2017-06-15

    The manuscript describes a concept of using off surface matrix modified with capturing biomolecule for on-chip electrochemical biosensing. 3D matrix made by laser engraving of polymethyl methacrylate (PMMA) sheet as off surface matrix was integrated in very close vicinity of the electrode surface. Laser engraving and holes in PMMA along with spacing from surface provide fluidic channel and incubation chamber. Covalent binding of capturing biomolecule (anti-TNF-α antibody) on off-surface matrix was achieved via azide group activity of 4-fluoro-3-nitro-azidobenzene (FNAB), which act as cross-linker and further covalently binds to anti-TNF-α antibody via thermal reaction. Anti-TNF-α/FNAB/PMMA matrix was then integrated over comb structured gold electrode array based sensor chip. Separate surface modification followed by integration of sensor helped to prevent the sensor chip surface from fouling during functionalization. Nonspecific binding was prevented using starting block T20 (PBS). Results for estimating protein biomarker (TNF-α) in undiluted serum using Anti-TNF-α/FNAB/PMMA/Au reveal that system can detect TNF-α in 100pg/ml to 100ng/ml range with high sensitivity of 119nA/(ng/ml), with negligible interference from serum proteins and other cytokines. Thus, use of off surface matrix may provide the opportunity to electrochemically sense biomarkers sensitively to ng/ml range with negligible nonspecific binding and false signal in undiluted serum.

  18. Stop codons in the hepatitis B surface proteins are enriched during antiviral therapy and are associated with host cell apoptosis.

    Science.gov (United States)

    Colledge, Danielle; Soppe, Sally; Yuen, Lilly; Selleck, Lucy; Walsh, Renae; Locarnini, Stephen; Warner, Nadia

    2017-01-15

    Premature stop codons in the hepatitis B virus (HBV) surface protein can be associated with nucleos(t)ide analogue resistance due to overlap of the HBV surface and polymerase genes. The aim of this study was to determine the effect of the replication of three common surface stop codon variants on the hepatocyte. Cell lines were transfected with infectious HBV clones encoding surface stop codons rtM204I/sW196*, rtA181T/sW172*, rtV191I/sW182*, and a panel of substitutions in the surface proteins. HBsAg was measured by Western blotting. Proliferation and apoptosis were measured using flow cytometry. All three surface stop codon variants were defective in HBsAg secretion. Cells transfected with these variants were less proliferative and had higher levels of apoptosis than those transfected with variants that did not encode surface stop codons. The most cytopathic variant was rtM204I/sW196*. Replication of HBV encoding surface stop codons was toxic to the cell and promoted apoptosis, exacerbating disease progression.

  19. Moderation of prekallkrein-factor XII interactions in surface activation of coagulation by protein-adsorption competition.

    Science.gov (United States)

    Chatterjee, Kaushik; Thornton, Jennifer L; Bauer, James W; Vogler, Erwin A; Siedlecki, Christopher A

    2009-10-01

    Traditional biochemistry of contact activation of blood coagulation suggesting that anionic hydrophilic surfaces are specific activators of the cascade is inconsistent with known trends in protein adsorption. To investigate contact activation reactions, a chromogenic assay was used to measure prekallkrein (PK) hydrolysis to kallikrein (Kal) by activated factor XII (FXIIa) at test hydrophilic (clean glass) and hydrophobic (silanized glass) surfaces in the presence of bovine serum albumin (BSA). Hydrolysis of PK by FXIIa is detected after contact of the zymogen FXII with a test hydrophobic surface only if putatively-adsorbed FXIIa is competitively displaced by BSA. By contrast, FXIIa activity is detected spontaneously following FXII activation by a hydrophilic surface and requires no adsorption displacement. These results (i) show that an anionic hydrophilic surface is not a necessary cofactor for FXIIa-mediated hydrolysis of PK, (ii) indicate that PK hydrolysis does not need to occur by an activation complex assembled directly on an anionic, activating surface, (iii) confirms that contact activation of FXII (autoactivation) is not specific to anionic hydrophilic surfaces, and (iv) demonstrates that protein-adsorption competition is an essential feature that must be included in any comprehensive mechanism of surface-induced blood coagulation.

  20. Bovine serum albumin surface imprinted polymer fabricated by surface grafting copolymerization on zinc oxide rods and its application for protein recognition.

    Science.gov (United States)

    Li, Xiangjie; Zhou, Jingjing; Tian, Lei; Li, Wei; Zhang, Baoliang; Zhang, Hepeng; Zhang, Qiuyu

    2015-10-01

    A novel bovine serum albumin (BSA) surface imprinted polymer based on ZnO rods was synthesized by surface grafting copolymerization. It exhibited an excellent recognition performance to bovine serum albumin. The adsorption capacity and imprinting factor of bovine serum albumin could reach 89.27 mg/g and 2.35, respectively. Furthermore, the fluorescence property of ZnO was used for tracing the process of protein imprinting and it implied the excellent optical sensing property of this material. More importantly, the hypothesis that the surface charge of carrier could affect the imprinting process was confirmed. That is, ZnO with positive surface charge could not only improve the recognition specificity of binding sites to template proteins (pI 7). It was also important that the reusability of ZnO@BSA molecularly imprinted polymers was satisfactory. This implied that the poor mechanical/chemical stability of traditional zinc oxide sensors could be solved by the introduction of surface grafting copolymerization. These results revealed that the ZnO@BSA molecularly imprinted polymers are a promising optical/electrochemical sensor element.

  1. A study on poly (N-vinyl-2-pyrrolidone covalently bonded NiTi surface for inhibiting protein adsorption

    Directory of Open Access Journals (Sweden)

    Hongyan Yu

    2016-12-01

    Full Text Available Near equiatomic NiTi alloys have been extensively applied as biomaterials owing to its unique shape memory effect, superelasticity and biocompatibility. It has been demonstrated that surfaces capable of preventing plasma protein adsorption could reduce the reactivity of biomaterials with human blood. This motivated a lot of researches on the surface modification of NiTi alloy. In the present work, following heat and alkaline treatment and silanization by trichlorovinylsilane (TCVS, coating of poly (N-vinyl-2-pyrrolidone (PVP was produced on the NiTi alloy by gamma ray induced chemical bonding. The structures and properties of modified NiTi were characterized and in vitro biocompatibility of plasma protein adsorption was investigated. The results indicated that heat treatment at 823 K for 1 h could result in the formation of a protective TiO2 layer with “Ni-free” zone on NiTi surface. It was found that PVP was covalently bonded on NiTi surface to create a hydrophilic layer for inhibiting protein adsorption on the surface. The present work offers a green approach to introduce a bioorganic surface on metal and other polymeric or inorganic substrates by gamma irradiation.

  2. PEP-SiteFinder: a tool for the blind identification of peptide binding sites on protein surfaces.

    Science.gov (United States)

    Saladin, Adrien; Rey, Julien; Thévenet, Pierre; Zacharias, Martin; Moroy, Gautier; Tufféry, Pierre

    2014-07-01

    Peptide-protein interactions are important to many processes of life, particularly for signal transmission or regulatory mechanisms. When no information is known about the interaction between a protein and a peptide, it is of interest to propose candidate sites of interaction at the protein surface, to assist the design of biological experiments to probe the interaction, or to serve as a starting point for more focused in silico approaches. PEP-SiteFinder is a tool that will, given the structure of a protein and the sequence of a peptide, identify protein residues predicted to be at peptide-protein interface. PEP-SiteFinder relies on the 3D de novo generation of peptide conformations given its sequence. These conformations then undergo a fast blind rigid docking on the complete protein surface, and we have found, as the result of a benchmark over 41 complexes, that the best poses overlap to some extent the experimental patch of interaction for close to 90% complexes. In addition, PEP-SiteFinder also returns a propensity index we have found informative about the confidence of the prediction. The PEP-SiteFinder web server is available at http://bioserv.rpbs.univ-paris-diderot.fr/PEP-SiteFinder.

  3. A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins.

    Directory of Open Access Journals (Sweden)

    Anthony Ryan

    2011-06-01

    Full Text Available Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4⁻/⁻ and Myd88⁻/⁻, but not TRIF⁻/⁻ mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

  4. Protein and carbohydrate exopolymer particles in the sea surface microlayer (SML

    Directory of Open Access Journals (Sweden)

    Daniel Conrad Ogilvie Thornton

    2016-08-01

    Full Text Available Exchanges of matter and energy between ocean and atmosphere occur through the sea surface microlayer (SML. The SML is the thin surface layer of the ocean at the ocean-atmosphere interface that has distinctive physical, chemical and biological properties compared with the underlying water. We measured the concentration of two types of exopolymer particles in the SML and underlying water in the Pacific Ocean off the coast of Oregon (United States during July 2011. Transparent exopolymer particles (TEP are defined by their acidic polysaccharide content, whereas Coomassie staining particles (CSP are composed of protein. TEP and CSP were ubiquitous in the SML. TEP were not significantly enriched in the SML compared with the underlying water. CSP were significantly enriched in the SML, with an enrichment factor (EF of 1.4 to 2.4. The distribution of exopolymer particles in the water and microscopic imaging indicated that TEP and CSP are distinct populations of particles rather than different chemical components of the same particles. Dissolved polysaccharides were not enriched in the SML, whereas monosaccharides had an EF of 1.2 to 1.8. Sampling occurred during the collapse of a diatom bloom, and diatoms were found both in the water column and SML. While there were living diatoms in the samples, most of the diatoms were dead and there were abundant empty frustules covered in layer of TEP. The collapsing diatom bloom was probably the source of exopolymer particles to both the SML and underlying water. Exopolymer particles are a component of the SML that may play a significant role in the marine carbon and nitrogen cycles, and the exchange of material between ocean and atmosphere.

  5. Molecular Characterizations of Surface Proteins Hemagglutinin and Neuraminidase from Recent H5Nx Avian Influenza Viruses

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hua; Carney, Paul J.; Mishin, Vasiliy P.; Guo, Zhu; Chang, Jessie C.; Wentworth, David E.; Gubareva, Larisa V.; Stevens, James; Schultz-Cherry, S.

    2016-04-06

    ABSTRACT

    During 2014, a subclade 2.3.4.4 highly pathogenic avian influenza (HPAI) A(H5N8) virus caused poultry outbreaks around the world. In late 2014/early 2015, the virus was detected in wild birds in Canada and the United States, and these viruses also gave rise to reassortant progeny, composed of viral RNA segments (vRNAs) from both Eurasian and North American lineages. In particular, viruses were found with N1, N2, and N8 neuraminidase vRNAs, and these are collectively referred to as H5Nx viruses. In the United States, more than 48 million domestic birds have been affected. Here we present a detailed structural and biochemical analysis of the surface antigens of H5N1, H5N2, and H5N8 viruses in addition to those of a recent human H5N6 virus. Our results with recombinant hemagglutinin reveal that these viruses have a strict avian receptor binding preference, while recombinantly expressed neuraminidases are sensitive to FDA-approved and investigational antivirals. Although H5Nx viruses currently pose a low risk to humans, it is important to maintain surveillance of these circulating viruses and to continually assess future changes that may increase their pandemic potential.

    IMPORTANCEThe H5Nx viruses emerging in North America, Europe, and Asia pose a great public health concern. Here we report a molecular and structural study of the major surface proteins of several H5Nx influenza viruses. Our results improve the understanding of these new viruses and provide important information on their receptor preferences and susceptibilities to antivirals, which are central to pandemic risk assessment.

  6. A Role for TLR4 in Clostridium difficile Infection and the Recognition of Surface Layer Proteins.

    LENUS (Irish Health Repository)

    Ryan, Anthony

    2011-06-01

    Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H\\/HeN and C3H\\/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H\\/HeJ mice and failed to induce a subsequent Th cell response. TLR4(-\\/-) and Myd88(-\\/-), but not TRIF(-\\/-) mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

  7. Platelet adhesion and plasma protein adsorption control of collagen surfaces by He{sup +} ion implantation

    Energy Technology Data Exchange (ETDEWEB)

    Kurotobi, K. E-mail: kurotobi@postman.riken.go.jp; Suzuki, Y.; Nakajima, H.; Suzuki, H.; Iwaki, M

    2003-05-01

    He{sup +} ion implanted collagen-coated tubes with a fluence of 1 x 10{sup 14} ions/cm{sup 2} were exhibited antithrombogenicity. To investigate the mechanisms of antithrombogenicity of these samples, plasma protein adsorption assay and platelet adhesion experiments were performed. The adsorption of fibrinogen (Fg) and von Willebrand factor (vWf) was minimum on the He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2}. Platelet adhesion (using platelet rich plasma) was inhibited on the He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2} and was accelerated on the untreated collagen and ion implanted collagen with fluences of 1 x 10{sup 13}, 1 x 10{sup 15} and 1 x 10{sup 16} ions/cm{sup 2}. Platelet activation with washed platelets was observed on untreated collagen and He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2} and was inhibited with fluences of 1 x 10{sup 13}, 1 x 10{sup 15} and 1 x 10{sup 16} ions/cm{sup 2}. Generally, platelets can react with a specific ligand inside the collagen (GFOGER sequence). The results of platelets adhesion experiments using washed platelets indicated that there were no ligands such as GFOGER on the He{sup +} ion implanted collagen over a fluence of 1 x 10{sup 13} ions/cm{sup 2}. On the 1 x 10{sup 14} ions/cm{sup 2} implanted collagen, no platelet activation was observed due to the influence of plasma proteins. >From the above, it is concluded that the decrease of adsorbed Fg and vWf caused the antithrombogenicity of He{sup +} ion implanted collagen with a fluence of 1 x 10{sup 14} ions/cm{sup 2} and that plasma protein adsorption took an important role repairing the graft surface.

  8. Characterisation of the protein corona using tunable resistive pulse sensing: determining the change and distribution of a particle's surface charge.

    Science.gov (United States)

    Blundell, Emma L C J; Healey, Matthew J; Holton, Elizabeth; Sivakumaran, Muttuswamy; Manstana, Sarabjit; Platt, Mark

    2016-08-01

    The zeta potential of the protein corona around carboxyl particles has been measured using tunable resistive pulse sensing (TRPS). A simple and rapid assay for characterising zeta potentials within buffer, serum and plasma is presented monitoring the change, magnitude and distribution of proteins on the particle surface. First, we measure the change in zeta potential of carboxyl-functionalised nanoparticles in solutions that contain biologically relevant concentrations of individual proteins, typically constituted in plasma and serum, and observe a significant difference in distributions and zeta values between room temperature and 37 °C assays. The effect is protein dependent, and the largest difference between the two temperatures is recorded for the γ-globulin protein where the mean zeta potential changes from -16.7 to -9.0 mV for 25 and 37 °C, respectively. This method is further applied to monitor particles placed into serum and/or plasma. A temperature-dependent change is again observed with serum showing a 4.9 mV difference in zeta potential between samples incubated at 25 and 37 °C; this shift was larger than that observed for samples in plasma (0.4 mV). Finally, we monitor the kinetics of the corona reorientation for particles initially placed into serum and then adding 5 % (V/V) plasma. The technology presented offers an interesting insight into protein corona structure and kinetics of formation measured in biologically relevant solutions, i.e. high protein, high salt levels, and its particle-by-particle analysis gives a measure of the distribution of particle zeta potential that may offer a better understanding of the behaviour of nanoparticles in solution. Graphical Abstract The relative velocity of a nanoparticle as it traverses a nanopore can be used to determine its zeta potential. Monitoring the changes in translocation speeds can therefore be used to follow changes to the surface chemistry/composition of 210 nm particles that were placed

  9. Surface-induced intramolecular electron transfer in multi-centre redox metalloproteins: the di-haem protein cytochrome c4 in homogeneous solution and at electrochemical surfaces

    Science.gov (United States)

    Chi, Qijin; Zhang, Jingdong; Jensen, Palle S.; Nazmudtinov, Renat R.; Ulstrup, Jens

    2008-09-01

    Intramolecular electron transfer (ET) between transition metal centres is a core feature of biological ET and redox enzyme function. The number of microscopic redox potentials and ET rate constants is, however, mostly prohibitive for experimental mapping, but two-centre proteins offer simple enough communication networks for complete mapping to be within reach. At the same time, multi-centre redox proteins operate in a membrane environment where conformational dynamics and ET patterns are quite different from the conditions in a homogeneous solution. The bacterial respiratory di-haem protein Pseudomonas stutzeri cytochrome c4 offers a prototype target for environmental gating of intra-haem ET. ET between P. stutzeri cyt c4 and small molecular reaction partners in solution appears completely dominated by intermolecular ET of each haem group/protein domain, with no competing intra-haem ET, for which accompanying propionate-mediated proton transfer is a further barrier. The protein can, however, be immobilized on single-crystal, modified Au(111) electrode surfaces with either the low-potential N terminal or the high-potential C terminal domain facing the surface, clearly with fast intramolecular ET as a key feature in the electrochemical two-ET process. This dual behaviour suggests a pattern for multi-centre redox metalloprotein function. In a homogeneous solution, which is not the natural environment of cyt c4, the two haem group domains operate largely independently with conformations prohibitive for intramolecular ET. Binding to a membrane or electrochemical surface, however, triggers conformational opening of intramolecular ET channels. The haem group orientation in P. stutzeri cyt c4 is finally noted to offer a case for orientation dependent electronic rectification between a substrate and a tip in electrochemical in situ scanning tunnelling microscopy or nanoscale electrode configurations.

  10. SIGNR3-dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis.

    Science.gov (United States)

    Lightfoot, Yaíma L; Selle, Kurt; Yang, Tao; Goh, Yong Jun; Sahay, Bikash; Zade