Two states or not two states: Single-molecule folding studies of protein L

Science.gov (United States)

Aviram, Haim Yuval; Pirchi, Menahem; Barak, Yoav; Riven, Inbal; Haran, Gilad

2018-03-01

Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, ˜7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal β strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.

Force spectroscopy studies on protein-ligand interactions: a single protein mechanics perspective.

Science.gov (United States)

Hu, Xiaotang; Li, Hongbin

2014-10-01

Protein-ligand interactions are ubiquitous and play important roles in almost every biological process. The direct elucidation of the thermodynamic, structural and functional consequences of protein-ligand interactions is thus of critical importance to decipher the mechanism underlying these biological processes. A toolbox containing a variety of powerful techniques has been developed to quantitatively study protein-ligand interactions in vitro as well as in living systems. The development of atomic force microscopy-based single molecule force spectroscopy techniques has expanded this toolbox and made it possible to directly probe the mechanical consequence of ligand binding on proteins. Many recent experiments have revealed how ligand binding affects the mechanical stability and mechanical unfolding dynamics of proteins, and provided mechanistic understanding on these effects. The enhancement effect of mechanical stability by ligand binding has been used to help tune the mechanical stability of proteins in a rational manner and develop novel functional binding assays for protein-ligand interactions. Single molecule force spectroscopy studies have started to shed new lights on the structural and functional consequence of ligand binding on proteins that bear force under their biological settings. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A mathematical model of T lymphocyte calcium dynamics derived from single transmembrane protein properties

Directory of Open Access Journals (Sweden)

Christine Dorothee Schmeitz

2013-09-01

Full Text Available Fate decision processes of T lymphocytes are crucial for health and disease. Whether a T lymphocyte is activated, divides, gets anergic or initiates apoptosis depends on extracellular triggers and intracellular signalling. Free cytosolic calcium dynamics plays an important role in this context. The relative contributions of store-derived calcium entry and calcium entry from extracellular space to T lymphocyte activation are still a matter of debate. Here we develop a quantitative mathematical model of T lymphocyte calcium dynamics in order to establish a tool which allows to disentangle cause-effect relationships between ion fluxes and observed calcium time courses. The model is based on single transmembrane protein characteristics which have been determined in independent experiments. This reduces the number of unknown parameters in the model to a minimum and ensures the predictive power of the model. Simulation results are subsequently used for an analysis of whole cell calcium dynamics measured under various experimental conditions. The model accounts for a variety of these conditions, which supports the suitability of the modelling approach. The simulation results suggest a model in which calcium dynamics dominantly relies on the opening of channels in calcium stores while calcium entry through calcium-release activated channels (CRAC is more associated with the maintenance of the T lymphocyte calcium levels and prevents the cell from calcium depletion. Our findings indicate that CRAC guarantees a long-term stable calcium level which is required for cell survival and sustained calcium enhancement.

Quality assessment of protein model-structures based on structural and functional similarities.

Science.gov (United States)

Konopka, Bogumil M; Nebel, Jean-Christophe; Kotulska, Malgorzata

2012-09-21

Experimental determination of protein 3D structures is expensive, time consuming and sometimes impossible. A gap between number of protein structures deposited in the World Wide Protein Data Bank and the number of sequenced proteins constantly broadens. Computational modeling is deemed to be one of the ways to deal with the problem. Although protein 3D structure prediction is a difficult task, many tools are available. These tools can model it from a sequence or partial structural information, e.g. contact maps. Consequently, biologists have the ability to generate automatically a putative 3D structure model of any protein. However, the main issue becomes evaluation of the model quality, which is one of the most important challenges of structural biology. GOBA--Gene Ontology-Based Assessment is a novel Protein Model Quality Assessment Program. It estimates the compatibility between a model-structure and its expected function. GOBA is based on the assumption that a high quality model is expected to be structurally similar to proteins functionally similar to the prediction target. Whereas DALI is used to measure structure similarity, protein functional similarity is quantified using standardized and hierarchical description of proteins provided by Gene Ontology combined with Wang's algorithm for calculating semantic similarity. Two approaches are proposed to express the quality of protein model-structures. One is a single model quality assessment method, the other is its modification, which provides a relative measure of model quality. Exhaustive evaluation is performed on data sets of model-structures submitted to the CASP8 and CASP9 contests. The validation shows that the method is able to discriminate between good and bad model-structures. The best of tested GOBA scores achieved 0.74 and 0.8 as a mean Pearson correlation to the observed quality of models in our CASP8 and CASP9-based validation sets. GOBA also obtained the best result for two targets of CASP8, and

Automated protein structure modeling with SWISS-MODEL Workspace and the Protein Model Portal.

Science.gov (United States)

Bordoli, Lorenza; Schwede, Torsten

2012-01-01

Comparative protein structure modeling is a computational approach to build three-dimensional structural models for proteins using experimental structures of related protein family members as templates. Regular blind assessments of modeling accuracy have demonstrated that comparative protein structure modeling is currently the most reliable technique to model protein structures. Homology models are often sufficiently accurate to substitute for experimental structures in a wide variety of applications. Since the usefulness of a model for specific application is determined by its accuracy, model quality estimation is an essential component of protein structure prediction. Comparative protein modeling has become a routine approach in many areas of life science research since fully automated modeling systems allow also nonexperts to build reliable models. In this chapter, we describe practical approaches for automated protein structure modeling with SWISS-MODEL Workspace and the Protein Model Portal.

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

Science.gov (United States)

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-03-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

The effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on protein-protein interactions.

Science.gov (United States)

Yates, Christopher M; Sternberg, Michael J E

2013-11-01

Non-synonymous single nucleotide polymorphisms (nsSNPs) are single base changes leading to a change to the amino acid sequence of the encoded protein. Many of these variants are associated with disease, so nsSNPs have been well studied, with studies looking at the effects of nsSNPs on individual proteins, for example, on stability and enzyme active sites. In recent years, the impact of nsSNPs upon protein-protein interactions has also been investigated, giving a greater insight into the mechanisms by which nsSNPs can lead to disease. In this review, we summarize these studies, looking at the various mechanisms by which nsSNPs can affect protein-protein interactions. We focus on structural changes that can impair interaction, changes to disorder, gain of interaction, and post-translational modifications before looking at some examples of nsSNPs at human-pathogen protein-protein interfaces and the analysis of nsSNPs from a network perspective. © 2013.

Prions: Beyond a Single Protein

Science.gov (United States)

Das, Alvin S.

2016-01-01

SUMMARY Since the term protein was first coined in 1838 and protein was discovered to be the essential component of fibrin and albumin, all cellular proteins were presumed to play beneficial roles in plants and mammals. However, in 1967, Griffith proposed that proteins could be infectious pathogens and postulated their involvement in scrapie, a universally fatal transmissible spongiform encephalopathy in goats and sheep. Nevertheless, this novel hypothesis had not been evidenced until 1982, when Prusiner and coworkers purified infectious particles from scrapie-infected hamster brains and demonstrated that they consisted of a specific protein that he called a “prion.” Unprecedentedly, the infectious prion pathogen is actually derived from its endogenous cellular form in the central nervous system. Unlike other infectious agents, such as bacteria, viruses, and fungi, prions do not contain genetic materials such as DNA or RNA. The unique traits and genetic information of prions are believed to be encoded within the conformational structure and posttranslational modifications of the proteins. Remarkably, prion-like behavior has been recently observed in other cellular proteins—not only in pathogenic roles but also serving physiological functions. The significance of these fascinating developments in prion biology is far beyond the scope of a single cellular protein and its related disease. PMID:27226089

Biophysics of DNA-Protein Interactions From Single Molecules to Biological Systems

CERN Document Server

Williams, Mark C

2011-01-01

This book presents a concise overview of current research on the biophysics of DNA-protein interactions. A wide range of new and classical methods are presented by authors investigating physical mechanisms by which proteins interact with DNA. For example, several chapters address the mechanisms by which proteins search for and recognize specific binding sites on DNA, a process critical for cellular function. Single molecule methods such as force spectroscopy as well as fluorescence imaging and tracking are described in these chapters as well as other parts of the book that address the dynamics of protein-DNA interactions. Other important topics include the mechanisms by which proteins engage DNA sequences and/or alter DNA structure. These simple but important model interactions are then placed in the broader biological context with discussion of larger protein-DNA complexes . Topics include replication forks, recombination complexes, DNA repair interactions, and ultimately, methods to understand the chromatin...

Errors in Computing the Normalized Protein Catabolic Rate due to Use of Single-pool Urea Kinetic Modeling or to Omission of the Residual Kidney Urea Clearance.

Science.gov (United States)

Daugirdas, John T

2017-07-01

The protein catabolic rate normalized to body size (PCRn) often is computed in dialysis units to obtain information about protein ingestion. However, errors can manifest when inappropriate modeling methods are used. We used a variable volume 2-pool urea kinetic model to examine the percent errors in PCRn due to use of a 1-pool urea kinetic model or after omission of residual urea clearance (Kru). When a single-pool model was used, 2 sources of errors were identified. The first, dependent on the ratio of dialyzer urea clearance to urea distribution volume (K/V), resulted in a 7% inflation of the PCRn when K/V was in the range of 6 mL/min per L. A second, larger error appeared when Kt/V values were below 1.0 and was related to underestimation of urea distribution volume (due to overestimation of effective clearance) by the single-pool model. A previously reported prediction equation for PCRn was valid, but data suggest that it should be modified using 2-pool eKt/V and V coefficients instead of single-pool values. A third source of error, this one unrelated to use of a single-pool model, namely omission of Kru, was shown to result in an underestimation of PCRn, such that each ml/minute Kru per 35 L of V caused a 5.6% underestimate in PCRn. Marked overestimation of PCRn can result due to inappropriate use of a single-pool urea kinetic model, particularly when Kt/V <1.0 (as in short daily dialysis), or after omission of residual native kidney clearance. Copyright © 2017 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Protein (multi-)location prediction: utilizing interdependencies via a generative model.

Science.gov (United States)

Simha, Ramanuja; Briesemeister, Sebastian; Kohlbacher, Oliver; Shatkay, Hagit

2015-06-15

Proteins are responsible for a multitude of vital tasks in all living organisms. Given that a protein's function and role are strongly related to its subcellular location, protein location prediction is an important research area. While proteins move from one location to another and can localize to multiple locations, most existing location prediction systems assign only a single location per protein. A few recent systems attempt to predict multiple locations for proteins, however, their performance leaves much room for improvement. Moreover, such systems do not capture dependencies among locations and usually consider locations as independent. We hypothesize that a multi-location predictor that captures location inter-dependencies can improve location predictions for proteins. We introduce a probabilistic generative model for protein localization, and develop a system based on it-which we call MDLoc-that utilizes inter-dependencies among locations to predict multiple locations for proteins. The model captures location inter-dependencies using Bayesian networks and represents dependency between features and locations using a mixture model. We use iterative processes for learning model parameters and for estimating protein locations. We evaluate our classifier MDLoc, on a dataset of single- and multi-localized proteins derived from the DBMLoc dataset, which is the most comprehensive protein multi-localization dataset currently available. Our results, obtained by using MDLoc, significantly improve upon results obtained by an initial simpler classifier, as well as on results reported by other top systems. MDLoc is available at: http://www.eecis.udel.edu/∼compbio/mdloc. © The Author 2015. Published by Oxford University Press.

Biological Evaluation of Single Cell Protein

International Nuclear Information System (INIS)

Hasan, I.A.; Mohamed, N.E.; El-Sayed, E.A.; Younis, N.A.

2011-01-01

In this study, the nutritional value of single cell protein (SCP) was evaluated as a non conventional protein source produced by fermenting fungal local strains of Trichoderma longibrachiatum, Aspergillus niger, Aspergillus terreus and Penicillium funiculosum with alkali treated sugar cane bagasse. Amino acid analysis revealed that the produced SCP contains essential and non essential amino acids. Male mice were fed on normal (basal) diet which contains 18% conventional protein and served as control group. In the second (T1) and the third (T2) group, the animals were fed on a diet in which 15% and 30% of conventional protein source were replaced by SCP, respectively. At intervals of 15, 30, 45 and 60 days, mice were sacrificed and the blood samples were collected for the biochemical evaluation. The daily averages of body weight were significantly higher with group T2 than group T1. Where as, the kidney weights in groups (T1) and (T2) were significantly increased as compared with control. A non significant difference between the tested groups in the enzyme activities of AST, ALT and GSH content of liver tissue were recorded. While, cholesterol and triglycerides contents showed a significant decrease in both (T1) and (T2) groups as compared with control. The recorded values of the serum hormone (T4), ALP activities, albumin and A/G ratio did not changed by the previous treatments. Serum levels of total protein, urea, creatinine and uric acid were higher for groups (T1) and (T2) than the control group. In conclusion, partial substitution of soy bean protein in mice diet with single cell protein (15%) improved the mice growth without any adverse effects on some of the physiological functions tested

Protein determination in single corns

International Nuclear Information System (INIS)

Knorr, J.; Schiekel, M.; Franke, W.; Focke, F.

1994-01-01

Determination of protein content in food materials is usually done by analyzing the nitrogen amount by wet chemical Kjeldahl method. An improved accuracy accompanied by smaller analyzing intervals can be achieved using nondestructive neutron activation. Analyses have been performed using 14 MeV neutrons to determine the content of N and P in single wheat corns. Irradiation parameters have been optimized to prevent serious radiation damage in grains. About 200 single corns have been investigated with total net weights ranging from 30 to 70 mg. The tested arrangement allows determination of nitrogen amount in a single corn down to 0.3 mg with an accuracy of better than 4 %. Mean nitrogen concentrations in the range from 9 to 19% per corn have been detected. (author) 5 refs.; 6 figs

Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells

Directory of Open Access Journals (Sweden)

Spyros Darmanis

2016-01-01

Full Text Available Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells.

Science.gov (United States)

Darmanis, Spyros; Gallant, Caroline Julie; Marinescu, Voichita Dana; Niklasson, Mia; Segerman, Anna; Flamourakis, Georgios; Fredriksson, Simon; Assarsson, Erika; Lundberg, Martin; Nelander, Sven; Westermark, Bengt; Landegren, Ulf

2016-01-12

Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell's phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Protein logic: a statistical mechanical study of signal integration at the single-molecule level.

Science.gov (United States)

de Ronde, Wiet; Rein ten Wolde, Pieter; Mugler, Andrew

2012-09-05

Information processing and decision-making is based upon logic operations, which in cellular networks has been well characterized at the level of transcription. In recent years, however, both experimentalists and theorists have begun to appreciate that cellular decision-making can also be performed at the level of a single protein, giving rise to the notion of protein logic. Here we systematically explore protein logic using a well-known statistical mechanical model. As an example system, we focus on receptors that bind either one or two ligands, and their associated dimers. Notably, we find that a single heterodimer can realize any of the 16 possible logic gates, including the XOR gate, by variation of biochemical parameters. We then introduce what to our knowledge is a novel idea: that a set of receptors with fixed parameters can encode functionally unique logic gates simply by forming different dimeric combinations. An exhaustive search reveals that the simplest set of receptors (two single-ligand receptors and one double-ligand receptor) can realize several different groups of three unique gates, a result for which the parametric analysis of single receptors and dimers provides a clear interpretation. Both results underscore the surprising functional freedom readily available to cells at the single-protein level. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Protein (multi-)location prediction: utilizing interdependencies via a generative model

Science.gov (United States)

Shatkay, Hagit

2015-01-01

Motivation: Proteins are responsible for a multitude of vital tasks in all living organisms. Given that a protein’s function and role are strongly related to its subcellular location, protein location prediction is an important research area. While proteins move from one location to another and can localize to multiple locations, most existing location prediction systems assign only a single location per protein. A few recent systems attempt to predict multiple locations for proteins, however, their performance leaves much room for improvement. Moreover, such systems do not capture dependencies among locations and usually consider locations as independent. We hypothesize that a multi-location predictor that captures location inter-dependencies can improve location predictions for proteins. Results: We introduce a probabilistic generative model for protein localization, and develop a system based on it—which we call MDLoc—that utilizes inter-dependencies among locations to predict multiple locations for proteins. The model captures location inter-dependencies using Bayesian networks and represents dependency between features and locations using a mixture model. We use iterative processes for learning model parameters and for estimating protein locations. We evaluate our classifier MDLoc, on a dataset of single- and multi-localized proteins derived from the DBMLoc dataset, which is the most comprehensive protein multi-localization dataset currently available. Our results, obtained by using MDLoc, significantly improve upon results obtained by an initial simpler classifier, as well as on results reported by other top systems. Availability and implementation: MDLoc is available at: http://www.eecis.udel.edu/∼compbio/mdloc. Contact: shatkay@udel.edu. PMID:26072505

Statistical inference in single molecule measurements of protein adsorption

Science.gov (United States)

Armstrong, Megan J.; Tsitkov, Stanislav; Hess, Henry

2018-02-01

Significant effort has been invested into understanding the dynamics of protein adsorption on surfaces, in particular to predict protein behavior at the specialized surfaces of biomedical technologies like hydrogels, nanoparticles, and biosensors. Recently, the application of fluorescent single molecule imaging to this field has permitted the tracking of individual proteins and their stochastic contribution to the aggregate dynamics of adsorption. However, the interpretation of these results is complicated by (1) the finite time available to observe effectively infinite adsorption timescales and (2) the contribution of photobleaching kinetics to adsorption kinetics. Here, we perform a protein adsorption simulation to introduce specific survival analysis methods that overcome the first complication. Additionally, we collect single molecule residence time data from the adsorption of fibrinogen to glass and use survival analysis to distinguish photobleaching kinetics from protein adsorption kinetics.

Automated Protein Structure Modeling with SWISS-MODEL Workspace and the Protein Model Portal

OpenAIRE

Bordoli, Lorenza; Schwede, Torsten

2012-01-01

Comparative protein structure modeling is a computational approach to build three-dimensional structural models for proteins using experimental structures of related protein family members as templates. Regular blind assessments of modeling accuracy have demonstrated that comparative protein structure modeling is currently the most reliable technique to model protein structures. Homology models are often sufficiently accurate to substitute for experimental structures in a wide variety of appl...

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

2014-01-01

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. PMID:24797261

Optical probing of single fluorescent molecules and proteins

NARCIS (Netherlands)

Garcia Parajo, M.F.; Veerman, J.A.; Bouwhuis, R.; Bouwhuis, Rudo; van Hulst, N.F.; Vallée, R.A.L.

2001-01-01

Single-molecule detection and analysis of organic fluorescent molecules and proteins are presented, with emphasis o­n the underlying principles methodology and the application of single-molecule analysis at room temperature. This Minireview is mainly focused o­n the application of confocal and

PRODt;CTION OF SINGLE CELL PROTEIN FROM BREWERY ...

African Journals Online (AJOL)

BSN

customary food and feed sources of protein (agriculnrre and fishery) to ocher sources like single cell protein (SCP); whose production from hydrocarbons is one ... origin is unicellular or simple multicellular organism such as bacteria, yeasts, fungi, algae. protozoa, mid even bacterinphagcs generally cultivated on substrates ...

Modeling complexes of modeled proteins.

Science.gov (United States)

Anishchenko, Ivan; Kundrotas, Petras J; Vakser, Ilya A

2017-03-01

Structural characterization of proteins is essential for understanding life processes at the molecular level. However, only a fraction of known proteins have experimentally determined structures. This fraction is even smaller for protein-protein complexes. Thus, structural modeling of protein-protein interactions (docking) primarily has to rely on modeled structures of the individual proteins, which typically are less accurate than the experimentally determined ones. Such "double" modeling is the Grand Challenge of structural reconstruction of the interactome. Yet it remains so far largely untested in a systematic way. We present a comprehensive validation of template-based and free docking on a set of 165 complexes, where each protein model has six levels of structural accuracy, from 1 to 6 Å C α RMSD. Many template-based docking predictions fall into acceptable quality category, according to the CAPRI criteria, even for highly inaccurate proteins (5-6 Å RMSD), although the number of such models (and, consequently, the docking success rate) drops significantly for models with RMSD > 4 Å. The results show that the existing docking methodologies can be successfully applied to protein models with a broad range of structural accuracy, and the template-based docking is much less sensitive to inaccuracies of protein models than the free docking. Proteins 2017; 85:470-478. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Transient intermediates are populated in the folding pathways of single-domain two-state folding protein L

Science.gov (United States)

Maity, Hiranmay; Reddy, Govardhan

2018-04-01

Small single-domain globular proteins, which are believed to be dominantly two-state folders, played an important role in elucidating various aspects of the protein folding mechanism. However, recent single molecule fluorescence resonance energy transfer experiments [H. Y. Aviram et al. J. Chem. Phys. 148, 123303 (2018)] on a single-domain two-state folding protein L showed evidence for the population of an intermediate state and it was suggested that in this state, a β-hairpin present near the C-terminal of the native protein state is unfolded. We performed molecular dynamics simulations using a coarse-grained self-organized-polymer model with side chains to study the folding pathways of protein L. In agreement with the experiments, an intermediate is populated in the simulation folding pathways where the C-terminal β-hairpin detaches from the rest of the protein structure. The lifetime of this intermediate structure increased with the decrease in temperature. In low temperature conditions, we also observed a second intermediate state, which is globular with a significant fraction of the native-like tertiary contacts satisfying the features of a dry molten globule.

An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.

Science.gov (United States)

Hamadani, Kambiz M; Howe, Jesse; Jensen, Madeleine K; Wu, Peng; Cate, Jamie H D; Marqusee, Susan

2017-09-22

Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational

Probing Enzyme-Surface Interactions via Protein Engineering and Single-Molecule Techniques

Science.gov (United States)

2017-06-26

SECURITY CLASSIFICATION OF: The overall objective of this research was to exploit protein engineering and fluorescence single-molecule methods to...enhance our understanding of the interaction of proteins and surfaces. Given this objective, the specific aims of this research were to: 1) exploit the...incorporation of unnatural amino acids in proteins to introduce single-molecule probes (i.e., fluorophores for fluorescence resonance energy transfer

Multiplex single-molecule interaction profiling of DNA barcoded proteins

Science.gov (United States)

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

2014-01-01

In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

A network model to correlate conformational change and the impedance spectrum of single proteins

Energy Technology Data Exchange (ETDEWEB)

Alfinito, Eleonora; Pennetta, Cecilia; Reggiani, Lino [Dipartimento di Ingegneria dell' Innovazione, Universita del Salento, Via Arnesano, Lecce (Italy); Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM) (Italy)

2008-02-13

Integrated nanodevices based on proteins or biomolecules are attracting increasing interest in today's research. In fact, it has been shown that proteins such as azurin and bacteriorhodopsin manifest some electrical properties that are promising for the development of active components of molecular electronic devices. Here we focus on two relevant kinds of protein: bovine rhodopsin, prototype of G-protein-coupled-receptor (GPCR) proteins, and the enzyme acetylcholinesterase (AChE), whose inhibition is one of the most qualified treatments of Alzheimer's disease. Both these proteins exert their function starting with a conformational change of their native structure. Our guess is that such a change should be accompanied with a detectable variation of their electrical properties. To investigate this conjecture, we present an impedance network model of proteins, able to estimate the different impedance spectra associated with the different configurations. The distinct types of conformational change of rhodopsin and AChE agree with their dissimilar electrical responses. In particular, for rhodopsin the model predicts variations of the impedance spectra up to about 30%, while for AChE the same variations are limited to about 10%, which supports the existence of a dynamical equilibrium between its native and complexed states.

Click strategies for single-molecule protein fluorescence.

Science.gov (United States)

Milles, Sigrid; Tyagi, Swati; Banterle, Niccolò; Koehler, Christine; VanDelinder, Virginia; Plass, Tilman; Neal, Adrian P; Lemke, Edward A

2012-03-21

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

International Nuclear Information System (INIS)

Shokri, Leila; Williams, Mark C; Rouzina, Ioulia

2009-01-01

Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork

Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA–Protein Interactions at the Single-Molecule Level

Directory of Open Access Journals (Sweden)

Huijun Xue

2017-10-01

Full Text Available Much of our knowledge in conventional biochemistry has derived from bulk assays. However, many stochastic processes and transient intermediates are hidden when averaged over the ensemble. The powerful technique of single-molecule fluorescence microscopy has made great contributions to the understanding of life processes that are inaccessible when using traditional approaches. In single-molecule studies, quantum dots (Qdots have several unique advantages over other fluorescent probes, such as high brightness, extremely high photostability, and large Stokes shift, thus allowing long-time observation and improved signal-to-noise ratios. So far, however, there is no convenient way to label proteins purified from budding yeast with Qdots. Based on BirA–Avi and biotin–streptavidin systems, we have established a simple method to acquire a Qdot-labeled protein and visualize its interaction with DNA using total internal reflection fluorescence microscopy. For proof-of-concept, we chose replication protein A (RPA and origin recognition complex (ORC as the proteins of interest. Proteins were purified from budding yeast with high biotinylation efficiency and rapidly labeled with streptavidin-coated Qdots. Interactions between proteins and DNA were observed successfully at the single-molecule level.

Semisynthetic protein nanoreactor for single-molecule chemistry

OpenAIRE

Lee, Joongoo; Bayley, Hagan

2015-01-01

The modulation of ionic current flowing through an individual protein pore provides information at the single-molecule level about chemical reactions occurring within the pore. However, chemistry investigated in this way has been largely confined to the reactions of thiolates, presented by the side chains of cysteine residues. The introduction of unnatural amino acids would provide a large variety of reactive side chains with which additional single-molecule chemistry could be investigated. H...

DockQ: A Quality Measure for Protein-Protein Docking Models.

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Sankar Basu

Full Text Available The state-of-the-art to assess the structural quality of docking models is currently based on three related yet independent quality measures: Fnat, LRMS, and iRMS as proposed and standardized by CAPRI. These quality measures quantify different aspects of the quality of a particular docking model and need to be viewed together to reveal the true quality, e.g. a model with relatively poor LRMS (>10Å might still qualify as 'acceptable' with a descent Fnat (>0.50 and iRMS (<3.0Å. This is also the reason why the so called CAPRI criteria for assessing the quality of docking models is defined by applying various ad-hoc cutoffs on these measures to classify a docking model into the four classes: Incorrect, Acceptable, Medium, or High quality. This classification has been useful in CAPRI, but since models are grouped in only four bins it is also rather limiting, making it difficult to rank models, correlate with scoring functions or use it as target function in machine learning algorithms. Here, we present DockQ, a continuous protein-protein docking model quality measure derived by combining Fnat, LRMS, and iRMS to a single score in the range [0, 1] that can be used to assess the quality of protein docking models. By using DockQ on CAPRI models it is possible to almost completely reproduce the original CAPRI classification into Incorrect, Acceptable, Medium and High quality. An average PPV of 94% at 90% Recall demonstrating that there is no need to apply predefined ad-hoc cutoffs to classify docking models. Since DockQ recapitulates the CAPRI classification almost perfectly, it can be viewed as a higher resolution version of the CAPRI classification, making it possible to estimate model quality in a more quantitative way using Z-scores or sum of top ranked models, which has been so valuable for the CASP community. The possibility to directly correlate a quality measure to a scoring function has been crucial for the development of scoring functions for

DockQ: A Quality Measure for Protein-Protein Docking Models

Science.gov (United States)

Basu, Sankar

2016-01-01

The state-of-the-art to assess the structural quality of docking models is currently based on three related yet independent quality measures: Fnat, LRMS, and iRMS as proposed and standardized by CAPRI. These quality measures quantify different aspects of the quality of a particular docking model and need to be viewed together to reveal the true quality, e.g. a model with relatively poor LRMS (>10Å) might still qualify as 'acceptable' with a descent Fnat (>0.50) and iRMS (iRMS to a single score in the range [0, 1] that can be used to assess the quality of protein docking models. By using DockQ on CAPRI models it is possible to almost completely reproduce the original CAPRI classification into Incorrect, Acceptable, Medium and High quality. An average PPV of 94% at 90% Recall demonstrating that there is no need to apply predefined ad-hoc cutoffs to classify docking models. Since DockQ recapitulates the CAPRI classification almost perfectly, it can be viewed as a higher resolution version of the CAPRI classification, making it possible to estimate model quality in a more quantitative way using Z-scores or sum of top ranked models, which has been so valuable for the CASP community. The possibility to directly correlate a quality measure to a scoring function has been crucial for the development of scoring functions for protein structure prediction, and DockQ should be useful in a similar development in the protein docking field. DockQ is available at http://github.com/bjornwallner/DockQ/ PMID:27560519

Protein dynamics revealed in the excitonic spectra of single LH2 complexes

International Nuclear Information System (INIS)

Valkunas, Leonas; Janusonis, Julius; Rutkauskas, Danielis; Grondelle, Rienk van

2007-01-01

The fluorescence emission spectrum of single peripheral light-harvesting (LH2) complexes of the photosynthetic purple bacterium Rhodopseudomonas acidophila exhibits remarkable dynamics on a time scale of several minutes. Often the spectral properties are quasi-stable; sometimes large spectral jumps to the blue or to the red are observed. To explain the dynamics, every pigment is proposed to be in two conformational substates with different excitation energies, which originate from the conformational state of the protein as a result of pigment-protein interaction. Due to the excitonic coupling in the ring of 18 pigments, the two-state assumption generates a substantial amount of distinct spectroscopic states, which reflect part of the inhomogeneous distributed spectral properties of LH2. To describe the observed dynamics, spontaneous and light-induced transitions are introduced between the two states. For each 'realization of the disorder', the spectral properties are calculated using a disordered exciton model combined with the modified Redfield theory to obtain realistic spectral line shapes. The single-molecule fluorescence peak (FLP) distribution, the distribution dependence on the excitation intensity, and the FLP time traces are well described within the framework of this model

Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii

OpenAIRE

Stroud, A. L.

2012-01-01

Replication protein A (RPA) is a single-stranded DNA-binding protein that is present in all three domains of life. The roles of RPA include stabilising and protecting single- stranded DNA from nuclease degradation during DNA replication and repair. To achieve this, RPA uses an oligosaccharide-binding fold (OB fold) to bind single- stranded DNA. Haloferax volcanii encodes three RPAs – RPA1, RPA2 and RPA3, of which rpa1 and rpa3 are in operons with genes encoding associated proteins (APs). ...

Expression of membrane-associated proteins within single emulsion cell facsimiles.

Science.gov (United States)

Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

2012-07-07

MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

Folding 19 proteins to their native state and stability of large proteins from a coarse-grained model.

Science.gov (United States)

Kapoor, Abhijeet; Travesset, Alex

2014-03-01

We develop an intermediate resolution model, where the backbone is modeled with atomic resolution but the side chain with a single bead, by extending our previous model (Proteins (2013) DOI: 10.1002/prot.24269) to properly include proline, preproline residues and backbone rigidity. Starting from random configurations, the model properly folds 19 proteins (including a mutant 2A3D sequence) into native states containing β sheet, α helix, and mixed α/β. As a further test, the stability of H-RAS (a 169 residue protein, critical in many signaling pathways) is investigated: The protein is stable, with excellent agreement with experimental B-factors. Despite that proteins containing only α helices fold to their native state at lower backbone rigidity, and other limitations, which we discuss thoroughly, the model provides a reliable description of the dynamics as compared with all atom simulations, but does not constrain secondary structures as it is typically the case in more coarse-grained models. Further implications are described. Copyright © 2013 Wiley Periodicals, Inc.

Microencapsulation of single-cell protein from various microalgae species

Directory of Open Access Journals (Sweden)

Purnama Sukardi

2015-10-01

Full Text Available ABSTRACT The objective of the research was to evaluate nutritional values of microencapsulated diet made from single cell protein of microalgae. Complete randomized design was applied using three different types of microalgae for inclusion trials i.e. (A Nannochloropsis sp., (B Chlorella sp., and (C Spirulina sp. with five replications respectively. Microencapsulated diet was produced by a modification method based on thermal cross-linking with stable temperature. Phytoplankton was cultured in sea water for which fertilized by a modification of Walne and Guillard fertilizer. The results showed that the highest value of nutrition content was Spirulina sp. and the average composition of protein, crude lipid, carbohydrate, ash, nitrogen free extract, and water content was 34.80%, 0.30%, 18.53%, 20.09%, 26.29%, and 13.32%, respectively. Organoleptically, microcapsule showed that the color of capsule was dark green and smell fresh phytoplankton. Keywords: microcapsule, single-cell protein, thermal cross-linking, microalgae, phytoplankton  ABSTRAK Tujuan penelitian adalah mengevaluasi kandungan nutrisi pakan mikrokapsul protein sel tunggal (single cell protein yang berasal dari berbagai jenis mikroalga (fitoplankton. Rancangan percobaan yang digunakan adalah rancangan acak lengkap, dengan perlakuan inklusi mikrokapsul dari jenis fitoplankton (A Nannochloropsis sp., (B Chlorella sp., dan (C Spirulina sp., masing-masing diulang lima kali. Pembuatan mikrokapsul dilakukan dengan menggunakan modifikasi metode dasar thermal cross-linking, serta menerapkan teknik pengeringan suhu konstan. Proses pembuatan mikrokapsul protein diawali dengan kultur fitoplankton jenis Nannochloropsis sp., Chlorella sp., dan Spirulina sp. Kultur dilakukan di dalam laboratorium menggunakan media air laut dan modifikasi pupuk Walne dan Guillard. Hasil penelitian menunjukkan bahwa kandungan nutrisi tertinggi terdapat pada jenis mikrokapsul protein sel tunggal yang berasal dari

Single proteins that serve linked functions in intracellular and extracellular microenvironments

Energy Technology Data Exchange (ETDEWEB)

Radisky, Derek C.; Stallings-Mann, Melody; Hirai, Yohei; Bissell, Mina J.

2009-06-03

Maintenance of organ homeostasis and control of appropriate response to environmental alterations requires intimate coordination of cellular function and tissue organization. An important component of this coordination may be provided by proteins that can serve distinct, but linked, functions on both sides of the plasma membrane. Here we present a novel hypothesis in which non-classical secretion can provide a mechanism through which single proteins can integrate complex tissue functions. Single genes can exert a complex, dynamic influence through a number of different processes that act to multiply the function of the gene product(s). Alternative splicing can create many different transcripts that encode proteins of diverse, even antagonistic, function from a single gene. Posttranslational modifications can alter the stability, activity, localization, and even basic function of proteins. A protein can exist in different subcellular localizations. More recently, it has become clear that single proteins can function both inside and outside the cell. These proteins often lack defined secretory signal sequences, and transit the plasma membrane by mechanisms separate from the classical ER/Golgi secretory process. When examples of such proteins are examined individually, the multifunctionality and lack of a signal sequence are puzzling - why should a protein with a well known function in one context function in such a distinct fashion in another? We propose that one reason for a single protein to perform intracellular and extracellular roles is to coordinate organization and maintenance of a global tissue function. Here, we describe in detail three specific examples of proteins that act in this fashion, outlining their specific functions in the extracellular space and in the intracellular space, and we discuss how these functions may be linked. We present epimorphin/syntaxin-2, which may coordinate morphogenesis of secretory organs (as epimorphin) with control of

A single peroxisomal targeting signal mediates matrix protein import in diatoms.

Directory of Open Access Journals (Sweden)

Nicola H Gonzalez

Full Text Available Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.

Coarse-grain modelling of protein-protein interactions

NARCIS (Netherlands)

Baaden, Marc; Marrink, Siewert J.

2013-01-01

Here, we review recent advances towards the modelling of protein-protein interactions (PPI) at the coarse-grained (CG) level, a technique that is now widely used to understand protein affinity, aggregation and self-assembly behaviour. PPI models of soluble proteins and membrane proteins are

Evaluation of yeast single cell protein (SCP) diets on growth ...

African Journals Online (AJOL)

An investigation was carried out on the possibility of replacing fishmeal with graded levels of yeast single cell protein (SCP; 10, 20, 30, 40 and 50%) in isonitrogenous feed formulations (30% protein) in the diet of Oreochromis niloticus fingerlings for a period of 12 weeks. The control diet had fishmeal as the primary protein ...

Protein Data Bank (PDB): The Single Global Macromolecular Structure Archive.

Science.gov (United States)

Burley, Stephen K; Berman, Helen M; Kleywegt, Gerard J; Markley, John L; Nakamura, Haruki; Velankar, Sameer

2017-01-01

The Protein Data Bank (PDB)--the single global repository of experimentally determined 3D structures of biological macromolecules and their complexes--was established in 1971, becoming the first open-access digital resource in the biological sciences. The PDB archive currently houses ~130,000 entries (May 2017). It is managed by the Worldwide Protein Data Bank organization (wwPDB; wwpdb.org), which includes the RCSB Protein Data Bank (RCSB PDB; rcsb.org), the Protein Data Bank Japan (PDBj; pdbj.org), the Protein Data Bank in Europe (PDBe; pdbe.org), and BioMagResBank (BMRB; www.bmrb.wisc.edu). The four wwPDB partners operate a unified global software system that enforces community-agreed data standards and supports data Deposition, Biocuration, and Validation of ~11,000 new PDB entries annually (deposit.wwpdb.org). The RCSB PDB currently acts as the archive keeper, ensuring disaster recovery of PDB data and coordinating weekly updates. wwPDB partners disseminate the same archival data from multiple FTP sites, while operating complementary websites that provide their own views of PDB data with selected value-added information and links to related data resources. At present, the PDB archives experimental data, associated metadata, and 3D-atomic level structural models derived from three well-established methods: crystallography, nuclear magnetic resonance spectroscopy (NMR), and electron microscopy (3DEM). wwPDB partners are working closely with experts in related experimental areas (small-angle scattering, chemical cross-linking/mass spectrometry, Forster energy resonance transfer or FRET, etc.) to establish a federation of data resources that will support sustainable archiving and validation of 3D structural models and experimental data derived from integrative or hybrid methods.

Single-molecule protein sequencing through fingerprinting: computational assessment

Science.gov (United States)

Yao, Yao; Docter, Margreet; van Ginkel, Jetty; de Ridder, Dick; Joo, Chirlmin

2015-10-01

Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences.

Single-molecule protein sequencing through fingerprinting: computational assessment

International Nuclear Information System (INIS)

Yao, Yao; Docter, Margreet; Van Ginkel, Jetty; Joo, Chirlmin; De Ridder, Dick

2015-01-01

Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences. (paper)

Compressive Force Spectroscopy: From Living Cells to Single Proteins.

Science.gov (United States)

Wang, Jiabin; Liu, Meijun; Shen, Yi; Sun, Jielin; Shao, Zhifeng; Czajkowsky, Daniel Mark

2018-03-23

One of the most successful applications of atomic force microscopy (AFM) in biology involves monitoring the effect of force on single biological molecules, often referred to as force spectroscopy. Such studies generally entail the application of pulling forces of different magnitudes and velocities upon individual molecules to resolve individualistic unfolding/separation pathways and the quantification of the force-dependent rate constants. However, a less recognized variation of this method, the application of compressive force, actually pre-dates many of these "tensile" force spectroscopic studies. Further, beyond being limited to the study of single molecules, these compressive force spectroscopic investigations have spanned samples as large as living cells to smaller, multi-molecular complexes such as viruses down to single protein molecules. Correspondingly, these studies have enabled the detailed characterization of individual cell states, subtle differences between seemingly identical viral structures, as well as the quantification of rate constants of functionally important, structural transitions in single proteins. Here, we briefly review some of the recent achievements that have been obtained with compressive force spectroscopy using AFM and highlight exciting areas of its future development.

MICROORGANISMS: A MARVELOUS SOURCE OF SINGLE CELL PROTEINS

Directory of Open Access Journals (Sweden)

Agam Nangul

2013-08-01

Full Text Available The increasing global population living below the poverty line is driving the scientific community to search for non-conventional protein sources that can replace conventional expensive ones. Microbial proteins, or single-cell protein (SCP, represent a potential future nutrient source for human food and animal feed. These microbial proteins can be grown rapidly on substrates with minimum dependence on soil, water and climate conditions. They can be produced from algae, fungi and bacteria the chief sources of SCP. It is convenient to use microorganisms for production of SCP as they grow rapidly and have high protein content. Industrially, they can be produced from algal biomass, yeast, fungi. There are several other ways of getting SCP as well. Despite numerous advantages of SCP, they have disadvantages and toxic effects too, especially related to mycotoxins and bacterial toxins.

Single-molecule mechanics of protein-labelled DNA handles

Directory of Open Access Journals (Sweden)

Vivek S. Jadhav

2016-01-01

Full Text Available DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular

The Protein Model Portal

OpenAIRE

Arnold, Konstantin; Kiefer, Florian; Kopp, J?rgen; Battey, James N. D.; Podvinec, Michael; Westbrook, John D.; Berman, Helen M.; Bordoli, Lorenza; Schwede, Torsten

2008-01-01

Structural Genomics has been successful in determining the structures of many unique proteins in a high throughput manner. Still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. Homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. Thereby, experimental structure determination efforts and homology modeling complement each other in the exploratio...

Conversion of Food waste to Single Cell Protein using Aspergillus ...

African Journals Online (AJOL)

The utilization of food waste into products like single cell protein is an alternative solution to global protein shortage and to alleviate pollution problems. This investigation was carried out with food wastes such as orange, pineapple, banana, watermelon and cucumber waste as growth media for A. niger using standard ...

PRODt;CTION OF SINGLE CELL PROTEIN FROM BREWERY ...

African Journals Online (AJOL)

BSN

origin is unicellular or simple multicellular organism such as bacteria, yeasts, fungi, ... Pilot plant produe1io11 of single cell proteins now take place in several centre.ii in ... animal feed but little or no information has been documented as per its ...

Atomic force microscopy and spectroscopy to probe single membrane proteins in lipid bilayers.

Science.gov (United States)

Sapra, K Tanuj

2013-01-01

The atomic force microscope (AFM) has opened vast avenues hitherto inaccessible to the biological scientist. The high temporal (millisecond) and spatial (nanometer) resolutions of the AFM are suited for studying many biological processes in their native conditions. The AFM cantilever stylus is aptly termed as a "lab on a tip" owing to its versatility as an imaging tool as well as a handle to manipulate single bonds and proteins. Recent examples assert that the AFM can be used to study the mechanical properties and monitor processes of single proteins and single cells, thus affording insight into important mechanistic details. This chapter specifically focuses on practical and analytical protocols of single-molecule AFM methodologies related to high-resolution imaging and single-molecule force spectroscopy of membrane proteins. Both these techniques are operator oriented, and require specialized working knowledge of the instrument, theoretical, and practical skills.

Analysis and Ranking of Protein-Protein Docking Models Using Inter-Residue Contacts and Inter-Molecular Contact Maps

KAUST Repository

Oliva, Romina; Chermak, Edrisse; Cavallo, Luigi

2015-01-01

In view of the increasing interest both in inhibitors of protein-protein interactions and in protein drugs themselves, analysis of the three-dimensional structure of protein-protein complexes is assuming greater relevance in drug design. In the many cases where an experimental structure is not available, protein-protein docking becomes the method of choice for predicting the arrangement of the complex. However, reliably scoring protein-protein docking poses is still an unsolved problem. As a consequence, the screening of many docking models is usually required in the analysis step, to possibly single out the correct ones. Here, making use of exemplary cases, we review our recently introduced methods for the analysis of protein complex structures and for the scoring of protein docking poses, based on the use of inter-residue contacts and their visualization in inter-molecular contact maps. We also show that the ensemble of tools we developed can be used in the context of rational drug design targeting protein-protein interactions.

Analysis and Ranking of Protein-Protein Docking Models Using Inter-Residue Contacts and Inter-Molecular Contact Maps

KAUST Repository

Oliva, Romina

2015-07-01

In view of the increasing interest both in inhibitors of protein-protein interactions and in protein drugs themselves, analysis of the three-dimensional structure of protein-protein complexes is assuming greater relevance in drug design. In the many cases where an experimental structure is not available, protein-protein docking becomes the method of choice for predicting the arrangement of the complex. However, reliably scoring protein-protein docking poses is still an unsolved problem. As a consequence, the screening of many docking models is usually required in the analysis step, to possibly single out the correct ones. Here, making use of exemplary cases, we review our recently introduced methods for the analysis of protein complex structures and for the scoring of protein docking poses, based on the use of inter-residue contacts and their visualization in inter-molecular contact maps. We also show that the ensemble of tools we developed can be used in the context of rational drug design targeting protein-protein interactions.

Membrane Compartmentalization Reducing the Mobility of Lipids and Proteins within a Model Plasma Membrane.

Science.gov (United States)

Koldsø, Heidi; Reddy, Tyler; Fowler, Philip W; Duncan, Anna L; Sansom, Mark S P

2016-09-01

The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate

Directory of Open Access Journals (Sweden)

Jeong Y

2015-11-01

Full Text Available Yoon Jeong,1,2 Kwan Hong Lee,1,2 Hansoo Park,3 Jonghoon Choi1,2 1Department of Bionano Technology, Graduate School, Hanyang University, Seoul, 2Department of Bionano Engineering, Hanyang University ERICA, Ansan, 3School of Integrative Engineering, Chung-Ang University, Seoul, South Korea Abstract: We present an evaluation of protein-G-terminated glass slides that may contain a suitable substrate for aligning the orientation of antibodies to obtain better binding moiety to the target antigen. The results of the protein-G-terminated slides were compared with those obtained with epoxy-based slides to evaluate signal enhancement for human immunoglobulin G (IgG targets, and an increase in the average fluorescence intensity was observed for the lowest measurable amount of IgG target in the assay using protein-G-terminated slides. Applying this strategy for signal amplification to single-cell assays improves the limits of detection for human IgG protein and cytokines (interleukin-2 and interferon-γ captured from hybridomas. Our data indicate that protein-G-terminated slides have a higher binding capacity for antigens and have better spot-to-spot consistency than that of traditional epoxy-based slides. These properties would be beneficial in the detection of fine amounts of single-cell-secreted proteins, which may provide key insights into cell–cell communication and immune responses. Keywords: microwell array, antibody’s orientation, single cell analysis, secreted cytokine, protein-G-terminated surface

Zinc(II) and the single-stranded DNA binding protein of bacteriophage T4

International Nuclear Information System (INIS)

Gauss, P.; Krassa, K.B.; McPheeters, D.S.; Nelson, M.A.; Gold, L.

1987-01-01

The DNA binding domain of the gene 32 protein of the bacteriophage T4 contains a single zinc-finger sequence. The gene 32 protein is an extensively studied member of a class of proteins that bind relatively nonspecifically to single-stranded DNA. The authors have sequenced and characterized mutations in gene 32 whose defective proteins are activated by increasing the Zn(II) concentration in the growth medium. The results identify a role for the gene 32 protein in activation of T4 late transcription. Several eukaryotic proteins with zinc fingers participate in activation of transcription, and the gene 32 protein of T4 should provide a simple, well-characterized system in which genetics can be utilized to study the role of a zinc finger in nucleic acid binding and gene expression

Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

Science.gov (United States)

Ullal, Adeeti V; Weissleder, Ralph

2015-01-01

We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

Anomalous diffusion in neutral evolution of model proteins

Science.gov (United States)

Nelson, Erik D.; Grishin, Nick V.

2015-06-01

Protein evolution is frequently explored using minimalist polymer models, however, little attention has been given to the problem of structural drift, or diffusion. Here, we study neutral evolution of small protein motifs using an off-lattice heteropolymer model in which individual monomers interact as low-resolution amino acids. In contrast to most earlier models, both the length and folded structure of the polymers are permitted to change. To describe structural change, we compute the mean-square distance (MSD) between monomers in homologous folds separated by n neutral mutations. We find that structural change is episodic, and, averaged over lineages (for example, those extending from a single sequence), exhibits a power-law dependence on n . We show that this exponent depends on the alignment method used, and we analyze the distribution of waiting times between neutral mutations. The latter are more disperse than for models required to maintain a specific fold, but exhibit a similar power-law tail.

Quantification of protein based on single-molecule counting by total internal reflection fluorescence microscopy with adsorption equilibrium

International Nuclear Information System (INIS)

Wang Lei; Xu Guang; Shi Zhikun; Jiang Wei; Jin Wenrui

2007-01-01

We developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy with adsorption equilibrium. In this method, the adsorption equilibrium of protein was achieved between solution and glass substrate. Then, fluorescence images of protein molecules in a evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally, the number of fluorescent spots corresponding to the protein molecules in the images was counted. Alexa Fluor 488-labeled goat anti-rat IgG(H + L) was chosen as the model protein. The spot number showed an excellent linear relationship with protein concentration. The concentration linear range was 5.4 x 10 -11 to 8.1 x 10 -10 mol L -1

Concentrated Solutions of Single-Chain Nanoparticles: A Simple Model for Intrinsically Disordered Proteins under Crowding Conditions.

Science.gov (United States)

Moreno, Angel J; Lo Verso, Federica; Arbe, Arantxa; Pomposo, José A; Colmenero, Juan

2016-03-03

By means of large-scale computer simulations and small-angle neutron scattering (SANS), we investigate solutions of single-chain nanoparticles (SCNPs), covering the whole concentration range from infinite dilution to melt density. The analysis of the conformational properties of the SCNPs reveals that these synthetic nano-objects share basic ingredients with intrinsically disordered proteins (IDPs), as topological polydispersity, generally sparse conformations, and locally compact domains. We investigate the role of the architecture of the SCNPs in their collapse behavior under macromolecular crowding. Unlike in the case of linear macromolecules, which experience the usual transition from self-avoiding to Gaussian random-walk conformations, crowding leads to collapsed conformations of SCNPs resembling those of crumpled globules. This behavior is already found at volume fractions (about 30%) that are characteristic of crowding in cellular environments. The simulation results are confirmed by the SANS experiments. Our results for SCNPs--a model system free of specific interactions--propose a general scenario for the effect of steric crowding on IDPs: collapse from sparse conformations at high dilution to crumpled globular conformations in cell environments.

Modeling the Activity of Single Genes

Science.gov (United States)

Mjolsness, Eric; Gibson, Michael

1999-01-01

the key questions in gene regulation are: What genes are expressed in a certain cell at a certain time? How does gene expression differ from cell to cell in a multicellular organism? Which proteins act as transcription factors, i.e., are important in regulating gene expression? From questions like these, we hope to understand which genes are important for various macroscopic processes. Nearly all of the cells of a multicellular organism contain the same DNA. Yet this same genetic information yields a large number of different cell types. The fundamental difference between a neuron and a liver cell, for example, is which genes are expressed. Thus understanding gene regulation is an important step in understanding development. Furthermore, understanding the usual genes that are expressed in cells may give important clues about various diseases. Some diseases, such as sickle cell anemia and cystic fibrosis, are caused by defects in single, non-regulatory genes; others, such as certain cancers, are caused when the cellular control circuitry malfunctions - an understanding of these diseases will involve pathways of multiple interacting gene products. There are numerous challenges in the area of understanding and modeling gene regulation. First and foremost, biologists would like to develop a deeper understanding of the processes involved, including which genes and families of genes are important, how they interact, etc. From a computation point of view, there has been embarrassingly little work done. In this chapter there are many areas in which we can phrase meaningful, non-trivial computational questions, but questions that have not been addressed. Some of these are purely computational (what is a good algorithm for dealing with a model of type X) and others are more mathematical (given a system with certain characteristics, what sort of model can one use? How does one find biochemical parameters from system-level behavior using as few experiments as possible?). In

Electronic transport in single-helical protein molecules: Effects of multiple charge conduction pathways and helical symmetry

Energy Technology Data Exchange (ETDEWEB)

Kundu, Sourav, E-mail: sourav.kunduphy@gmail.com; Karmakar, S.N.

2016-07-15

We propose a tight-binding model to investigate electronic transport properties of single helical protein molecules incorporating both the helical symmetry and the possibility of multiple charge transfer pathways. Our study reveals that due to existence of both the multiple charge transfer pathways and helical symmetry, the transport properties are quite rigid under influence of environmental fluctuations which indicates that these biomolecules can serve as better alternatives in nanoelectronic devices than its other biological counterparts e.g., single-stranded DNA.

Shedding Light on Protein Folding, Structural and Functional Dynamics by Single Molecule Studies

Directory of Open Access Journals (Sweden)

Krutika Bavishi

2014-11-01

Full Text Available The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions.

Protein-Nanocrystal Conjugates Support a Single Filament Polymerization Model in R1 Plasmid Segregation

Energy Technology Data Exchange (ETDEWEB)

Choi, Charina L.; Claridge, Shelley A.; Garner, Ethan C.; Alivisatos, A. Paul; Mullins, R. Dyche

2008-07-15

To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. Wefound that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmidssegregating as a unit.

Single cell protein from mandarin orange peel

Energy Technology Data Exchange (ETDEWEB)

Mishio, M.; Magai, J.

1981-01-01

As the hydrolysis of mandarin orange peel with macerating enzyme (40 degrees C, 24 h) produced 0.59 g g-1 reducing sugar per dry peel compared to 0.36 by acid-hydrolysis (15 min at 120 degrees C with 0.8 N H2S04), the production of single cell protein (SCP) from orange peel was studied mostly using enzymatically hydrolyzed orange peel. When the enzymatically hydrolyzed peel media were used, the utilization efficiency of reducing sugars (%) and the growth yield from reducing sugars (g g-1) were: 63 and 0.51 for Saccharomyces cerevisiae; 56 and 0.48 for Candida utilis; 74 and 0.69 for Debaryomyces hansenii and 64 and 0.70 for Rhodotorula glutinis. SCP production from orange peel by D. hansenii and R. glutinis were further studied. Batch cultures for 24 h at 30 degrees C using 100g dried orange peel produced 45 g of dried cultivated peel (protein content, 33%) with D. hansenii and 34 g (protein content, 50%) with R. glutinis, and 38 g (protein content, 44%) with a mixture of both yeasts. (Refs. 12).

Exploring abiotic stress on asynchronous protein metabolism in single kernels of wheat studied by NMR spectroscopy and chemometrics

DEFF Research Database (Denmark)

Winning, H.; Viereck, N.; Wollenweber, B.

2009-01-01

at the vegetative growth stage had little effect on the parameters investigated. For the first time, H-1 HR-MAS NMR spectra of grains taken during grain-filling were analysed by an advanced multiway model. In addition to the results from the chemical protein analysis and the H-1 HR-MAS NMR spectra of single kernels...... was to examine the implications of different drought treatments on the protein fractions in grains of winter wheat using H-1 nuclear magnetic resonance spectroscopy followed by chemometric analysis. Triticum aestivum L. cv. Vinjett was studied in a semi-field experiment and subjected to drought episodes either...... at terminal spikelet, during grain-filling or at both stages. Principal component trajectories of the total protein content and the protein fractions of flour as well as the H-1 NMR spectra of single wheat kernels, wheat flour, and wheat methanol extracts were analysed to elucidate the metabolic development...

Stochastic lattice model of synaptic membrane protein domains.

Science.gov (United States)

Li, Yiwei; Kahraman, Osman; Haselwandter, Christoph A

2017-05-01

Neurotransmitter receptor molecules, concentrated in synaptic membrane domains along with scaffolds and other kinds of proteins, are crucial for signal transmission across chemical synapses. In common with other membrane protein domains, synaptic domains are characterized by low protein copy numbers and protein crowding, with rapid stochastic turnover of individual molecules. We study here in detail a stochastic lattice model of the receptor-scaffold reaction-diffusion dynamics at synaptic domains that was found previously to capture, at the mean-field level, the self-assembly, stability, and characteristic size of synaptic domains observed in experiments. We show that our stochastic lattice model yields quantitative agreement with mean-field models of nonlinear diffusion in crowded membranes. Through a combination of analytic and numerical solutions of the master equation governing the reaction dynamics at synaptic domains, together with kinetic Monte Carlo simulations, we find substantial discrepancies between mean-field and stochastic models for the reaction dynamics at synaptic domains. Based on the reaction and diffusion properties of synaptic receptors and scaffolds suggested by previous experiments and mean-field calculations, we show that the stochastic reaction-diffusion dynamics of synaptic receptors and scaffolds provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the observed single-molecule trajectories, and spatial heterogeneity in the effective rates at which receptors and scaffolds are recycled at the cell membrane. Our work sheds light on the physical mechanisms and principles linking the collective properties of membrane protein domains to the stochastic dynamics that rule their molecular components.

The Protein Model Portal--a comprehensive resource for protein structure and model information.

Science.gov (United States)

Haas, Juergen; Roth, Steven; Arnold, Konstantin; Kiefer, Florian; Schmidt, Tobias; Bordoli, Lorenza; Schwede, Torsten

2013-01-01

The Protein Model Portal (PMP) has been developed to foster effective use of 3D molecular models in biomedical research by providing convenient and comprehensive access to structural information for proteins. Both experimental structures and theoretical models for a given protein can be searched simultaneously and analyzed for structural variability. By providing a comprehensive view on structural information, PMP offers the opportunity to apply consistent assessment and validation criteria to the complete set of structural models available for proteins. PMP is an open project so that new methods developed by the community can contribute to PMP, for example, new modeling servers for creating homology models and model quality estimation servers for model validation. The accuracy of participating modeling servers is continuously evaluated by the Continuous Automated Model EvaluatiOn (CAMEO) project. The PMP offers a unique interface to visualize structural coverage of a protein combining both theoretical models and experimental structures, allowing straightforward assessment of the model quality and hence their utility. The portal is updated regularly and actively developed to include latest methods in the field of computational structural biology. Database URL: http://www.proteinmodelportal.org.

The Protein Model Portal—a comprehensive resource for protein structure and model information

Science.gov (United States)

Haas, Juergen; Roth, Steven; Arnold, Konstantin; Kiefer, Florian; Schmidt, Tobias; Bordoli, Lorenza; Schwede, Torsten

2013-01-01

The Protein Model Portal (PMP) has been developed to foster effective use of 3D molecular models in biomedical research by providing convenient and comprehensive access to structural information for proteins. Both experimental structures and theoretical models for a given protein can be searched simultaneously and analyzed for structural variability. By providing a comprehensive view on structural information, PMP offers the opportunity to apply consistent assessment and validation criteria to the complete set of structural models available for proteins. PMP is an open project so that new methods developed by the community can contribute to PMP, for example, new modeling servers for creating homology models and model quality estimation servers for model validation. The accuracy of participating modeling servers is continuously evaluated by the Continuous Automated Model EvaluatiOn (CAMEO) project. The PMP offers a unique interface to visualize structural coverage of a protein combining both theoretical models and experimental structures, allowing straightforward assessment of the model quality and hence their utility. The portal is updated regularly and actively developed to include latest methods in the field of computational structural biology. Database URL: http://www.proteinmodelportal.org PMID:23624946

Whey utilization for single-cell protein production

Energy Technology Data Exchange (ETDEWEB)

Barraquio, V; Silverio, L G; Revilleza, R P; Fernadez, W L

1980-01-01

The production of single-cell protein by yeast assimilation of lactose in soft cheese whey was studied using Candida pseudotropicalis as a test organism. Under shake-flask cultivation conditions with deproteinized whey as the medium, lactose (initially 4.20%) was completely assimilated in 48h; cell mass was 5.56 mg/mL after 72h; and average protein content of the dried mass was approximately 11.8%. Batch cultivation using undeproteinized whey resulted in a faster lactose utilization rate from an initial 3.93% to a residual 0.56% in 12 h; cell mass was 8.41 mg/mL in 10 h; and average protein was approximately 37.7%. In a semicontinuous culture with 10 to the power of 7 viable cells/mL as initial cell concentration, 15.69 mg/mL cell mass with a mean protein content of approximately 21.4% could be produced and lactose could be considerably consumed (from an initial 4.75% to a residual 0.42%) within 13-14 h. Supplementation with (NH/sub 4/)/sub 2/S0/sub 4/ and KH/sub 2/P0/sub 4/ did not increase cell mass (12.47 mg/mL in 12 h) and hasten lactose assimulation (from initial 4.49% to residual 0.3% in 12 h). Average protein content was approximately 31%. Cell mass yield was established as 0.29 mg yeast cell/mg lactose consumed. Factors that might have affected protein content are also discussed.

Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range.

Science.gov (United States)

Milles, Sigrid; Koehler, Christine; Gambin, Yann; Deniz, Ashok A; Lemke, Edward A

2012-10-01

Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins.

Observation of Single-Protein and DNA Macromolecule Collisions on Ultramicroelectrodes.

Science.gov (United States)

Dick, Jeffrey E; Renault, Christophe; Bard, Allen J

2015-07-08

Single-molecule detection is the ultimate sensitivity in analytical chemistry and has been largely unavailable in electrochemical analysis. Here, we demonstrate the feasibility of detecting electrochemically inactive single biomacromolecules, such as enzymes, antibodies, and DNA, by blocking a solution redox reaction when molecules adsorb and block electrode sites. By oxidizing a large concentration of potassium ferrocyanide on an ultramicroelectrode (UME, radius ≤150 nm), time-resolved, discrete adsorption events of antibodies, enzymes, DNA, and polystyrene nanospheres can be differentiated from the background by their "footprint". Further, by assuming that the mass transport of proteins to the electrode surface is controlled mainly by diffusion, a size estimate using the Stokes-Einstein relationship shows good agreement of electrochemical data with known protein sizes.

The PMDB Protein Model Database

Science.gov (United States)

Castrignanò, Tiziana; De Meo, Paolo D'Onorio; Cozzetto, Domenico; Talamo, Ivano Giuseppe; Tramontano, Anna

2006-01-01

The Protein Model Database (PMDB) is a public resource aimed at storing manually built 3D models of proteins. The database is designed to provide access to models published in the scientific literature, together with validating experimental data. It is a relational database and it currently contains >74 000 models for ∼240 proteins. The system is accessible at and allows predictors to submit models along with related supporting evidence and users to download them through a simple and intuitive interface. Users can navigate in the database and retrieve models referring to the same target protein or to different regions of the same protein. Each model is assigned a unique identifier that allows interested users to directly access the data. PMID:16381873

Mechanical strength of 17,134 model proteins and cysteine slipknots.

Directory of Open Access Journals (Sweden)

Mateusz Sikora

2009-10-01

Full Text Available A new theoretical survey of proteins' resistance to constant speed stretching is performed for a set of 17,134 proteins as described by a structure-based model. The proteins selected have no gaps in their structure determination and consist of no more than 250 amino acids. Our previous studies have dealt with 7510 proteins of no more than 150 amino acids. The proteins are ranked according to the strength of the resistance. Most of the predicted top-strength proteins have not yet been studied experimentally. Architectures and folds which are likely to yield large forces are identified. New types of potent force clamps are discovered. They involve disulphide bridges and, in particular, cysteine slipknots. An effective energy parameter of the model is estimated by comparing the theoretical data on characteristic forces to the corresponding experimental values combined with an extrapolation of the theoretical data to the experimental pulling speeds. These studies provide guidance for future experiments on single molecule manipulation and should lead to selection of proteins for applications. A new class of proteins, involving cysteine slipknots, is identified as one that is expected to lead to the strongest force clamps known. This class is characterized through molecular dynamics simulations.

Trichoderma Reesei single cell protein production from rice straw pulp in solid state fermentation

Science.gov (United States)

Zaki, M.; Said, S. D.

2018-04-01

The dependency on fish meal as a major protein source for animal feed can lead toit priceinstability in line with the increasing in meat production and consumption in Indonesia. In order todeal with this problem, an effort to produce an alternative protein sources production is needed. This scenario is possible due to the abundantavailability of agricultural residues such as rice straw whichcould be utilized as substrate for production of single cell proteins as an alternative proteinsource. This work investigated the potential utilization of rice straw pulp and urea mixture as substrate for the production of local Trichoderma reesei single cell protein in solid state fermentation system. Some parameters have been analyzed to evaluate the effect of ratio of rice straw pulp to urea on mixed single cell protein biomass (mixed SCP biomass) composition, such as total crude protein (analyzed by kjedhal method) and lignin content (TAPPI method).The results showed that crude protein content in mixed SCP biomassincreases with the increasing in fermentation time, otherwise it decreases with the increasing insubstrate carbon to nitrogen (C/N) ratio. Residual lignin content in mixed SCP biomass decreases from 7% to 0.63% during fermentationproceeded of 21 days. The highest crude protein content in mixed SCP biomasswas obtained at substrate C/N ratio 20:1 of 25%.

A high-throughput 2D-analytical technique to obtain single protein parameters from complex cell lysates for in silico process development of ion exchange chromatography.

Science.gov (United States)

Kröner, Frieder; Elsäßer, Dennis; Hubbuch, Jürgen

2013-11-29

The accelerating growth of the market for biopharmaceutical proteins, the market entry of biosimilars and the growing interest in new, more complex molecules constantly pose new challenges for bioseparation process development. In the presented work we demonstrate the application of a multidimensional, analytical separation approach to obtain the relevant physicochemical parameters of single proteins in a complex mixture for in silico chromatographic process development. A complete cell lysate containing a low titre target protein was first fractionated by multiple linear salt gradient anion exchange chromatography (AEC) with varying gradient length. The collected fractions were subsequently analysed by high-throughput capillary gel electrophoresis (HT-CGE) after being desalted and concentrated. From the obtained data of the 2D-separation the retention-volumes and the concentration of the single proteins were determined. The retention-volumes of the single proteins were used to calculate the related steric-mass action model parameters. In a final evaluation experiment the received parameters were successfully applied to predict the retention behaviour of the single proteins in salt gradient AEC. Copyright © 2013 Elsevier B.V. All rights reserved.

Single vector system for efficient N-myristoylation of recombinant proteins in E. coli.

Directory of Open Access Journals (Sweden)

Julian M Glück

Full Text Available BACKGROUND: N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT. Prokaryotes are lacking endogenous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium. CONCLUSIONS/SIGNIFICANCE: The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.

Modeling disordered regions in proteins using Rosetta.

Directory of Open Access Journals (Sweden)

Ray Yu-Ruei Wang

Full Text Available Protein structure prediction methods such as Rosetta search for the lowest energy conformation of the polypeptide chain. However, the experimentally observed native state is at a minimum of the free energy, rather than the energy. The neglect of the missing configurational entropy contribution to the free energy can be partially justified by the assumption that the entropies of alternative folded states, while very much less than unfolded states, are not too different from one another, and hence can be to a first approximation neglected when searching for the lowest free energy state. The shortcomings of current structure prediction methods may be due in part to the breakdown of this assumption. Particularly problematic are proteins with significant disordered regions which do not populate single low energy conformations even in the native state. We describe two approaches within the Rosetta structure modeling methodology for treating such regions. The first does not require advance knowledge of the regions likely to be disordered; instead these are identified by minimizing a simple free energy function used previously to model protein folding landscapes and transition states. In this model, residues can be either completely ordered or completely disordered; they are considered disordered if the gain in entropy outweighs the loss of favorable energetic interactions with the rest of the protein chain. The second approach requires identification in advance of the disordered regions either from sequence alone using for example the DISOPRED server or from experimental data such as NMR chemical shifts. During Rosetta structure prediction calculations the disordered regions make only unfavorable repulsive contributions to the total energy. We find that the second approach has greater practical utility and illustrate this with examples from de novo structure prediction, NMR structure calculation, and comparative modeling.

Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.

Science.gov (United States)

Parvez, Saba; Fu, Yuan; Li, Jiayang; Long, Marcus J C; Lin, Hong-Yu; Lee, Dustin K; Hu, Gene S; Aye, Yimon

2015-01-14

Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains untested. Herein, using T-REX (targetable reactive electrophiles and oxidants), an approach aimed at selectively flipping a single redox switch in cells at a precise time, we show that a modest level (∼34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology.

Towards single-molecule observation of protein synthesis

International Nuclear Information System (INIS)

Dulin, David; Le Gall, Antoine; Bouyer, Philippe; Perronet, Karen; Westbrook, Nathalie; Soler, Nicolas; Fourmy, Dominique; Yoshizawa, Satoko

2009-01-01

The ribosome is the molecular motor responsible for the protein synthesis within all cells. Ribosome motions along the messenger RNA (mRNA) to read the genetic code are asynchronous and occur along multiple kinetic paths. Consequently, a study at the single macromolecule level is desirable to unravel the complex dynamics involved. In this communication, we present the development of an advanced surface chemistry to attach an active ribosome to the microscope coverslip and follow the amino-acid incorporation by fluorescence microscopy. The ribosome is labeled with a quantum dot (QD) in order to localize it on the surface while a specific amino acid (lysine) is marked with Bodipy-FL. This fluorescent dye is small enough to enter the ribosomal channel thus leaving intact ribosomal activity. It should then be possible to observe the protein synthesis in real time as the labeled amino acids are incorporated into the polypeptide chain. (Author)

Protein model discrimination using mutational sensitivity derived from deep sequencing.

Science.gov (United States)

Adkar, Bharat V; Tripathi, Arti; Sahoo, Anusmita; Bajaj, Kanika; Goswami, Devrishi; Chakrabarti, Purbani; Swarnkar, Mohit K; Gokhale, Rajesh S; Varadarajan, Raghavan

2012-02-08

A major bottleneck in protein structure prediction is the selection of correct models from a pool of decoys. Relative activities of ∼1,200 individual single-site mutants in a saturation library of the bacterial toxin CcdB were estimated by determining their relative populations using deep sequencing. This phenotypic information was used to define an empirical score for each residue (RankScore), which correlated with the residue depth, and identify active-site residues. Using these correlations, ∼98% of correct models of CcdB (RMSD ≤ 4Å) were identified from a large set of decoys. The model-discrimination methodology was further validated on eleven different monomeric proteins using simulated RankScore values. The methodology is also a rapid, accurate way to obtain relative activities of each mutant in a large pool and derive sequence-structure-function relationships without protein isolation or characterization. It can be applied to any system in which mutational effects can be monitored by a phenotypic readout. Copyright © 2012 Elsevier Ltd. All rights reserved.

Single cell protein production from mandarin orange peel

Energy Technology Data Exchange (ETDEWEB)

Nishio, N.; Nagai, S.

1981-01-01

As the hydrolysis of mandarin orange peel with macerating enzyme (40/sup 0/C,24 h)produced 0.59 g g/sup -1/ reducing sugar per dry peel compared to 0.36 by acid-hydrolysis (15 min at 120/sup 0/C with 0.8 N H/sub 2/SO/sub 4/), the production of single cell protein (SCP) from orange peel was studied mostly using enzymatically hydrolyzed orange peel. When the enzymatically hydrolyzed peel media were used, the utilization efficiency of reducing sugars (%) and the growth yield from reducing sugars (gg/sup -1/)were: 63 and 0.51 for Saccharomyces cerevisiae; 56 and 0.48 for Candida utilis; 74 and 0.69 for Debaryomyces hansenii and 64 and 0.70 for Rhodotorula glutinis. SCP production from orange peel by D. hansenii and R. glutinis were further studied. Batch cultures for 24 h at 30/sup 0/C using 100 g dried orange peel produced 45 g of dried cultivated peel (protein content, 33%) with D. hansenii and 34 g (protein content, 50%) with R. glutinis, and 38 g (protein content, 44%) with a mixture of both yeasts.

Expression, purification and biochemical characterization of a single-stranded DNA binding protein from Herbaspirillum seropedicae.

Science.gov (United States)

Vernal, Javier; Serpa, Viviane I; Tavares, Carolina; Souza, Emanuel M; Pedrosa, Fábio O; Terenzi, Hernán

2007-05-01

An open reading frame encoding a protein similar in size and sequence to the Escherichia coli single-stranded DNA binding protein (SSB protein) was identified in the Herbaspirillum seropedicae genome. This open reading frame was cloned into the expression plasmid pET14b. The SSB protein from H. seropedicae, named Hs_SSB, was overexpressed in E. coli strain BL21(DE3) and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein. The apparent molecular mass of the native Hs_SSB was estimated by gel filtration, suggesting that the native protein is a tetramer made up of four similar subunits. The purified protein binds to single-stranded DNA (ssDNA) in a similar manner to other SSB proteins. The production of this recombinant protein in good yield opens up the possibility of obtaining its 3D-structure and will help further investigations into DNA metabolism.

Protein buffering in model systems and in whole human saliva.

Directory of Open Access Journals (Sweden)

Andreas Lamanda

Full Text Available The aim of this study was to quantify the buffer attributes (value, power, range and optimum of two model systems for whole human resting saliva, the purified proteins from whole human resting saliva and single proteins. Two model systems, the first containing amyloglucosidase and lysozyme, and the second containing amyloglucosidase and alpha-amylase, were shown to provide, in combination with hydrogencarbonate and di-hydrogenphosphate, almost identical buffer attributes as whole human resting saliva. It was further demonstrated that changes in the protein concentration as small as 0.1% may change the buffer value of a buffer solution up to 15 times. Additionally, it was shown that there was a protein concentration change in the same range (0.16% between saliva samples collected at the time periods of 13:00 and others collected at 9:00 am and 17:00. The mode of the protein expression changed between these samples corresponded to the change in basic buffer power and the change of the buffer value at pH 6.7. Finally, SDS Page and Ruthenium II tris (bathophenantroline disulfonate staining unveiled a constant protein expression in all samples except for one 50 kDa protein band. As the change in the expression pattern of that 50 kDa protein band corresponded to the change in basic buffer power and the buffer value at pH 6.7, it was reasonable to conclude that this 50 kDa protein band may contain the protein(s belonging to the protein buffer system of human saliva.

[Compared Markov with fractal models by using single-channel experimental and simulation data].

Science.gov (United States)

Lan, Tonghan; Wu, Hongxiu; Lin, Jiarui

2006-10-01

The gating mechanical kinetical of ion channels has been modeled as a Markov process. In these models it is assumed that the channel protein has a small number of discrete conformational states and kinetic rate constants connecting these states are constant, the transition rate constants among the states is independent both of time and of the previous channel activity. It is assumed in Liebovitch's fractal model that the channel exists in an infinite number of energy states, consequently, transitions from one conductance state to another would be governed by a continuum of rate constants. In this paper, a statistical comparison is presented of Markov and fractal models of ion channel gating, the analysis is based on single-channel data from ion channel voltage-dependence K+ single channel of neuron cell and simulation data from three-states Markov model.

Single Molecule Spectroscopy on Photosynthetic Pigment-Protein Complexes

CERN Document Server

Jelezko, F; Schuler, S; Thews, E; Tietz, C; Wechsler, A; Wrachtrup, J

2001-01-01

Single molecule spectroscopy was applied to unravel the energy transfer pathway in photosynthetic pigment-protein complexes. Detailed analysis of excitation and fluorescence emission spectra has been made for peripheral plant antenna LHC II and Photosystem I from cyanobacterium Synechococcus elongatus. Optical transitions of individual pigments were resolved under nonselective excitation of antenna chlorophylls. High-resolution fluorescence spectroscopy of individual plant antenna LHC II indicates that at low temperatures, the excitation energy is localized on the red-most Chl a pool absorbing at 680 nm. More than one pigment molecule is responsible for the fluorescence emission of the LHC II trimer. The spectral lines of single Chl a molecules absorbing at 675 nm are broadened because of the Foerster energy transfer towards the red-most pigments. Low-temperature spectroscopy on single PS I trimers indicates that two subgroups of pigments, which are present in the red antenna pool, differ by the strength of t...

Hidden Markov model approach for identifying the modular framework of the protein backbone.

Science.gov (United States)

Camproux, A C; Tuffery, P; Chevrolat, J P; Boisvieux, J F; Hazout, S

1999-12-01

The hidden Markov model (HMM) was used to identify recurrent short 3D structural building blocks (SBBs) describing protein backbones, independently of any a priori knowledge. Polypeptide chains are decomposed into a series of short segments defined by their inter-alpha-carbon distances. Basically, the model takes into account the sequentiality of the observed segments and assumes that each one corresponds to one of several possible SBBs. Fitting the model to a database of non-redundant proteins allowed us to decode proteins in terms of 12 distinct SBBs with different roles in protein structure. Some SBBs correspond to classical regular secondary structures. Others correspond to a significant subdivision of their bounding regions previously considered to be a single pattern. The major contribution of the HMM is that this model implicitly takes into account the sequential connections between SBBs and thus describes the most probable pathways by which the blocks are connected to form the framework of the protein structures. Validation of the SBBs code was performed by extracting SBB series repeated in recoding proteins and examining their structural similarities. Preliminary results on the sequence specificity of SBBs suggest promising perspectives for the prediction of SBBs or series of SBBs from the protein sequences.

Protein Signaling Networks from Single Cell Fluctuations and Information Theory Profiling

Science.gov (United States)

Shin, Young Shik; Remacle, F.; Fan, Rong; Hwang, Kiwook; Wei, Wei; Ahmad, Habib; Levine, R.D.; Heath, James R.

2011-01-01

Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network. PMID:21575571

Single-molecule resolution of protein dynamics on polymeric membrane surfaces: the roles of spatial and population heterogeneity.

Science.gov (United States)

Langdon, Blake B; Mirhossaini, Roya B; Mabry, Joshua N; Sriram, Indira; Lajmi, Ajay; Zhang, Yanxia; Rojas, Orlando J; Schwartz, Daniel K

2015-02-18

Although polymeric membranes are widely used in the purification of protein pharmaceuticals, interactions between biomolecules and membrane surfaces can lead to reduced membrane performance and damage to the product. In this study, single-molecule fluorescence microscopy provided direct observation of bovine serum albumin (BSA) and human monoclonal antibody (IgG) dynamics at the interface between aqueous buffer and polymeric membrane materials including regenerated cellulose and unmodified poly(ether sulfone) (PES) blended with either polyvinylpyrrolidone (PVP), polyvinyl acetate-co-polyvinylpyrrolidone (PVAc-PVP), or polyethylene glycol methacrylate (PEGM) before casting. These polymer surfaces were compared with model surfaces composed of hydrophilic bare fused silica and hydrophobic trimethylsilane-coated fused silica. At extremely dilute protein concentrations (10(-3)-10(-7) mg/mL), protein surface exchange was highly dynamic with protein monomers desorbing from the surface within ∼1 s after adsorption. Protein oligomers (e.g., nonspecific dimers, trimers, or larger aggregates), although less common, remained on the surface for 5 times longer than monomers. Using newly developed super-resolution methods, we could localize adsorption sites with ∼50 nm resolution and quantify the spatial heterogeneity of the various surfaces. On a small anomalous subset of the adsorption sites, proteins adsorbed preferentially and tended to reside for significantly longer times (i.e., on "strong" sites). Proteins resided for shorter times overall on surfaces that were more homogeneous and exhibited fewer strong sites (e.g., PVAc-PVP/PES). We propose that strong surface sites may nucleate protein aggregation, initiated preferentially by protein oligomers, and accelerate ultrafiltration membrane fouling. At high protein concentrations (0.3-1.0 mg/mL), fewer strong adsorption sites were observed, and surface residence times were reduced. This suggests that at high concentrations

Addressable droplet microarrays for single cell protein analysis.

Science.gov (United States)

Salehi-Reyhani, Ali; Burgin, Edward; Ces, Oscar; Willison, Keith R; Klug, David R

2014-11-07

Addressable droplet microarrays are potentially attractive as a way to achieve miniaturised, reduced volume, high sensitivity analyses without the need to fabricate microfluidic devices or small volume chambers. We report a practical method for producing oil-encapsulated addressable droplet microarrays which can be used for such analyses. To demonstrate their utility, we undertake a series of single cell analyses, to determine the variation in copy number of p53 proteins in cells of a human cancer cell line.

The dynamics of p53 in single cells: physiologically based ODE and reaction–diffusion PDE models

International Nuclear Information System (INIS)

Eliaš, Ján; Clairambault, Jean; Dimitrio, Luna; Natalini, Roberto

2014-01-01

The intracellular signalling network of the p53 protein plays important roles in genome protection and the control of cell cycle phase transitions. Recently observed oscillatory behaviour in single cells under stress conditions has inspired several research groups to simulate and study the dynamics of the protein with the aim of gaining a proper understanding of the physiological meanings of the oscillations. We propose compartmental ODE and PDE models of p53 activation and regulation in single cells following DNA damage and we show that the p53 oscillations can be retrieved by plainly involving p53–Mdm2 and ATM–p53–Wip1 negative feedbacks, which are sufficient for oscillations experimentally, with no further need to introduce any delays into the protein responses and without considering additional positive feedback. (paper)

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies

Science.gov (United States)

Pant, Kiran; Anderson, Brian; Perdana, Hendrik; Malinowski, Matthew A.; Win, Aye T.; Williams, Mark C.

2018-01-01

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292–296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes. PMID:29634784

Enhanced Single Seed Trait Predictions in Soybean (Glycine max) and Robust Calibration Model Transfer with Near-Infrared Reflectance Spectroscopy.

Science.gov (United States)

Hacisalihoglu, Gokhan; Gustin, Jeffery L; Louisma, Jean; Armstrong, Paul; Peter, Gary F; Walker, Alejandro R; Settles, A Mark

2016-02-10

Single seed near-infrared reflectance (NIR) spectroscopy predicts soybean (Glycine max) seed quality traits of moisture, oil, and protein. We tested the accuracy of transferring calibrations between different single seed NIR analyzers of the same design by collecting NIR spectra and analytical trait data for globally diverse soybean germplasm. X-ray microcomputed tomography (μCT) was used to collect seed density and shape traits to enhance the number of soybean traits that can be predicted from single seed NIR. Partial least-squares (PLS) regression gave accurate predictive models for oil, weight, volume, protein, and maximal cross-sectional area of the seed. PLS models for width, length, and density were not predictive. Although principal component analysis (PCA) of the NIR spectra showed that black seed coat color had significant signal, excluding black seeds from the calibrations did not impact model accuracies. Calibrations for oil and protein developed in this study as well as earlier calibrations for a separate NIR analyzer of the same design were used to test the ability to transfer PLS regressions between platforms. PLS models built from data collected on one NIR analyzer had minimal differences in accuracy when applied to spectra collected from a sister device. Model transfer was more robust when spectra were trimmed from 910 to 1679 nm to 955-1635 nm due to divergence of edge wavelengths between the two devices. The ability to transfer calibrations between similar single seed NIR spectrometers facilitates broader adoption of this high-throughput, nondestructive, seed phenotyping technology.

Green Fluorescent Protein as a Model for Protein Crystal Growth Studies

Science.gov (United States)

Agena, Sabine; Smith, Lori; Karr, Laurel; Pusey, Marc

1998-01-01

Green fluorescent protein (GFP) from jellyfish Aequorea Victoria has become a popular marker for e.g. mutagenesis work. Its fluorescent property, which originates from a chromophore located in the center of the molecule, makes it widely applicable as a research too]. GFP clones have been produced with a variety of spectral properties, such as blue and yellow emitting species. The protein is a single chain of molecular weight 27 kDa and its structure has been determined at 1.9 Angstrom resolution. The combination of GFP's fluorescent property, the knowledge of its several crystallization conditions, and its increasing use in biophysical and biochemical studies, all led us to consider it as a model material for macromolecular crystal growth studies. Initial preparations of GFP were from E.coli with yields of approximately 5 mg/L of culture media. Current yields are now in the 50 - 120 mg/L range, and we hope to further increase this by expression of the GFP gene in the Pichia system. The results of these efforts and of preliminary crystal growth studies will be presented.

Improved healing of transected rabbit Achilles tendon after a single injection of cartilage-derived morphogenetic protein-2.

Science.gov (United States)

Forslund, Carina; Aspenberg, Per

2003-01-01

Achilles tendon ruptures in humans might be treated more efficiently with the help of a growth factor. Cartilage-derived morphogenetic protein-2 has been shown to induce formation of tendon-like tissue. Cartilage-derived morphogenetic protein-2 has a positive effect on mechanical parameters for tendon healing in a rabbit model with Achilles tendon transection. Controlled laboratory study. The right Achilles tendon of 40 rabbits was transected without tendon suture. Cartilage-derived morphogenetic protein-2 (10 micro g) or vehicle control (acetate buffer) was injected locally 2 hours postoperatively. All tendons were tested biomechanically at 8 and 14 days, and treated tendons were histologically and radiographically evaluated at 56 days. At 14 days, both failure load and stiffness of treated tendons were increased by 35%. The treated tendons had significantly larger callus size at 8 and 14 days. Histologic and radiographic examination showed no signs of ossification in the treated tendons after 56 days. A single injection of cartilage-derived morphogenetic protein-2 led to a stronger and stiffer tendon callus than that in the controls without inducing bone formation. Similar results from a larger animal model would suggest a possible future use of cartilage-derived morphogenetic protein-2 in the treatment of human Achilles tendon ruptures.

Single molecule force spectroscopy data and BD- and MD simulations on the blood protein von Willebrand factor

Directory of Open Access Journals (Sweden)

Sandra Posch

2016-09-01

Full Text Available We here give information for a deeper understanding of single molecule force spectroscopy (SMFS data through the example of the blood protein von Willebrand factor (VWF. It is also shown, how fitting of rupture forces versus loading rate profiles in the molecular dynamics (MD loading-rate range can be used to demonstrate the qualitative agreement between SMFS and MD simulations. The recently developed model by Bullerjahn, Sturm, and Kroy (BSK was used for this demonstration. Further, Brownian dynamics (BD simulations, which can be utilized to estimate the lifetimes of intramolecular VWF interactions under physiological shear, are described. For interpretation and discussion of the methods and data presented here, we would like to directly point the reader to the related research paper, “Mutual A domain interactions in the force sensing protein von Willebrand Factor” (Posch et al., 2016 [1]. Keywords: Atomic force microscopy, Single molecule force spectroscopy, Molecular dynamics simulation, Brownian dynamics simulation, von Willebrand factor

Non-uniform binding of single-stranded DNA binding proteins to hybrids of single-stranded DNA and single-walled carbon nanotubes observed by atomic force microscopy in air and in liquid

Energy Technology Data Exchange (ETDEWEB)

Umemura, Kazuo, E-mail: meicun2006@163.com; Ishizaka, Kei; Nii, Daisuke; Izumi, Katsuki

2016-12-01

Highlights: • Conjugates of protein, DNA, and SWNTs were observed by AFM in liquid. • Non-uniform binding of proteins was visualized in liquid. • Thickness of DNA molecules on SWNT surfaces was well characterized in liquid. - Abstract: Using atomic force spectroscopy (AFM), we observed hybrids of single-stranded DNA (ssDNA) and single-walled carbon nanotubes (SWNTs) with or without protein molecules in air and in an aqueous solution. This is the first report of ssDNA–SWNT hybrids with proteins in solution analyzed by AFM. In the absence of protein, the height of the ssDNA–SWNT hybrids was 1.1 ± 0.3 nm and 2.4 ± 0.6 nm in air and liquid, respectively, suggesting that the ssDNA molecules adopted a flexible structure on the SWNT surface. In the presence of single-stranded DNA binding (SSB) proteins, the heights of the hybrids in air and liquid increased to 6.4 ± 3.1 nm and 10.0 ± 4.5 nm, respectively. The AFM images clearly showed binding of the SSB proteins to the ssDNA–SWNT hybrids. The morphology of the SSB–ssDNA–SWNT hybrids was non-uniform, particularly in aqueous solution. The variance of hybrid height was quantitatively estimated by cross-section analysis along the long-axis of each hybrid. The SSB–ssDNA–SWNT hybrids showed much larger variance than the ssDNA–SWNT hybrids.

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions.

Science.gov (United States)

Götz, Markus; Wortmann, Philipp; Schmid, Sonja; Hugel, Thorsten

2018-01-30

Single-molecule Förster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail. Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates. By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Single-tier city logistics model for single product

Science.gov (United States)

Saragih, N. I.; Nur Bahagia, S.; Suprayogi; Syabri, I.

2017-11-01

This research develops single-tier city logistics model which consists of suppliers, UCCs, and retailers. The problem that will be answered in this research is how to determine the location of UCCs, to allocate retailers to opened UCCs, to assign suppliers to opened UCCs, to control inventory in the three entities involved, and to determine the route of the vehicles from opened UCCs to retailers. This model has never been developed before. All the decisions will be simultaneously optimized. Characteristic of the demand is probabilistic following a normal distribution, and the number of product is single.

Protein modelling of triterpene synthase genes from mangrove plants using Phyre2 and Swiss-model

Science.gov (United States)

Basyuni, M.; Wati, R.; Sulistiyono, N.; Hayati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.

2018-03-01

Molecular cloning of five oxidosqualene cyclases (OSC) genes from Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa had previously been cloned, characterized, and encoded mono and -multi triterpene synthases. The present study analyzed protein modelling of triterpene synthase genes from mangrove using Phyre2 and Swiss-model. The diversity was noted within protein modelling of triterpene synthases using Phyre2 from sequence identity (38-43%) and residue (696-703). RsM2 was distinguishable from others for template structure; it used lanosterol synthase as a template (PDB ID: w6j.1.A). By contrast, other genes used human lanosterol synthase (1w6k.1.A). The predicted bind sites were correlated with the product of triterpene synthase, the product of BgbAS was β-amyrin, while RsM1 contained a significant amount of β-amyrin. Similarly BgLUS and KcMS, both main products was lupeol, on the other hand, RsM2 with the outcome of taraxerol. Homology modelling revealed that 696 residues of BgbAS, BgLUS, RsM1, and RsM2 (91-92% of the amino acid sequence) had been modelled with 100% confidence by the single highest scoring template using Phyre2. This coverage was higher than Swiss-model (85-90%). The present study suggested that molecular cloning of triterpene genes provides useful tools for studying the protein modelling related regulation of isoprenoids biosynthesis in mangrove forests.

Completion of autobuilt protein models using a database of protein fragments

International Nuclear Information System (INIS)

Cowtan, Kevin

2012-01-01

Two developments in the process of automated protein model building in the Buccaneer software are described: the use of a database of protein fragments in improving the model completeness and the assembly of disconnected chain fragments into complete molecules. Two developments in the process of automated protein model building in the Buccaneer software are presented. A general-purpose library for protein fragments of arbitrary size is described, with a highly optimized search method allowing the use of a larger database than in previous work. The problem of assembling an autobuilt model into complete chains is discussed. This involves the assembly of disconnected chain fragments into complete molecules and the use of the database of protein fragments in improving the model completeness. Assembly of fragments into molecules is a standard step in existing model-building software, but the methods have not received detailed discussion in the literature

Computational exploration of single-protein mechanics by steered molecular dynamics

Energy Technology Data Exchange (ETDEWEB)

Sotomayor, Marcos [Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio (United States)

2015-12-31

Hair cell mechanotransduction happens in tens of microseconds, involves forces of a few picoNewtons, and is mediated by nanometer-scale molecular conformational changes. As proteins involved in this process become identified and their high resolution structures become available, multiple tools are being used to explore their “single-molecule responses” to force. Optical tweezers and atomic force microscopy offer exquisite force and extension resolution, but cannot reach the high loading rates expected for high frequency auditory stimuli. Molecular dynamics (MD) simulations can reach these fast time scales, and also provide a unique view of the molecular events underlying protein mechanics, but its predictions must be experimentally verified. Thus a combination of simulations and experiments might be appropriate to study the molecular mechanics of hearing. Here I review the basics of MD simulations and the different methods used to apply force and study protein mechanics in silico. Simulations of tip link proteins are used to illustrate the advantages and limitations of this method.

Fluctuations in protein synthesis from a single RNA template: stochastic kinetics of ribosomes.

Science.gov (United States)

Garai, Ashok; Chowdhury, Debashish; Ramakrishnan, T V

2009-01-01

Proteins are polymerized by cyclic machines called ribosomes, which use their messenger RNA (mRNA) track also as the corresponding template, and the process is called translation. We explore, in depth and detail, the stochastic nature of the translation. We compute various distributions associated with the translation process; one of them--namely, the dwell time distribution--has been measured in recent single-ribosome experiments. The form of the distribution, which fits best with our simulation data, is consistent with that extracted from the experimental data. For our computations, we use a model that captures both the mechanochemistry of each individual ribosome and their steric interactions. We also demonstrate the effects of the sequence inhomogeneities of real genes on the fluctuations and noise in translation. Finally, inspired by recent advances in the experimental techniques of manipulating single ribosomes, we make theoretical predictions on the force-velocity relation for individual ribosomes. In principle, all our predictions can be tested by carrying out in vitro experiments.

A Method for Extracting the Free Energy Surface and Conformational Dynamics of Fast-Folding Proteins from Single Molecule Photon Trajectories

Science.gov (United States)

2015-01-01

Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-state to one-state downhill folding, as a function of relevant experimental variables such as photon count rate, amount of input data, and background noise. The tests demonstrate that the analysis can accurately retrieve the original one-dimensional free energy surface and microsecond folding dynamics in spite of the sub-megahertz photon count rates and significant background noise levels of current single molecule fluorescence experiments. Therefore, our approach provides a powerful tool for the quantitative analysis of single molecule FRET experiments of fast protein folding that is also potentially extensible to the analysis of any other biomolecular process governed by sub-millisecond conformational dynamics. PMID:25988351

Coupled aggregation of mitochondrial single-strand DNA-binding protein tagged with Eos fluorescent protein visualizes synchronized activity of mitochondrial nucleoids

Czech Academy of Sciences Publication Activity Database

Olejár, Tomáš; Pajuelo-Reguera, David; Alán, Lukáš; Dlasková, Andrea; Ježek, Petr

2015-01-01

Roč. 12, č. 4 (2015), s. 5185-5190 ISSN 1791-2997 R&D Projects: GA ČR(CZ) GAP302/10/0346; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : mitochondrial nucleoid * single-stranded DNA-binding protein * photoconvertible fluorescent protein Eos Subject RIV: EA - Cell Biology Impact factor: 1.559, year: 2015

Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

DEFF Research Database (Denmark)

Mijakovic, Ivan; Petranovic, Dina; Macek, B

2006-01-01

for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (Sc......SSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically...... by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

Study of molasses / vinasse waste ratio for single cell protein and total microorganisms

Directory of Open Access Journals (Sweden)

Marcia Luciana Cazetta

2006-02-01

Full Text Available Different molasses/ vinasse ratio were used as substrate to investigate single cell protein and total lipids production by five microorganisms: four yeasts strains: Candida lipolytica, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, a yeast isolated from vinasse lake (denominated LLV98 and a bacterium strain, Corynebacterium glutamicum. The media utilized were: a 50% molasses and 50% vinasse; b 25% molasses and 75% vinasse and c 75% molasses and 25% vinasse. The objective of this work was to study the growth of microorganisms and also evaluate protein and lipids content in the biomass obtained from these by-products. The highest single cell protein production was obtained by S. cerevisiae, 50.35%, followed by R. mucilaginosa, 41.96%. The lowest productions were obtained by C. glutamicum. The higher total lipids productions, more than 26%, were founded in molasses plus vinasse at 50%/50% by S. cerevisiae and C. glutamicum.

In silico modeling of the yeast protein and protein family interaction network

Science.gov (United States)

Goh, K.-I.; Kahng, B.; Kim, D.

2004-03-01

Understanding of how protein interaction networks of living organisms have evolved or are organized can be the first stepping stone in unveiling how life works on a fundamental ground. Here we introduce an in silico ``coevolutionary'' model for the protein interaction network and the protein family network. The essential ingredient of the model includes the protein family identity and its robustness under evolution, as well as the three previously proposed: gene duplication, divergence, and mutation. This model produces a prototypical feature of complex networks in a wide range of parameter space, following the generalized Pareto distribution in connectivity. Moreover, we investigate other structural properties of our model in detail with some specific values of parameters relevant to the yeast Saccharomyces cerevisiae, showing excellent agreement with the empirical data. Our model indicates that the physical constraints encoded via the domain structure of proteins play a crucial role in protein interactions.

Functionalization of single-walled carbon nanotubes with protein by click chemistry as sensing platform for sensitized electrochemical immunoassay

International Nuclear Information System (INIS)

Qi Honglan; Ling Chen; Huang Ru; Qiu Xiaoying; Shangguan Li; Gao Qiang; Zhang Chengxiao

2012-01-01

Highlights: ► Single-walled carbon nanotubes were functionalized with protein by click chemistry. ► The SWNTs conjugated with protein showed excellent dispersion in water and kept good bioacitvity. ► A competitive electrochemical immunoassay for the determination of anti-IgG was developed with high sensitivity and good stability. - Abstract: The application of the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition to the functionalization of single-walled carbon nanotubes (SWNTs) with the protein and the use of the artificial SWNTs as a sensing platform for sensitive immunoassay were reported. Covalent functionalization of azide decorated SWNTs with alkyne modified protein was firstly accomplished by the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition. FT-IR spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and transmission electron micrograph were used to characterize the protein-functionalized SWNTs. It was found that the SWNTs conjugated with the proteins showed excellent dispersion in water and kept good bioacitivity when immunoglobulin (IgG) and horseradish peroxidase (HRP) were chosen as model proteins. As a proof-of-concept, IgG-functionalized SWNTs were immobilized onto the surface of a glassy carbon electrode by simple casting method as immunosensing platform and a sensitive competitive electrochemical immunoassay was developed for the determination of anti-immunoglobulin (anti-IgG) using HRP as enzyme label. The fabrication of the immunosensor were characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the redox probe [Fe(CN) 6 ] 3−/4− . The SWNTs as immobilization platform showed better sensitizing effect, a detection limit of 30 pg mL −1 (S/N = 3) was obtained for anti-IgG. The proposed strategy provided a stable immobilization method and sensitized recognition platform for analytes. This work demonstrated that the click coupling of SWNTs with protein was an effective

Motion of single MreB bacterial actin proteins in Caulobacter show treadmilling in vivo

Science.gov (United States)

Moerner, W. E.; Kim, Soyeon; Gitai, Zemer; Kinkhabwala, Anika; McAdams, Harley; Shapiro, Lucy

2006-03-01

Ensemble imaging of a bacterial actin homologue, the MreB protein, suggests that the MreB proteins form a dynamic filamentous spiral along the long axis of the cell in Caulobacter crescentus. MreB contracts and expands along the cell axis and plays an important role in cell shape and polarity maintenance, as well as chromosome segregation and translocation of the origin of replication during cell division. In this study we investigated the real-time polymerization of MreB in Caulobacter crescentus using single-molecule fluorescence imaging. With time-lapse imaging, polymerized MreB could be distinguished from cytoplasmic MreB monomers, because single monomeric MreB showed fast motion characteristic of Brownian diffusion, while single polymerized MreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer implies that treadmilling is the predominant mechanism in MreB filament formation. These single-molecule imaging experiments provide the first available information on the velocity of bacterial actin polymerization in a living cell.

Single Event Resolution of Plant Plasma Membrane Protein Endocytosis by TIRF Microscopy.

Science.gov (United States)

Johnson, Alexander; Vert, Grégory

2017-01-01

Endocytosis is a key process in the internalization of extracellular materials and plasma membrane proteins, such as receptors and transporters, thereby controlling many aspects of cell signaling and cellular homeostasis. Endocytosis in plants has an essential role not only for basic cellular functions but also for growth and development, nutrient delivery, toxin avoidance, and pathogen defense. The precise mechanisms of endocytosis in plants remain quite elusive. The lack of direct visualization and examination of single events of endocytosis has greatly hampered our ability to precisely monitor the cell surface lifetime and the recruitment profile of proteins driving endocytosis or endocytosed cargos in plants. Here, we discuss the necessity to systematically implement total internal reflection fluorescence microcopy (TIRF) in the Plant Cell Biology community and present reliable protocols for high spatial and temporal imaging of endocytosis in plants using clathrin-mediated endocytosis as a test case, since it represents the major route for internalization of cell-surface proteins in plants. We developed a robust method to directly visualize cell surface proteins using TIRF microscopy combined to a high throughput, automated and unbiased analysis pipeline to determine the temporal recruitment profile of proteins to single sites of endocytosis, using the departure of clathrin as a physiological reference for scission. Using this 'departure assay', we assessed the recruitment of two different AP-2 subunits, alpha and mu, to the sites of endocytosis and found that AP2A1 was recruited in concert with clathrin, while AP2M was not. This validated approach therefore offers a powerful solution to better characterize the plant endocytic machinery and the dynamics of one's favorite cargo protein.

Building alternate protein structures using the elastic network model.

Science.gov (United States)

Yang, Qingyi; Sharp, Kim A

2009-02-15

We describe a method for efficiently generating ensembles of alternate, all-atom protein structures that (a) differ significantly from the starting structure, (b) have good stereochemistry (bonded geometry), and (c) have good steric properties (absence of atomic overlap). The method uses reconstruction from a series of backbone framework structures that are obtained from a modified elastic network model (ENM) by perturbation along low-frequency normal modes. To ensure good quality backbone frameworks, the single force parameter ENM is modified by introducing two more force parameters to characterize the interaction between the consecutive carbon alphas and those within the same secondary structure domain. The relative stiffness of the three parameters is parameterized to reproduce B-factors, while maintaining good bonded geometry. After parameterization, violations of experimental Calpha-Calpha distances and Calpha-Calpha-Calpha pseudo angles along the backbone are reduced to less than 1%. Simultaneously, the average B-factor correlation coefficient improves to R = 0.77. Two applications illustrate the potential of the approach. (1) 102,051 protein backbones spanning a conformational space of 15 A root mean square deviation were generated from 148 nonredundant proteins in the PDB database, and all-atom models with minimal bonded and nonbonded violations were produced from this ensemble of backbone structures using the SCWRL side chain building program. (2) Improved backbone templates for homology modeling. Fifteen query sequences were each modeled on two targets. For each of the 30 target frameworks, dozens of improved templates could be produced In all cases, improved full atom homology models resulted, of which 50% could be identified blind using the D-Fire statistical potential. (c) 2008 Wiley-Liss, Inc.

Low-temperature protein dynamics of the B800 molecules in the LH2 light-harvesting complex: spectral hole burning study and comparison with single photosynthetic complex spectroscopy.

Science.gov (United States)

Grozdanov, Daniel; Herascu, Nicoleta; Reinot, Tõnu; Jankowiak, Ryszard; Zazubovich, Valter

2010-03-18

Previously published and new spectral hole burning (SHB) data on the B800 band of LH2 light-harvesting antenna complex of Rps. acidophila are analyzed in light of recent single photosynthetic complex spectroscopy (SPCS) results (for a review, see Berlin et al. Phys. Life Rev. 2007, 4, 64.). It is demonstrated that, in general, SHB-related phenomena observed for the B800 band are in qualitative agreement with the SPCS data and the protein models involving multiwell multitier protein energy landscapes. Regarding the quantitative agreement, we argue that the single-molecule behavior associated with the fastest spectral diffusion (smallest barrier) tier of the protein energy landscape is inconsistent with the SHB data. The latter discrepancy can be attributed to SPCS probing not only the dynamics of of the protein complex per se, but also that of the surrounding amorphous host and/or of the host-protein interface. It is argued that SHB (once improved models are developed) should also be able to provide the average magnitudes and probability distributions of light-induced spectral shifts and could be used to determine whether SPCS probes a set of protein complexes that are both intact and statistically relevant. SHB results are consistent with the B800 --> B850 energy-transfer models including consideration of the whole B850 density of states.

A Mesoscopic Model for Protein-Protein Interactions in Solution

OpenAIRE

Lund, Mikael; Jönsson, Bo

2003-01-01

Protein self-association may be detrimental in biological systems, but can be utilized in a controlled fashion for protein crystallization. It is hence of considerable interest to understand how factors like solution conditions prevent or promote aggregation. Here we present a computational model describing interactions between protein molecules in solution. The calculations are based on a molecular description capturing the detailed structure of the protein molecule using x-ray or nuclear ma...

Effects of the daily consumption of protein enriched bread and protein enriched drinking yoghurt on the total protein intake in older adults in a rehabilitation centre: A single blind randomised controlled trial

NARCIS (Netherlands)

van Til, A.J.; Naumann, E.; Cox-Claessens, I.J.H.M.; Kremer, S.; Boelsma, E.; van Bokhorst-de van der Schueren, M.A.E.

2015-01-01

Objectives: To investigate the effects of protein enriched bread and drinking yoghurt, substituting regular products, on the total protein intake and the distribution of protein intake over the day in older adults.Design: A single blind randomised controlled trial.Setting: Rehabilitation

Ultra-fast optical manipulation of single proteins binding to the actin cytoskeleton

Science.gov (United States)

Capitanio, Marco; Gardini, Lucia; Pavone, Francesco Saverio

2014-02-01

In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

Prediction of protein-protein interactions between viruses and human by an SVM model

Directory of Open Access Journals (Sweden)

Cui Guangyu

2012-05-01

Full Text Available Abstract Background Several computational methods have been developed to predict protein-protein interactions from amino acid sequences, but most of those methods are intended for the interactions within a species rather than for interactions across different species. Methods for predicting interactions between homogeneous proteins are not appropriate for finding those between heterogeneous proteins since they do not distinguish the interactions between proteins of the same species from those of different species. Results We developed a new method for representing a protein sequence of variable length in a frequency vector of fixed length, which encodes the relative frequency of three consecutive amino acids of a sequence. We built a support vector machine (SVM model to predict human proteins that interact with virus proteins. In two types of viruses, human papillomaviruses (HPV and hepatitis C virus (HCV, our SVM model achieved an average accuracy above 80%, which is higher than that of another SVM model with a different representation scheme. Using the SVM model and Gene Ontology (GO annotations of proteins, we predicted new interactions between virus proteins and human proteins. Conclusions Encoding the relative frequency of amino acid triplets of a protein sequence is a simple yet powerful representation method for predicting protein-protein interactions across different species. The representation method has several advantages: (1 it enables a prediction model to achieve a better performance than other representations, (2 it generates feature vectors of fixed length regardless of the sequence length, and (3 the same representation is applicable to different types of proteins.

Effect of the daily consumption of protein enriched bread and protein enriched drinking yoghurt on the total protein intake in older adults in a rehabilitation centre: a single blind randomised controlled trial

NARCIS (Netherlands)

Til, van A.J.; Naumann, E.; Cox-Claessens, I.J.H.M.; Kremer, S.; Boelsma, E.; Schueren, van der D.E.

2015-01-01

Objectives To investigate the effects of protein enriched bread and drinking yoghurt, substituting regular products, on the total protein intake and the distribution of protein intake over the day in older adults. Design A single blind randomised controlled trial. Setting Rehabilitation centre.

Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

Science.gov (United States)

Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn

2016-05-10

Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

A mathematical model of single target site location by Brownian movement in subcellular compartments.

Science.gov (United States)

Kuthan, Hartmut

2003-03-07

The location of distinct sites is mandatory for many cellular processes. In the subcompartments of the cell nucleus, only very small numbers of diffusing macromolecules and specific target sites of some types may be present. In this case, we are faced with the Brownian movement of individual macromolecules and their "random search" for single/few specific target sites, rather than bulk-averaged diffusion and multiple sites. In this article, I consider the location of a distant central target site, e.g. a globular protein, by individual macromolecules executing unbiased (i.e. drift-free) random walks in a spherical compartment. For this walk-and-capture model, the closed-form analytic solution of the first passage time probability density function (p.d.f.) has been obtained as well as the first and second moment. In the limit of a large ratio of the radii of the spherical diffusion space and central target, well-known relations for the variance and the first two moments for the exponential p.d.f. were found to hold with high accuracy. These calculations reinforce earlier numerical results and Monte Carlo simulations. A major implication derivable from the model is that non-directed random movement is an effective means for locating single sites in submicron-sized compartments, even when the diffusion coefficients are comparatively small and the diffusing species are present in one copy only. These theoretical conclusions are underscored numerically for effective diffusion constants ranging from 0.5 to 10.0 microm(2) s(-1), which have been reported for a couple of nuclear proteins in their physiological environment. Spherical compartments of submicron size are, for example, the Cajal bodies (size: 0.1-1.0 microm), which are present in 1-5 copies in the cell nucleus. Within a small Cajal body of radius 0.1 microm a single diffusing protein molecule (with D=0.5 microm(2) s(-1)) would encounter a medium-sized protein of radius 2.5 nm within 1 s with a probability near

Use of multiple-trait animal models for genetic evaluation of milk, fat and protein lactation yields of dairy cattle in Belgium

Directory of Open Access Journals (Sweden)

Pierre Coenraets

1997-01-01

Full Text Available Comparison of computation time between single-trait and multiple-trait evaluations showed that with the use of the canonicat transformation associated with multiple diagonalization of (covariance matrices, multiple-trait analysis for milk, fat and protein yields is not more expensive than three single-trait analyzes. Rank correlations between breeding values for 54,820 cows with records (for their 1,406 sires estimated with the single-trait and multiple-trait models were over .98 (.99 in fat yield and over .99 (.99 in milk and protein yields. The relative gain expressed as reduction in mean prediction error variance was 3% (1% in milk yield, 6% (3% in fat yield, and .4% (.2% in protein yield for cows (for sires. Relative genetic gains were 3% (1%, 6% (2% and .5% (.2% respectively in milk, fat and protein yields for cows (for sires. The use of multiple-trait models bas therefore the advantages of improved precision and reduced selection bics. Multiple-trait analysis could be extended for the analyzes of test-day records. Results show that this or similar multiple-trait animal model could be implemented immediately in Belgium at low computing cost, using the proposed algorithme and could be the first step to new, more advanced evaluation methods.

Caenorhabditis elegans as a model system for studying non-cell-autonomous mechanisms in protein-misfolding diseases

Directory of Open Access Journals (Sweden)

Carmen I. Nussbaum-Krammer

2014-01-01

Full Text Available Caenorhabditis elegans has a number of distinct advantages that are useful for understanding the basis for cellular and organismal dysfunction underlying age-associated diseases of protein misfolding. Although protein aggregation, a key feature of human neurodegenerative diseases, has been typically explored in vivo at the single-cell level using cells in culture, there is now increasing evidence that proteotoxicity has a non-cell-autonomous component and is communicated between cells and tissues in a multicellular organism. These discoveries have opened up new avenues for the use of C. elegans as an ideal animal model system to study non-cell-autonomous proteotoxicity, prion-like propagation of aggregation-prone proteins, and the organismal regulation of stress responses and proteostasis. This Review focuses on recent evidence that C. elegans has mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism. These findings emphasize the potential of C. elegans to provide insights into non-cell-autonomous proteotoxic mechanisms underlying age-related protein-misfolding diseases.

On the involvement of single-bond rotation in the primary photochemistry of photoactive yellow protein

NARCIS (Netherlands)

Stahl, A.D.; Hospes, M.; Singhal, K.; van Stokkum, I.; van Grondelle, R.; Groot, M.L.; Hellingwerf, K.J.

2011-01-01

Prior experimental observations, as well as theoretical considerations, have led to the proposal that C4-C7 single-bond rotation may play an important role in the primary photochemistry of photoactive yellow protein (PYP). We therefore synthesized an analog of this protein's 4-hydroxy-cinnamic acid

Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches

Science.gov (United States)

Wu, Jincheng; Tzanakakis, Emmanuel S.

2014-01-01

Isogenic stem cell populations display cell-to-cell variations in a multitude of attributes including gene or protein expression, epigenetic state, morphology, proliferation and proclivity for differentiation. The origins of the observed heterogeneity and its roles in the maintenance of pluripotency and the lineage specification of stem cells remain unclear. Addressing pertinent questions will require the employment of single-cell analysis methods as traditional cell biochemical and biomolecular assays yield mostly population-average data. In addition to time-lapse microscopy and flow cytometry, recent advances in single-cell genomic, transcriptomic and proteomic profiling are reviewed. The application of multiple displacement amplification, next generation sequencing, mass cytometry and spectrometry to stem cell systems is expected to provide a wealth of information affording unprecedented levels of multiparametric characterization of cell ensembles under defined conditions promoting pluripotency or commitment. Establishing connections between single-cell analysis information and the observed phenotypes will also require suitable mathematical models. Stem cell self-renewal and differentiation are orchestrated by the coordinated regulation of subcellular, intercellular and niche-wide processes spanning multiple time scales. Here, we discuss different modeling approaches and challenges arising from their application to stem cell populations. Integrating single-cell analysis with computational methods will fill gaps in our knowledge about the functions of heterogeneity in stem cell physiology. This combination will also aid the rational design of efficient differentiation and reprogramming strategies as well as bioprocesses for the production of clinically valuable stem cell derivatives. PMID:24035899

Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

Science.gov (United States)

Fischer, Jana; Kleinau, Gunnar; Rutz, Claudia; Zwanziger, Denise; Khajavi, Noushafarin; Müller, Anne; Rehders, Maren; Brix, Klaudia; Worth, Catherine L; Führer, Dagmar; Krude, Heiko; Wiesner, Burkhard; Schülein, Ralf; Biebermann, Heike

2018-06-01

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

Single Molecule Effects of Osteogenesis Imperfecta Mutations in Tropocollagen Protein Domains

Science.gov (United States)

2008-12-02

Single molecule effects of osteogenesis imperfecta mutations in tropocollagen protein domains Alfonso Gautieri,1,2 Simone Vesentini,2 Alberto...2008 proteinscience.org Abstract: Osteogenesis imperfecta (OI) is a genetic disease characterized by fragile bones, skeletal deformities and, in severe...diagnosis and treatment, an effort referred to as materiomics. Keywords: steered molecular dynamics; osteogenesis imperfecta ; Young’s modulus; collagen

Chemo-mechanical pushing of proteins along single-stranded DNA.

Science.gov (United States)

Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto; Lohman, Timothy M

2016-05-31

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

Protein folding simulations: from coarse-grained model to all-atom model.

Science.gov (United States)

Zhang, Jian; Li, Wenfei; Wang, Jun; Qin, Meng; Wu, Lei; Yan, Zhiqiang; Xu, Weixin; Zuo, Guanghong; Wang, Wei

2009-06-01

Protein folding is an important and challenging problem in molecular biology. During the last two decades, molecular dynamics (MD) simulation has proved to be a paramount tool and was widely used to study protein structures, folding kinetics and thermodynamics, and structure-stability-function relationship. It was also used to help engineering and designing new proteins, and to answer even more general questions such as the minimal number of amino acid or the evolution principle of protein families. Nowadays, the MD simulation is still undergoing rapid developments. The first trend is to toward developing new coarse-grained models and studying larger and more complex molecular systems such as protein-protein complex and their assembling process, amyloid related aggregations, and structure and motion of chaperons, motors, channels and virus capsides; the second trend is toward building high resolution models and explore more detailed and accurate pictures of protein folding and the associated processes, such as the coordination bond or disulfide bond involved folding, the polarization, charge transfer and protonate/deprotonate process involved in metal coupled folding, and the ion permeation and its coupling with the kinetics of channels. On these new territories, MD simulations have given many promising results and will continue to offer exciting views. Here, we review several new subjects investigated by using MD simulations as well as the corresponding developments of appropriate protein models. These include but are not limited to the attempt to go beyond the topology based Gō-like model and characterize the energetic factors in protein structures and dynamics, the study of the thermodynamics and kinetics of disulfide bond involved protein folding, the modeling of the interactions between chaperonin and the encapsulated protein and the protein folding under this circumstance, the effort to clarify the important yet still elusive folding mechanism of protein BBL

Bisimulation for Single-Agent Plausibility Models

DEFF Research Database (Denmark)

Andersen, Mikkel Birkegaard; Bolander, Thomas; van Ditmarsch, H.

2013-01-01

define a proper notion of bisimulation, and prove that bisimulation corresponds to logical equivalence on image-finite models. We relate our results to other epistemic notions, such as safe belief and degrees of belief. Our results imply that there are only finitely many non-bisimilar single......-agent epistemic plausibility models on a finite set of propositions. This gives decidability for single-agent epistemic plausibility planning....

Quantitative assessment of protein activity in orphan tissues and single cells using the metaVIPER algorithm. | Office of Cancer Genomics

Science.gov (United States)

We and others have shown that transition and maintenance of biological states is controlled by master regulator proteins, which can be inferred by interrogating tissue-specific regulatory models (interactomes) with transcriptional signatures, using the VIPER algorithm. Yet, some tissues may lack molecular profiles necessary for interactome inference (orphan tissues), or, as for single cells isolated from heterogeneous samples, their tissue context may be undetermined.

Fungal protein from corn waste effluents : a model study

NARCIS (Netherlands)

Schellart, J.A.

1975-01-01

The purpose of this investigation was to study the microbiological aspects of the production of microbial protein ('single cell protein'; SCP) from corn waste effluents with simultaneous reduction of the COD of these effluents.

For practical reasons the corn waste water itself was

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time.

Science.gov (United States)

Wollman, Adam J M; Leake, Mark C

2015-01-01

We present a single-molecule tool called the CoPro (concentration of proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrate its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a ∼4-fold shift towards higher values in the concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, and which is almost exclusively localized in the nucleus under high and low external glucose levels. CoPro facilitates time-resolved quantification of

Single cell protein production of Chlorella sp. using food processing waste as a cultivation medium

Science.gov (United States)

Putri, D.; Ulhidayati, A.; Musthofa, I. A.; Wardani, A. K.

2018-03-01

The aim of this study was to investigate the effect of various food processing wastes on the production of single cell protein by Chlorella sp. Three various food processing wastes i.e. tofu waste, tempeh waste and cheese whey waste were used as cultivation medium for Chlorella sp. growth. Sea water was used as a control of cultivation medium. The addition of waste into cultivation medium was 10%, 20%, 30%, 40%, and 50%. The result showed that the highest yield of cell mass and protein content was found in 50% tofu waste cultivation medium was 47.8 × 106 cell/ml with protein content was 52.24%. The 50% tofu waste medium showed improved cell yield as nearly as 30% than tempeh waste medium. The yield of biomass and protein content when 30% tempeh waste was used as cultivation medium was 37.1 × 106 cell/ml and 52%, respectively. Thus, food processing waste especially tofu waste would be a promising candidate for cultivation medium for single cell production from Chlorella sp. Moreover, the utilization of waste can reduce environmental pollution and increase protein supply for food supplement or animal feed.

Room temperature phosphorescence study on the structural flexibility of single tryptophan containing proteins

Science.gov (United States)

Kowalska-Baron, Agnieszka; Gałęcki, Krystian; Wysocki, Stanisław

2015-01-01

In this study, we have undertaken efforts to find correlation between phosphorescence lifetimes of single tryptophan containing proteins and some structural indicators of protein flexibility/rigidity, such as the degree of tryptophan burial or its exposure to solvent, protein secondary and tertiary structure of the region of localization of tryptophan as well as B factors for tryptophan residue and its immediate surroundings. Bearing in mind that, apart from effective local viscosity of the protein/solvent matrix, the other factor that concur in determining room temperature tryptophan phosphorescence (RTTP) lifetime in proteins is the extent of intramolecular quenching by His, Cys, Tyr and Trp side chains, the crystallographic structures derived from the Brookhaven Protein Data Bank were also analyzed concentrating on the presence of potentially quenching amino acid side chains in the close proximity of the indole chromophore. The obtained results indicated that, in most cases, the phosphorescence lifetimes of tryptophan containing proteins studied tend to correlate with the above mentioned structural indicators of protein rigidity/flexibility. This correlation is expected to provide guidelines for the future development of phosphorescence lifetime-based method for the prediction of structural flexibility of proteins, which is directly linked to their biological function.

High-throughput single-molecule force spectroscopy for membrane proteins

Science.gov (United States)

Bosshart, Patrick D.; Casagrande, Fabio; Frederix, Patrick L. T. M.; Ratera, Merce; Bippes, Christian A.; Müller, Daniel J.; Palacin, Manuel; Engel, Andreas; Fotiadis, Dimitrios

2008-09-01

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ~400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ~200 (AdiC) and ~400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

High-throughput single-molecule force spectroscopy for membrane proteins

International Nuclear Information System (INIS)

Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios; Ratera, Merce; Palacin, Manuel; Bippes, Christian A; Mueller, Daniel J

2008-01-01

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ∼400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ∼200 (AdiC) and ∼400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications

High-throughput single-molecule force spectroscopy for membrane proteins

Energy Technology Data Exchange (ETDEWEB)

Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios [M E Mueller Institute for Structural Biology, Biozentrum of the University of Basel, CH-4056 Basel (Switzerland); Ratera, Merce; Palacin, Manuel [Institute for Research in Biomedicine, Barcelona Science Park, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona and Centro de Investigacion Biomedica en Red de Enfermedades Raras, E-08028 Barcelona (Spain); Bippes, Christian A; Mueller, Daniel J [BioTechnology Center, Technical University, Tatzberg 47, D-01307 Dresden (Germany)], E-mail: andreas.engel@unibas.ch, E-mail: dimitrios.fotiadis@mci.unibe.ch

2008-09-24

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether {approx}400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with {approx}200 (AdiC) and {approx}400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

What can we learn by computing 13Cα chemical shifts for X-ray protein models?

International Nuclear Information System (INIS)

Arnautova, Yelena A.; Vila, Jorge A.; Martin, Osvaldo A.; Scheraga, Harold A.

2009-01-01

The room-temperature X-ray structures of two proteins, solved at 1.8 and 1.9 Å resolution, are used to investigate whether a set of conformations, rather than a single X-ray structure, provides better agreement with both the X-ray data and the observed 13 C α chemical shifts in solution. The room-temperature X-ray structures of ubiquitin and of the RNA-binding domain of nonstructural protein 1 of influenza A virus solved at 1.8 and 1.9 Å resolution, respectively, were used to investigate whether a set of conformations rather than a single X-ray structure provides better agreement with both the X-ray data and the observed 13 C α chemical shifts in solution. For this purpose, a set of new conformations for each of these proteins was generated by fitting them to the experimental X-ray data deposited in the PDB. For each of the generated structures, which show R and R free factors similar to those of the deposited X-ray structure, the 13 C α chemical shifts of all residues in the sequence were computed at the DFT level of theory. The sets of conformations were then evaluated by their ability to reproduce the observed 13 C α chemical shifts by using the conformational average root-mean-square-deviation (ca-r.m.s.d.). For ubiquitin, the computed set of conformations is a better representation of the observed 13 C α chemical shifts in terms of the ca-r.m.s.d. than a single X-ray-derived structure. However, for the RNA-binding domain of nonstructural protein 1 of influenza A virus, consideration of an ensemble of conformations does not improve the agreement with the observed 13 C α chemical shifts. Whether an ensemble of conformations rather than any single structure is a more accurate representation of a protein structure in the crystal as well as of the observed 13 C α chemical shifts is determined by the dispersion of coordinates, in terms of the all-atom r.m.s.d. among the generated models; these generated models satisfy the experimental X-ray data with

Modeling the formation of cell-matrix adhesions on a single 3D matrix fiber.

Science.gov (United States)

Escribano, J; Sánchez, M T; García-Aznar, J M

2015-11-07

Cell-matrix adhesions are crucial in different biological processes like tissue morphogenesis, cell motility, and extracellular matrix remodeling. These interactions that link cell cytoskeleton and matrix fibers are built through protein clutches, generally known as adhesion complexes. The adhesion formation process has been deeply studied in two-dimensional (2D) cases; however, the knowledge is limited for three-dimensional (3D) cases. In this work, we simulate different local extracellular matrix properties in order to unravel the fundamental mechanisms that regulate the formation of cell-matrix adhesions in 3D. We aim to study the mechanical interaction of these biological structures through a three dimensional discrete approach, reproducing the transmission pattern force between the cytoskeleton and a single extracellular matrix fiber. This numerical model provides a discrete analysis of the proteins involved including spatial distribution, interaction between them, and study of the different phenomena, such as protein clutches unbinding or protein unfolding. Copyright © 2015 Elsevier Ltd. All rights reserved.

See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins

NARCIS (Netherlands)

van Mameren, J.; Peterman, E.J.G.; Wuite, G.J.L.

2008-01-01

Direct visualization of DNA and proteins allows researchers to investigate DNA-protein interactions with great detail. Much progress has been made in this area as a result of increasingly sensitive single-molecule fluorescence techniques. At the same time, methods that control the conformation of

Modelling of proteins in membranes

DEFF Research Database (Denmark)

Sperotto, Maria Maddalena; May, S.; Baumgaertner, A.

2006-01-01

This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly ...

Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization

OpenAIRE

Fan, Beiyuan; Li, Xiufeng; Chen, Deyong; Peng, Hongshang; Wang, Junbo; Chen, Jian

2016-01-01

This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research oppor...

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

International Nuclear Information System (INIS)

Neto, J.L. Siqueira; Lira, C.B.B.; Giardini, M.A.; Khater, L.; Perez, A.M.; Peroni, L.A.; Reis, J.R.R. dos; Freitas-Junior, L.H.; Ramos, C.H.I.; Cano, M.I.N.

2007-01-01

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres

Shedding light on protein folding, structural and functional dynamics by single molecule studies

DEFF Research Database (Denmark)

Bavishi, Krutika; Hatzakis, Nikos

2014-01-01

property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out...

Assessing Model Characterization of Single Source ...

Science.gov (United States)

Aircraft measurements made downwind from specific coal fired power plants during the 2013 Southeast Nexus field campaign provide a unique opportunity to evaluate single source photochemical model predictions of both O3 and secondary PM2.5 species. The model did well at predicting downwind plume placement. The model shows similar patterns of an increasing fraction of PM2.5 sulfate ion to the sum of SO2 and PM2.5 sulfate ion by distance from the source compared with ambient based estimates. The model was less consistent in capturing downwind ambient based trends in conversion of NOX to NOY from these sources. Source sensitivity approaches capture near-source O3 titration by fresh NO emissions, in particular subgrid plume treatment. However, capturing this near-source chemical feature did not translate into better downwind peak estimates of single source O3 impacts. The model estimated O3 production from these sources but often was lower than ambient based source production. The downwind transect ambient measurements, in particular secondary PM2.5 and O3, have some level of contribution from other sources which makes direct comparison with model source contribution challenging. Model source attribution results suggest contribution to secondary pollutants from multiple sources even where primary pollutants indicate the presence of a single source. The National Exposure Research Laboratory (NERL) Computational Exposure Division (CED) develops and evaluates data, deci

Assessment of nutritional value of single-cell protein from waste-activated sludge as a protein supplement in poultry feed.

Science.gov (United States)

Nkhalambayausi-Chirwa, Evans M; Lebitso, Moses T

2012-12-01

The amount of protein wasted through sludge in Gauteng, South Africa, amounts to 95 000 metric tonne/yr, with the order of magnitude of the national protein requirement of approximately 145 000 metric tonne/yr. Waste-activated sludge (WAS) from wastewater treatment plants (WWTPs) that treat domestic wastewater contains protein in a ratio of 2:1 against fishmeal. This protein source has not been utilized because of the high content of toxic heavy metals and other potential carcinogenic pollutants in the sludge. In this study, a pretreatment method of modified aqua regia dilute acid wash was used to lower the metal content by approximately 60%. However, this resulted in a 33% loss of amino acids in the acid-washed WAS. A feed substitution test in poultry with different fishmeal-sludge ratios (0%, 25%, 50%, 75%, and 100% WAS as percent substitution of fishmeal) showed no impact of sludge single-cell protein (SCP) on mortality rate. However, sludge substitution in the feed yielded weight gains and cost savings up to 46%.

Looping and clustering model for the organization of protein-DNA complexes on the bacterial genome

Science.gov (United States)

Walter, Jean-Charles; Walliser, Nils-Ole; David, Gabriel; Dorignac, Jérôme; Geniet, Frédéric; Palmeri, John; Parmeggiani, Andrea; Wingreen, Ned S.; Broedersz, Chase P.

2018-03-01

The bacterial genome is organized by a variety of associated proteins inside a structure called the nucleoid. These proteins can form complexes on DNA that play a central role in various biological processes, including chromosome segregation. A prominent example is the large ParB-DNA complex, which forms an essential component of the segregation machinery in many bacteria. ChIP-Seq experiments show that ParB proteins localize around centromere-like parS sites on the DNA to which ParB binds specifically, and spreads from there over large sections of the chromosome. Recent theoretical and experimental studies suggest that DNA-bound ParB proteins can interact with each other to condense into a coherent 3D complex on the DNA. However, the structural organization of this protein-DNA complex remains unclear, and a predictive quantitative theory for the distribution of ParB proteins on DNA is lacking. Here, we propose the looping and clustering model, which employs a statistical physics approach to describe protein-DNA complexes. The looping and clustering model accounts for the extrusion of DNA loops from a cluster of interacting DNA-bound proteins that is organized around a single high-affinity binding site. Conceptually, the structure of the protein-DNA complex is determined by a competition between attractive protein interactions and loop closure entropy of this protein-DNA cluster on the one hand, and the positional entropy for placing loops within the cluster on the other. Indeed, we show that the protein interaction strength determines the ‘tightness’ of the loopy protein-DNA complex. Thus, our model provides a theoretical framework for quantitatively computing the binding profiles of ParB-like proteins around a cognate (parS) binding site.

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

Science.gov (United States)

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

Science.gov (United States)

Zhou, Li; Wang, Hsuan-Yao; Tong, Shanshan; Okamoto, Curtis T; Shen, Wei-Chiang; Zaro, Jennica L

2017-02-01

Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc) 2 ), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc) 2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc) 2 -mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc) 2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein adsorption on nanoparticles: model development using computer simulation

International Nuclear Information System (INIS)

Shao, Qing; Hall, Carol K

2016-01-01

The adsorption of proteins on nanoparticles results in the formation of the protein corona, the composition of which determines how nanoparticles influence their biological surroundings. We seek to better understand corona formation by developing models that describe protein adsorption on nanoparticles using computer simulation results as data. Using a coarse-grained protein model, discontinuous molecular dynamics simulations are conducted to investigate the adsorption of two small proteins (Trp-cage and WW domain) on a model nanoparticle of diameter 10.0 nm at protein concentrations ranging from 0.5 to 5 mM. The resulting adsorption isotherms are well described by the Langmuir, Freundlich, Temkin and Kiselev models, but not by the Elovich, Fowler–Guggenheim and Hill–de Boer models. We also try to develop a generalized model that can describe protein adsorption equilibrium on nanoparticles of different diameters in terms of dimensionless size parameters. The simulation results for three proteins (Trp-cage, WW domain, and GB3) on four nanoparticles (diameter  =  5.0, 10.0, 15.0, and 20.0 nm) illustrate both the promise and the challenge associated with developing generalized models of protein adsorption on nanoparticles. (paper)

Connecting protein and mRNA burst distributions for stochastic models of gene expression

International Nuclear Information System (INIS)

Elgart, Vlad; Jia, Tao; Fenley, Andrew T; Kulkarni, Rahul

2011-01-01

The intrinsic stochasticity of gene expression can lead to large variability in protein levels for genetically identical cells. Such variability in protein levels can arise from infrequent synthesis of mRNAs which in turn give rise to bursts of protein expression. Protein expression occurring in bursts has indeed been observed experimentally and recent studies have also found evidence for transcriptional bursting, i.e. production of mRNAs in bursts. Given that there are distinct experimental techniques for quantifying the noise at different stages of gene expression, it is of interest to derive analytical results connecting experimental observations at different levels. In this work, we consider stochastic models of gene expression for which mRNA and protein production occurs in independent bursts. For such models, we derive analytical expressions connecting protein and mRNA burst distributions which show how the functional form of the mRNA burst distribution can be inferred from the protein burst distribution. Additionally, if gene expression is repressed such that observed protein bursts arise only from single mRNAs, we show how observations of protein burst distributions (repressed and unrepressed) can be used to completely determine the mRNA burst distribution. Assuming independent contributions from individual bursts, we derive analytical expressions connecting means and variances for burst and steady-state protein distributions. Finally, we validate our general analytical results by considering a specific reaction scheme involving regulation of protein bursts by small RNAs. For a range of parameters, we derive analytical expressions for regulated protein distributions that are validated using stochastic simulations. The analytical results obtained in this work can thus serve as useful inputs for a broad range of studies focusing on stochasticity in gene expression

Replication protein A (RPA hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

Directory of Open Access Journals (Sweden)

Artem G Lada

Full Text Available Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G, restricts retroviruses, and Activation Induced Deaminase (AID generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA, the eukaryotic single-stranded DNA (ssDNA binding protein, severely inhibits the deamination activity and processivity of A3G.We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast.Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

Replication protein A (RPA) hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

Science.gov (United States)

Lada, Artem G; Waisertreiger, Irina S-R; Grabow, Corinn E; Prakash, Aishwarya; Borgstahl, Gloria E O; Rogozin, Igor B; Pavlov, Youri I

2011-01-01

Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast. Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

Variance Function Partially Linear Single-Index Models1.

Science.gov (United States)

Lian, Heng; Liang, Hua; Carroll, Raymond J

2015-01-01

We consider heteroscedastic regression models where the mean function is a partially linear single index model and the variance function depends upon a generalized partially linear single index model. We do not insist that the variance function depend only upon the mean function, as happens in the classical generalized partially linear single index model. We develop efficient and practical estimation methods for the variance function and for the mean function. Asymptotic theory for the parametric and nonparametric parts of the model is developed. Simulations illustrate the results. An empirical example involving ozone levels is used to further illustrate the results, and is shown to be a case where the variance function does not depend upon the mean function.

Measuring the force of single protein molecule detachment from surfaces with AFM.

Science.gov (United States)

Tsapikouni, Theodora S; Missirlis, Yannis F

2010-01-01

Atomic force microscopy (AFM) was used to measure the non-specific detachment force of single fibrinogen molecules from glass surfaces. The identification of single unbinding events was based on the characteristics of the parabolic curves, recorded during the stretching of protein molecules. Fibrinogen molecules were covalently bound to Si(3)N(4) AFM tips, previously modified with 3-aminopropyl-dimethyl-ethoxysilane, through a homobifunctional poly(ethylene glycol) linker bearing two hydroxysulfosuccinimide esters. The most probable detachment force was found to be 210 pN, when the tip was retracting with a velocity of 1400 nm/s, while the distribution of the detachment distances indicated that the fibrinogen chain can be elongated beyond the length of the physical conformation before detachment. The dependence of the most probable detachment force on the loading rate was examined and the dynamics of fibrinogen binding to the surface were found amenable to the simple expression of the Bell-Evans theory. The theory's expansion, however, by incorporating the concept of the rupture of parallel residue-surface bonds could only describe the detachment of fibrinogen for a small number of such bonds. Finally, the mathematical expression of the Worm-Like Chain model was used to fit the stretching curves before rupture and two interpretations are suggested for the description of the AFM curves with multiple detachment events.

Exploring the role of internal friction in the dynamics of unfolded proteins using simple polymer models

Science.gov (United States)

Cheng, Ryan R.; Hawk, Alexander T.; Makarov, Dmitrii E.

2013-02-01

Recent experiments showed that the reconfiguration dynamics of unfolded proteins are often adequately described by simple polymer models. In particular, the Rouse model with internal friction (RIF) captures internal friction effects as observed in single-molecule fluorescence correlation spectroscopy (FCS) studies of a number of proteins. Here we use RIF, and its non-free draining analog, Zimm model with internal friction, to explore the effect of internal friction on the rate with which intramolecular contacts can be formed within the unfolded chain. Unlike the reconfiguration times inferred from FCS experiments, which depend linearly on the solvent viscosity, the first passage times to form intramolecular contacts are shown to display a more complex viscosity dependence. We further describe scaling relationships obeyed by contact formation times in the limits of high and low internal friction. Our findings provide experimentally testable predictions that can serve as a framework for the analysis of future studies of contact formation in proteins.

Equivalence of two models in single-phase multicomponent flow simulations

KAUST Repository

Wu, Yuanqing

2016-02-28

In this work, two models to simulate the single-phase multicomponent flow in reservoirs are introduced: single-phase multicomponent flow model and two-phase compositional flow model. Because the single-phase multicomponent flow is a special case of the two-phase compositional flow, the two-phase compositional flow model can also simulate the case. We compare and analyze the two models when simulating the single-phase multicomponent flow, and then demonstrate the equivalence of the two models mathematically. An experiment is also carried out to verify the equivalence of the two models.

Equivalence of two models in single-phase multicomponent flow simulations

KAUST Repository

Wu, Yuanqing; Sun, Shuyu

2016-01-01

In this work, two models to simulate the single-phase multicomponent flow in reservoirs are introduced: single-phase multicomponent flow model and two-phase compositional flow model. Because the single-phase multicomponent flow is a special case of the two-phase compositional flow, the two-phase compositional flow model can also simulate the case. We compare and analyze the two models when simulating the single-phase multicomponent flow, and then demonstrate the equivalence of the two models mathematically. An experiment is also carried out to verify the equivalence of the two models.

The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time

Science.gov (United States)

Hu, Xiaohu; Hong, Liang; Dean Smith, Micholas; Neusius, Thomas; Cheng, Xiaolin; Smith, Jeremy C.

2016-02-01

Internal motions of proteins are essential to their function. The time dependence of protein structural fluctuations is highly complex, manifesting subdiffusive, non-exponential behaviour with effective relaxation times existing over many decades in time, from ps up to ~102 s (refs ,,,). Here, using molecular dynamics simulations, we show that, on timescales from 10-12 to 10-5 s, motions in single proteins are self-similar, non-equilibrium and exhibit ageing. The characteristic relaxation time for a distance fluctuation, such as inter-domain motion, is observation-time-dependent, increasing in a simple, power-law fashion, arising from the fractal nature of the topology and geometry of the energy landscape explored. Diffusion over the energy landscape follows a non-ergodic continuous time random walk. Comparison with single-molecule experiments suggests that the non-equilibrium self-similar dynamical behaviour persists up to timescales approaching the in vivo lifespan of individual protein molecules.

Strand displacement by DNA polymerase III occurs through a tau-psi-chi link to single-stranded DNA-binding protein coating the lagging strand template.

Science.gov (United States)

Yuan, Quan; McHenry, Charles S

2009-11-13

In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of gamma-complex to support the reaction in the absence of tau. However, if gamma-complex is present to load beta(2), a truncated tau protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the alpha subunit of DNA polymerase (Pol) III and chipsi. This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where tau is only required to serve as a scaffold to hold Pol III and chi in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476-23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III-tau-psi-chi-SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k(cat)/K(m)) for the strand displacement reaction is approximately 300-fold less favorable than reactions on single-stranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (approximately 300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.

Quantitative chemogenomics: machine-learning models of protein-ligand interaction.

Science.gov (United States)

Andersson, Claes R; Gustafsson, Mats G; Strömbergsson, Helena

2011-01-01

Chemogenomics is an emerging interdisciplinary field that lies in the interface of biology, chemistry, and informatics. Most of the currently used drugs are small molecules that interact with proteins. Understanding protein-ligand interaction is therefore central to drug discovery and design. In the subfield of chemogenomics known as proteochemometrics, protein-ligand-interaction models are induced from data matrices that consist of both protein and ligand information along with some experimentally measured variable. The two general aims of this quantitative multi-structure-property-relationship modeling (QMSPR) approach are to exploit sparse/incomplete information sources and to obtain more general models covering larger parts of the protein-ligand space, than traditional approaches that focuses mainly on specific targets or ligands. The data matrices, usually obtained from multiple sparse/incomplete sources, typically contain series of proteins and ligands together with quantitative information about their interactions. A useful model should ideally be easy to interpret and generalize well to new unseen protein-ligand combinations. Resolving this requires sophisticated machine-learning methods for model induction, combined with adequate validation. This review is intended to provide a guide to methods and data sources suitable for this kind of protein-ligand-interaction modeling. An overview of the modeling process is presented including data collection, protein and ligand descriptor computation, data preprocessing, machine-learning-model induction and validation. Concerns and issues specific for each step in this kind of data-driven modeling will be discussed. © 2011 Bentham Science Publishers

Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair

International Nuclear Information System (INIS)

Warters, R.L.; Brizgys, L.M.; Lyons, B.W.

1987-01-01

The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 43 0 C and 45 0 C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 45 0 C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells were incubated at 37 0 C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 41 0 C a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks. (author)

Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction

Directory of Open Access Journals (Sweden)

Minsuk eKwak

2013-02-01

Full Text Available Secreted proteins including cytokines, chemokines and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g. in the immunological system, tumor microenvironment or stem cell niche. Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically-identical cell population can give rise to diverse phenotypic differences. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this Perspective Article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

Effects of the daily consumption of protein enriched bread and protein enriched drinking yoghurt on the total protein intake in older adults in a rehabilitation centre: a single blind randomised controlled trial.

Science.gov (United States)

van Til, A J; Naumann, E; Cox-Claessens, I J H M; Kremer, S; Boelsma, E; de van der Schueren, M A E

2015-05-01

To investigate the effects of protein enriched bread and drinking yoghurt, substituting regular products, on the total protein intake and the distribution of protein intake over the day in older adults. A single blind randomised controlled trial. Rehabilitation centre. Older adults (≥ 55 years) admitted to a rehabilitation centre after hospital discharge (n=34). Participants received a high protein diet (protein enriched bread and protein enriched drinking yoghurt; n=17) or a regular diet (regular bread and regular drinking yoghurt; n=17) for three consecutive weeks. Total protein intake and protein intake per meal, measured twice weekly over a three weeks period (six measurements per participant). Compared with controls, patients who received the protein enriched products had a significantly higher protein intake (115.3 g/d vs 72.5 g/d, Pconsumption of protein enriched products improves protein distribution over the day.

The presence of INA proteins on the surface of single cells of Pseudomonas syringae R10.79 isolated from rain

Science.gov (United States)

Šantl-Temkiv, Tina; Ling, Meilee; Holm, Stine; Finster, Kai; Boesen, Thomas

2016-04-01

One of the important open questions in atmospheric ice nucleation is the impact of bioaerosols on the ice content of mix phase clouds (DeMott and Prenni 2010). Biogenic ice nuclei have a unique capacity of facilitating ice formation at temperatures between -1 and -10 °C. The model biogenic ice nuclei are produced by a few species of plant-surface bacteria, such as Pseudomonas syringae, that are commonly transported through the atmosphere. These bacterial species have highly specialized proteins, the so-called ice nucleation active (INA) proteins, which are exposed at the outer membrane surface of the cell where they promote ice particle formation. The mechanisms behind the onset of INA protein synthesis in single bacterial cells are not well understood. We performed a laboratory study in order to (i) investigate the presence of INA proteins on single bacterial cells and (ii) understand the conditions that induce INA protein production. We previously isolated an INA-positive strain of Pseudomonas syringae from rain samples collected in Denmark. Bacterial cells initiated ice nucleation activity at temperatures ≤-2°C and the cell fragments at temperatures ≤-8°C (Šantl-Temkiv et al 2015). We determined the amino-acid sequence of the INA protein and used the sequence to produce custom-made antibodies (GenScript, Germany). These antibodies were used to specifically stain and visualize the INA protein on the surfaces of single cells, which can then be quantified by a technique called flow cytometry. The synthesis of INA proteins by individual cells was followed during a batch growth experiment. An unusually high proportion of cells that were adapting to the new conditions prior to growth produced INA proteins (~4.4% of all cells). A smaller fraction of actively growing cells was carrying INA proteins (~1.2 % of all cells). The cells that stopped growing due to unfavorable conditions had the lowest fraction of cells carrying INA proteins (~0.5 % of all cells). To

Physiologically Based Pharmacokinetic Modeling of Therapeutic Proteins.

Science.gov (United States)

Wong, Harvey; Chow, Timothy W

2017-09-01

Biologics or therapeutic proteins are becoming increasingly important as treatments for disease. The most common class of biologics are monoclonal antibodies (mAbs). Recently, there has been an increase in the use of physiologically based pharmacokinetic (PBPK) modeling in the pharmaceutical industry in drug development. We review PBPK models for therapeutic proteins with an emphasis on mAbs. Due to their size and similarity to endogenous antibodies, there are distinct differences between PBPK models for small molecules and mAbs. The high-level organization of a typical mAb PBPK model consists of a whole-body PBPK model with organ compartments interconnected by both blood and lymph flows. The whole-body PBPK model is coupled with tissue-level submodels used to describe key mechanisms governing mAb disposition including tissue efflux via the lymphatic system, elimination by catabolism, protection from catabolism binding to the neonatal Fc (FcRn) receptor, and nonlinear binding to specific pharmacological targets of interest. The use of PBPK modeling in the development of therapeutic proteins is still in its infancy. Further application of PBPK modeling for therapeutic proteins will help to define its developing role in drug discovery and development. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

ModFOLD6: an accurate web server for the global and local quality estimation of 3D protein models.

Science.gov (United States)

Maghrabi, Ali H A; McGuffin, Liam J

2017-07-03

Methods that reliably estimate the likely similarity between the predicted and native structures of proteins have become essential for driving the acceptance and adoption of three-dimensional protein models by life scientists. ModFOLD6 is the latest version of our leading resource for Estimates of Model Accuracy (EMA), which uses a pioneering hybrid quasi-single model approach. The ModFOLD6 server integrates scores from three pure-single model methods and three quasi-single model methods using a neural network to estimate local quality scores. Additionally, the server provides three options for producing global score estimates, depending on the requirements of the user: (i) ModFOLD6_rank, which is optimized for ranking/selection, (ii) ModFOLD6_cor, which is optimized for correlations of predicted and observed scores and (iii) ModFOLD6 global for balanced performance. The ModFOLD6 methods rank among the top few for EMA, according to independent blind testing by the CASP12 assessors. The ModFOLD6 server is also continuously automatically evaluated as part of the CAMEO project, where significant performance gains have been observed compared to our previous server and other publicly available servers. The ModFOLD6 server is freely available at: http://www.reading.ac.uk/bioinf/ModFOLD/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

Science.gov (United States)

2015-01-01

Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental

Suite of three protein crystallography beamlines with single superconducting bend magnet as the source

International Nuclear Information System (INIS)

MacDowell, Alastair A.; Celestre, Richard S.; Howells, Malcolm; McKinney, Wayne; Krupnick, James; Cambie, Daniella; Domning, Edward E; Duarte, Robert M.; Kelez, Nicholas; Plate, David W.; Cork, Carl W.; Earnest, Thomas N.; Dickert, Jeffery; Meigs, George; Ralston, Corie; Holton, James M.; Alber, Thomas; Berger, James M.; Agard, David A.; Padmore, Howard A.

2004-01-01

At the Advanced Light Source (ALS), three protein crystallography (PX) beamlines have been built that use as a source one of the three 6 Tesla single pole superconducting bending magnets (superbends) that were recently installed in the ring. The use of such single pole superconducting bend magnets enables the development of a hard x-ray program on a relatively low energy 1.9 GeV ring without taking up insertion device straight sections. The source is of relatively low power, but due to the small electron beam emittance, it has high brightness. X-ray optics are required to preserve the brightness and to match the illumination requirements for protein crystallography. This was achieved by means of a collimating premirror bent to a plane parabola, a double crystal monochromator followed by a toroidal mirror that focuses in the horizontal direction with a 2:1 demagnification. This optical arrangement partially balances aberrations from the collimating and toroidal mirrors such that a tight focused spot size is achieved. The optical properties of the beamline are an excellent match to those required by the small protein crystals that are typically measured. The design and performance of these new beamlines are described

Suite of three protein crystallography beamlines with single superconducting bend magnet as the source.

Science.gov (United States)

MacDowell, Alastair A; Celestre, Rich S; Howells, Malcolm; McKinney, Wayne; Krupnick, James; Cambie, Daniella; Domning, Edward E; Duarte, Robert M; Kelez, Nicholas; Plate, David W; Cork, Carl W; Earnest, Thomas N; Dickert, Jeffery; Meigs, George; Ralston, Corie; Holton, James M; Alber, Tom; Berger, James M; Agard, David A; Padmore, Howard A

2004-11-01

At the Advanced Light Source, three protein crystallography beamlines have been built that use as a source one of the three 6 T single-pole superconducting bending magnets (superbends) that were recently installed in the ring. The use of such single-pole superconducting bend magnets enables the development of a hard X-ray program on a relatively low-energy 1.9 GeV ring without taking up insertion-device straight sections. The source is of relatively low power but, owing to the small electron beam emittance, it has high brightness. X-ray optics are required to preserve the brightness and to match the illumination requirements for protein crystallography. This was achieved by means of a collimating premirror bent to a plane parabola, a double-crystal monochromator followed by a toroidal mirror that focuses in the horizontal direction with a 2:1 demagnification. This optical arrangement partially balances aberrations from the collimating and toroidal mirrors such that a tight focused spot size is achieved. The optical properties of the beamline are an excellent match to those required by the small protein crystals that are typically measured. The design and performance of these new beamlines are described.

Suite of three protein crystallography beamlines with single superconducting bend magnet as the source

Energy Technology Data Exchange (ETDEWEB)

MacDowell, Alastair A.; Celestre, Richard S.; Howells, Malcolm; McKinney, Wayne; Krupnick, James; Cambie, Daniella; Domning, Edward E; Duarte, Robert M.; Kelez, Nicholas; Plate, David W.; Cork, Carl W.; Earnest, Thomas N.; Dickert, Jeffery; Meigs, George; Ralston, Corie; Holton, James M.; Alber, Thomas; Berger, James M.; Agard, David A.; Padmore, Howard A.

2004-08-01

At the Advanced Light Source (ALS), three protein crystallography (PX) beamlines have been built that use as a source one of the three 6 Tesla single pole superconducting bending magnets (superbends) that were recently installed in the ring. The use of such single pole superconducting bend magnets enables the development of a hard x-ray program on a relatively low energy 1.9 GeV ring without taking up insertion device straight sections. The source is of relatively low power, but due to the small electron beam emittance, it has high brightness. X-ray optics are required to preserve the brightness and to match the illumination requirements for protein crystallography. This was achieved by means of a collimating premirror bent to a plane parabola, a double crystal monochromator followed by a toroidal mirror that focuses in the horizontal direction with a 2:1 demagnification. This optical arrangement partially balances aberrations from the collimating and toroidal mirrors such that a tight focused spot size is achieved. The optical properties of the beamline are an excellent match to those required by the small protein crystals that are typically measured. The design and performance of these new beamlines are described.

A model for variable phytoplankton stoichiometry based on cell protein regulation

Directory of Open Access Journals (Sweden)

J. A. Bonachela

2013-06-01

Full Text Available The elemental ratios of marine phytoplankton emerge from complex interactions between the biotic and abiotic components of the ocean, and reflect the plastic response of individuals to changes in their environment. The stoichiometry of phytoplankton is, thus, dynamic and dependent on the physiological state of the cell. We present a theoretical model for the dynamics of the carbon, nitrogen and phosphorus contents of a phytoplankton population. By representing the regulatory processes controlling nutrient uptake, and focusing on the relation between nutrient content and protein synthesis, our model qualitatively replicates existing experimental observations for nutrient content and ratios. The population described by our model takes up nutrients in proportions that match the input ratios for a broad range of growth conditions. In addition, there are two zones of single-nutrient limitation separated by a wide zone of co-limitation. Within the co-limitation zone, a single point can be identified where nutrients are supplied in an optimal ratio. When different species compete, the existence of a wide co-limitation zone implies a more complex pattern of coexistence and exclusion compared to previous model predictions. However, additional comprehensive laboratory experiments are needed to test our predictions. Our model contributes to the understanding of the global cycles of oceanic nitrogen and phosphorus, as well as the elemental ratios of these nutrients in phytoplankton populations.

Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

Science.gov (United States)

Ebert, Berit; Melle, Christian; Lieckfeldt, Elke; Zöller, Daniela; von Eggeling, Ferdinand; Fisahn, Joachim

2008-08-25

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

A hidden markov model derived structural alphabet for proteins.

Science.gov (United States)

Camproux, A C; Gautier, R; Tufféry, P

2004-06-04

Understanding and predicting protein structures depends on the complexity and the accuracy of the models used to represent them. We have set up a hidden Markov model that discretizes protein backbone conformation as series of overlapping fragments (states) of four residues length. This approach learns simultaneously the geometry of the states and their connections. We obtain, using a statistical criterion, an optimal systematic decomposition of the conformational variability of the protein peptidic chain in 27 states with strong connection logic. This result is stable over different protein sets. Our model fits well the previous knowledge related to protein architecture organisation and seems able to grab some subtle details of protein organisation, such as helix sub-level organisation schemes. Taking into account the dependence between the states results in a description of local protein structure of low complexity. On an average, the model makes use of only 8.3 states among 27 to describe each position of a protein structure. Although we use short fragments, the learning process on entire protein conformations captures the logic of the assembly on a larger scale. Using such a model, the structure of proteins can be reconstructed with an average accuracy close to 1.1A root-mean-square deviation and for a complexity of only 3. Finally, we also observe that sequence specificity increases with the number of states of the structural alphabet. Such models can constitute a very relevant approach to the analysis of protein architecture in particular for protein structure prediction.

Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes

Directory of Open Access Journals (Sweden)

Berkhout Ben

2006-12-01

Full Text Available Abstract Background The genome of coronaviruses contains structural and non-structural genes, including several so-called accessory genes. All group 1b coronaviruses encode a single accessory protein between the spike and envelope genes, except for human coronavirus (HCoV 229E. The prototype virus has a split gene, encoding the putative ORF4a and ORF4b proteins. To determine whether primary HCoV-229E isolates exhibit this unusual genome organization, we analyzed the ORF4a/b region of five current clinical isolates from The Netherlands and three early isolates collected at the Common Cold Unit (CCU in Salisbury, UK. Results All Dutch isolates were identical in the ORF4a/b region at amino acid level. All CCU isolates are only 98% identical to the Dutch isolates at the nucleotide level, but more closely related to the prototype HCoV-229E (>98%. Remarkably, our analyses revealed that the laboratory adapted, prototype HCoV-229E has a 2-nucleotide deletion in the ORF4a/b region, whereas all clinical isolates carry a single ORF, 660 nt in size, encoding a single protein of 219 amino acids, which is a homologue of the ORF3 proteins encoded by HCoV-NL63 and PEDV. Conclusion Thus, the genome organization of the group 1b coronaviruses HCoV-NL63, PEDV and HCoV-229E is identical. It is possible that extensive culturing of the HCoV-229E laboratory strain resulted in truncation of ORF4. This may indicate that the protein is not essential in cell culture, but the highly conserved amino acid sequence of the ORF4 protein among clinical isolates suggests that the protein plays an important role in vivo.

Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA

DEFF Research Database (Denmark)

Thresher, RJ; Christiansen, Gunna; Griffith, JD

1988-01-01

We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional Rec......M in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein...... assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein...

Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line.

Directory of Open Access Journals (Sweden)

Eszter Lakatos

Full Text Available We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.

Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line.

Science.gov (United States)

Lakatos, Eszter; Salehi-Reyhani, Ali; Barclay, Michael; Stumpf, Michael P H; Klug, David R

2017-01-01

We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.

Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.

Science.gov (United States)

Brewer, Laurence R; Friddle, Raymond; Noy, Aleksandr; Baldwin, Enoch; Martin, Shelley S; Corzett, Michele; Balhorn, Rod; Baskin, Ronald J

2003-10-01

Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.

Protein permeation through an electrically tunable membrane

International Nuclear Information System (INIS)

Jou, Ining A; Melnikov, Dmitriy V; Gracheva, Maria E

2016-01-01

Protein filtration is important in many fields of science and technology such as medicine, biology, chemistry, and engineering. Recently, protein separation and filtering with nanoporous membranes has attracted interest due to the possibility of fast separation and high throughput volume. This, however, requires understanding of the protein’s dynamics inside and in the vicinity of the nanopore. In this work, we utilize a Brownian dynamics approach to study the motion of the model protein insulin in the membrane–electrolyte electrostatic potential. We compare the results of the atomic model of the protein with the results of a coarse-grained and a single-bead model, and find that the coarse-grained representation of protein strikes the best balance between the accuracy of the results and the computational effort required. Contrary to common belief, we find that to adequately describe the protein, a single-bead model cannot be utilized without a significant effort to tabulate the simulation parameters. Similar to results for nanoparticle dynamics, our findings also indicate that the electric field and the electro-osmotic flow due to the applied membrane and electrolyte biases affect the capture and translocation of the biomolecule by either attracting or repelling it to or from the nanopore. Our computational model can also be applied to other types of proteins and separation conditions. (paper)

Confined Diffusion Without Fences of a G-Protein-Coupled Receptor as Revealed by Single Particle Tracking

Science.gov (United States)

Daumas, Frédéric; Destainville, Nicolas; Millot, Claire; Lopez, André; Dean, David; Salomé, Laurence

2003-01-01

Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the μ-opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients. PMID:12524289

Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

International Nuclear Information System (INIS)

Mao, Lili; Inoue, Koichi; Tao, Yisong; Montelione, Gaetano T.; McDermott, Ann E.; Inouye, Masayori

2011-01-01

Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that 13 C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that 13 C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.

WINKLER'S SINGLE-PARAMETER SUBGRADE MODEL FROM ...

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Page 1 ... corresponding single-parameter Winkler model presented in this work. Keywords: Heterogeneous subgrade, Reissner's simplified continuum, Shear interaction, Simplified continuum, Winkler ... model in practical applications and its long time familiarity among practical engineers, its usage has endured to this date ...

Prediction of Water Binding to Protein Hydration Sites with a Discrete, Semiexplicit Solvent Model.

Science.gov (United States)

Setny, Piotr

2015-12-08

Buried water molecules are ubiquitous in protein structures and are found at the interface of most protein-ligand complexes. Determining their distribution and thermodynamic effect is a challenging yet important task, of great of practical value for the modeling of biomolecular structures and their interactions. In this study, we present a novel method aimed at the prediction of buried water molecules in protein structures and estimation of their binding free energies. It is based on a semiexplicit, discrete solvation model, which we previously introduced in the context of small molecule hydration. The method is applicable to all macromolecular structures described by a standard all-atom force field, and predicts complete solvent distribution within a single run with modest computational cost. We demonstrate that it indicates positions of buried hydration sites, including those filled by more than one water molecule, and accurately differentiates them from sterically accessible to water but void regions. The obtained estimates of water binding free energies are in fair agreement with reference results determined with the double decoupling method.

Single Molecule Spectroscopy of Fluorescent Proteins

NARCIS (Netherlands)

Blum, Christian; Subramaniam, Vinod

2009-01-01

The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

Single-cluster dynamics for the random-cluster model

NARCIS (Netherlands)

Deng, Y.; Qian, X.; Blöte, H.W.J.

2009-01-01

We formulate a single-cluster Monte Carlo algorithm for the simulation of the random-cluster model. This algorithm is a generalization of the Wolff single-cluster method for the q-state Potts model to noninteger values q>1. Its results for static quantities are in a satisfactory agreement with those

Models of protein-ligand crystal structures: trust, but verify.

Science.gov (United States)

Deller, Marc C; Rupp, Bernhard

2015-09-01

X-ray crystallography provides the most accurate models of protein-ligand structures. These models serve as the foundation of many computational methods including structure prediction, molecular modelling, and structure-based drug design. The success of these computational methods ultimately depends on the quality of the underlying protein-ligand models. X-ray crystallography offers the unparalleled advantage of a clear mathematical formalism relating the experimental data to the protein-ligand model. In the case of X-ray crystallography, the primary experimental evidence is the electron density of the molecules forming the crystal. The first step in the generation of an accurate and precise crystallographic model is the interpretation of the electron density of the crystal, typically carried out by construction of an atomic model. The atomic model must then be validated for fit to the experimental electron density and also for agreement with prior expectations of stereochemistry. Stringent validation of protein-ligand models has become possible as a result of the mandatory deposition of primary diffraction data, and many computational tools are now available to aid in the validation process. Validation of protein-ligand complexes has revealed some instances of overenthusiastic interpretation of ligand density. Fundamental concepts and metrics of protein-ligand quality validation are discussed and we highlight software tools to assist in this process. It is essential that end users select high quality protein-ligand models for their computational and biological studies, and we provide an overview of how this can be achieved.

Network single-walled carbon nanotube biosensors for fast and highly sensitive detection of proteins

International Nuclear Information System (INIS)

Hu Pingan; Zhang Jia; Wen Zhenzhong; Zhang Can

2011-01-01

Detection of proteins is powerfully assayed in the diagnosis of diseases. A strategy for the development of an ultrahigh sensitivity biosensor based on a network single-walled carbon nanotube (SWNT) field-effect transistor (FET) has been demonstrated. Metallic SWNTs (m-SWNTs) in the network nanotube FET were selectively removed or cut via a carefully controlled procedure of electrical break-down (BD), and left non-conducting m-SWNTs which magnified the Schottky barrier (SB) area. This nanotube FET exhibited ultrahigh sensitivity and fast response to biomolecules. The lowest detection limit of 0.5 pM was achieved by exploiting streptavidin (SA) or a biotin/SA pair as the research model, and BD-treated nanotube biosensors had a 2 x 10 4 -fold lower minimum detectable concentration than the device without BD treatment. The response time is in the range of 0.3-3 min.

Markov dynamic models for long-timescale protein motion.

KAUST Repository

Chiang, Tsung-Han

2010-06-01

Molecular dynamics (MD) simulation is a well-established method for studying protein motion at the atomic scale. However, it is computationally intensive and generates massive amounts of data. One way of addressing the dual challenges of computation efficiency and data analysis is to construct simplified models of long-timescale protein motion from MD simulation data. In this direction, we propose to use Markov models with hidden states, in which the Markovian states represent potentially overlapping probabilistic distributions over protein conformations. We also propose a principled criterion for evaluating the quality of a model by its ability to predict long-timescale protein motions. Our method was tested on 2D synthetic energy landscapes and two extensively studied peptides, alanine dipeptide and the villin headpiece subdomain (HP-35 NleNle). One interesting finding is that although a widely accepted model of alanine dipeptide contains six states, a simpler model with only three states is equally good for predicting long-timescale motions. We also used the constructed Markov models to estimate important kinetic and dynamic quantities for protein folding, in particular, mean first-passage time. The results are consistent with available experimental measurements.

Markov dynamic models for long-timescale protein motion.

KAUST Repository

Chiang, Tsung-Han; Hsu, David; Latombe, Jean-Claude

2010-01-01

Molecular dynamics (MD) simulation is a well-established method for studying protein motion at the atomic scale. However, it is computationally intensive and generates massive amounts of data. One way of addressing the dual challenges of computation efficiency and data analysis is to construct simplified models of long-timescale protein motion from MD simulation data. In this direction, we propose to use Markov models with hidden states, in which the Markovian states represent potentially overlapping probabilistic distributions over protein conformations. We also propose a principled criterion for evaluating the quality of a model by its ability to predict long-timescale protein motions. Our method was tested on 2D synthetic energy landscapes and two extensively studied peptides, alanine dipeptide and the villin headpiece subdomain (HP-35 NleNle). One interesting finding is that although a widely accepted model of alanine dipeptide contains six states, a simpler model with only three states is equally good for predicting long-timescale motions. We also used the constructed Markov models to estimate important kinetic and dynamic quantities for protein folding, in particular, mean first-passage time. The results are consistent with available experimental measurements.

Studying Validity of Single-Fluid Model in Inertial Confinement Fusion

International Nuclear Information System (INIS)

Gu Jian-Fa; Fan Zheng-Feng; Dai Zhen-Sheng; Ye Wen-Hua; Pei Wen-Bing; Zhu Shao-Ping

2014-01-01

The validity of single-fluid model in inertial confinement fusion simulations is studied by comparing the results of the multi- and single-fluid models. The multi-fluid model includes the effects of collision and interpenetration between fluid species. By simulating the collision of fluid species, steady-state shock propagation into the thin DT gas and expansion of hohlraum Au wall heated by lasers, the results show that the validity of single-fluid model is strongly dependent on the ratio of the characteristic length of the simulated system to the particle mean free path. When the characteristic length L is one order larger than the mean free path λ, the single-fluid model's results are found to be in good agreement with the multi-fluid model's simulations, and the modeling of single-fluid remains valid. If the value of L/λ is lower than 10, the interpenetration between fluid species is significant, and the single-fluid simulations show some unphysical results; while the multi-fluid model can describe well the interpenetration and mix phenomena, and give more reasonable results. (physics of gases, plasmas, and electric discharges)

Target specific proteochemometric model development for BACE1 - protein flexibility and structural water are critical in virtual screening.

Science.gov (United States)

Manoharan, Prabu; Chennoju, Kiranmai; Ghoshal, Nanda

2015-07-01

BACE1 is an attractive target in Alzheimer's disease (AD) treatment. A rational drug design effort for the inhibition of BACE1 is actively pursued by researchers in both academic and pharmaceutical industries. This continued effort led to the steady accumulation of BACE1 crystal structures, co-complexed with different classes of inhibitors. This wealth of information is used in this study to develop target specific proteochemometric models and these models are exploited for predicting the prospective BACE1 inhibitors. The models developed in this study have performed excellently in predicting the computationally generated poses, separately obtained from single and ensemble docking approaches. The simple protein-ligand contact (SPLC) model outperforms other sophisticated high end models, in virtual screening performance, developed during this study. In an attempt to account for BACE1 protein active site flexibility information in predictive models, we included the change in the area of solvent accessible surface and the change in the volume of solvent accessible surface in our models. The ensemble and single receptor docking results obtained from this study indicate that the structural water mediated interactions improve the virtual screening results. Also, these waters are essential for recapitulating bioactive conformation during docking study. The proteochemometric models developed in this study can be used for the prediction of BACE1 inhibitors, during the early stage of AD drug discovery.

Robust model-based analysis of single-particle tracking experiments with Spot-On

Science.gov (United States)

Grimm, Jonathan B; Lavis, Luke D

2018-01-01

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce ‘Spot-On’, an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants. PMID:29300163

Graphical models for inferring single molecule dynamics

Directory of Open Access Journals (Sweden)

Gonzalez Ruben L

2010-10-01

Full Text Available Abstract Background The recent explosion of experimental techniques in single molecule biophysics has generated a variety of novel time series data requiring equally novel computational tools for analysis and inference. This article describes in general terms how graphical modeling may be used to learn from biophysical time series data using the variational Bayesian expectation maximization algorithm (VBEM. The discussion is illustrated by the example of single-molecule fluorescence resonance energy transfer (smFRET versus time data, where the smFRET time series is modeled as a hidden Markov model (HMM with Gaussian observables. A detailed description of smFRET is provided as well. Results The VBEM algorithm returns the model’s evidence and an approximating posterior parameter distribution given the data. The former provides a metric for model selection via maximum evidence (ME, and the latter a description of the model’s parameters learned from the data. ME/VBEM provide several advantages over the more commonly used approach of maximum likelihood (ML optimized by the expectation maximization (EM algorithm, the most important being a natural form of model selection and a well-posed (non-divergent optimization problem. Conclusions The results demonstrate the utility of graphical modeling for inference of dynamic processes in single molecule biophysics.

Factors affecting GEBV accuracy with single-step Bayesian models.

Science.gov (United States)

Zhou, Lei; Mrode, Raphael; Zhang, Shengli; Zhang, Qin; Li, Bugao; Liu, Jian-Feng

2018-01-01

A single-step approach to obtain genomic prediction was first proposed in 2009. Many studies have investigated the components of GEBV accuracy in genomic selection. However, it is still unclear how the population structure and the relationships between training and validation populations influence GEBV accuracy in terms of single-step analysis. Here, we explored the components of GEBV accuracy in single-step Bayesian analysis with a simulation study. Three scenarios with various numbers of QTL (5, 50, and 500) were simulated. Three models were implemented to analyze the simulated data: single-step genomic best linear unbiased prediction (GBLUP; SSGBLUP), single-step BayesA (SS-BayesA), and single-step BayesB (SS-BayesB). According to our results, GEBV accuracy was influenced by the relationships between the training and validation populations more significantly for ungenotyped animals than for genotyped animals. SS-BayesA/BayesB showed an obvious advantage over SSGBLUP with the scenarios of 5 and 50 QTL. SS-BayesB model obtained the lowest accuracy with the 500 QTL in the simulation. SS-BayesA model was the most efficient and robust considering all QTL scenarios. Generally, both the relationships between training and validation populations and LD between markers and QTL contributed to GEBV accuracy in the single-step analysis, and the advantages of single-step Bayesian models were more apparent when the trait is controlled by fewer QTL.

Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies.

Science.gov (United States)

Padhiar, Arshad Ahmed; Chanda, Warren; Joseph, Thomson Patrick; Guo, Xuefang; Liu, Min; Sha, Li; Batool, Samana; Gao, Yifan; Zhang, Wei; Huang, Min; Zhong, Mintao

2018-03-01

The formation of inclusion bodies (IBs) is considered as an Achilles heel of heterologous protein expression in bacterial hosts. Wide array of techniques has been developed to recover biochemically challenging proteins from IBs. However, acquiring the active state even from the same protein family was found to be an independent of single established method. Here, we present a new strategy for the recovery of wide sub-classes of recombinant protein from harsh IBs. We found that numerous methods and their combinations for reducing IB formation and producing soluble proteins were not effective, if the inclusion bodies were harsh in nature. On the other hand, different practices with mild solubilization buffers were able to solubilize IBs completely, yet the recovery of active protein requires large screening of refolding buffers. With the integration of previously reported mild solubilization techniques, we proposed an improved method, which comprised low sarkosyl concentration, ranging from 0.05 to 0.1% coupled with slow freezing (- 1 °C/min) and fast thaw (room temperature), resulting in greater solubility and the integrity of solubilized protein. Dilution method was employed with single buffer to restore activity for every sub-class of recombinant protein. Results showed that the recovered protein's activity was significantly higher compared with traditional solubilization/refolding approach. Solubilization of IBs by the described method was proved milder in nature, which restored native-like conformation of proteins within IBs.

Nature of the protein universe

Science.gov (United States)

Levitt, Michael

2009-01-01

The protein universe is the set of all proteins of all organisms. Here, all currently known sequences are analyzed in terms of families that have single-domain or multidomain architectures and whether they have a known three-dimensional structure. Growth of new single-domain families is very slow: Almost all growth comes from new multidomain architectures that are combinations of domains characterized by ≈15,000 sequence profiles. Single-domain families are mostly shared by the major groups of organisms, whereas multidomain architectures are specific and account for species diversity. There are known structures for a quarter of the single-domain families, and >70% of all sequences can be partially modeled thanks to their membership in these families. PMID:19541617

Binding free energy analysis of protein-protein docking model structures by evERdock.

Science.gov (United States)

Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

2018-03-14

To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry.

Science.gov (United States)

Kim, Kyung Lock; Park, Kyeng Min; Murray, James; Kim, Kimoon; Ryu, Sung Ho

2018-05-23

Combinatorial post-translational modifications (PTMs), which can serve as dynamic "molecular barcodes", have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

Long-range protein electron transfer observed at the single-molecule level: In situ mapping of redox-gated tunneling resonance

DEFF Research Database (Denmark)

Chi, Qijin; Farver, O; Ulstrup, Jens

2005-01-01

on the redox potential. Maximum resonance appears around the equilibrium redox potential of azurin with an on/off current ratio of approximate to 9. Simulation analyses, based on a two-step interfacial ET model for the scanning tunneling microscopy redox process, were performed and provide quantitative......A biomimetic long-range electron transfer (ET) system consisting of the blue copper protein azurin, a tunneling barrier bridge, and a gold single-crystal electrode was designed on the basis of molecular wiring self-assembly principles. This system is sufficiently stable and sensitive in a quasi...... constants display tunneling features with distance-decay factors of 0.83 and 0.91 angstrom(-1) in H2O and D2O, respectively. Redox-gated tunneling resonance is observed in situ at the single-molecule level by using electrochemical scanning tunneling microscopy, exhibiting an asymmetric dependence...

PROCARB: A Database of Known and Modelled Carbohydrate-Binding Protein Structures with Sequence-Based Prediction Tools

Directory of Open Access Journals (Sweden)

Adeel Malik

2010-01-01

Full Text Available Understanding of the three-dimensional structures of proteins that interact with carbohydrates covalently (glycoproteins as well as noncovalently (protein-carbohydrate complexes is essential to many biological processes and plays a significant role in normal and disease-associated functions. It is important to have a central repository of knowledge available about these protein-carbohydrate complexes as well as preprocessed data of predicted structures. This can be significantly enhanced by tools de novo which can predict carbohydrate-binding sites for proteins in the absence of structure of experimentally known binding site. PROCARB is an open-access database comprising three independently working components, namely, (i Core PROCARB module, consisting of three-dimensional structures of protein-carbohydrate complexes taken from Protein Data Bank (PDB, (ii Homology Models module, consisting of manually developed three-dimensional models of N-linked and O-linked glycoproteins of unknown three-dimensional structure, and (iii CBS-Pred prediction module, consisting of web servers to predict carbohydrate-binding sites using single sequence or server-generated PSSM. Several precomputed structural and functional properties of complexes are also included in the database for quick analysis. In particular, information about function, secondary structure, solvent accessibility, hydrogen bonds and literature reference, and so forth, is included. In addition, each protein in the database is mapped to Uniprot, Pfam, PDB, and so forth.

Model for calculation of electrostatic contribution into protein stability

Science.gov (United States)

Kundrotas, Petras; Karshikoff, Andrey

2003-03-01

Existing models of the denatured state of proteins consider only one possible spatial distribution of protein charges and therefore are applicable to a limited number of cases. In this presentation a more general framework for the modeling of the denatured state is proposed. It is based on the assumption that the titratable groups of an unfolded protein can adopt a quasi-random distribution, restricted by the protein sequence. The model was tested on two proteins, barnase and N-terminal domain of the ribosomal protein L9. The calculated free energy of denaturation, Δ G( pH), reproduces the experimental data essentially better than the commonly used null approximation (NA). It was demonstrated that the seemingly good agreement with experimental data obtained by NA originates from the compensatory effect between the pair-wise electrostatic interactions and the desolvation energy of the individual sites. It was also found that the ionization properties of denatured proteins are influenced by the protein sequence.

Discrete persistent-chain model for protein binding on DNA.

Science.gov (United States)

Lam, Pui-Man; Zhen, Yi

2011-04-01

We describe and solve a discrete persistent-chain model of protein binding on DNA, involving an extra σ(i) at a site i of the DNA. This variable takes the value 1 or 0, depending on whether or not the site is occupied by a protein. In addition, if the site is occupied by a protein, there is an extra energy cost ɛ. For a small force, we obtain analytic expressions for the force-extension curve and the fraction of bound protein on the DNA. For higher forces, the model can be solved numerically to obtain force-extension curves and the average fraction of bound proteins as a function of applied force. Our model can be used to analyze experimental force-extension curves of protein binding on DNA, and hence deduce the number of bound proteins in the case of nonspecific binding. ©2011 American Physical Society

A novel multimodal chromatography based single step purification process for efficient manufacturing of an E. coli based biotherapeutic protein product.

Science.gov (United States)

Bhambure, Rahul; Gupta, Darpan; Rathore, Anurag S

2013-11-01

Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pHproduct variants using the combined pH and salt based elution gradient and removal of the host cell impurities in flow-through are the key novel features of the developed multimodal chromatographic purification step. Copyright © 2013 Elsevier B.V. All rights reserved.

Modeling Mercury in Proteins

Energy Technology Data Exchange (ETDEWEB)

Smith, Jeremy C [ORNL; Parks, Jerry M [ORNL

2016-01-01

Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively non-toxic, other forms such as Hg2+ and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg2+ can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg2+ to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed picture and circumvent issues associated with toxicity. Here we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intra-protein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confers mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multi-scale model of environmental mercury cycling.

Production of single cell protein (SCP) from food and agricultural waste by using Saccharomyces cerevisiae.

Science.gov (United States)

Gervasi, Teresa; Pellizzeri, Vito; Calabrese, Giorgio; Di Bella, Giuseppa; Cicero, Nicola; Dugo, Giacomo

2018-03-01

Food waste is the single-largest component of the waste stream, in order to protect and safeguard the public health, useful and innovative recycling methods are investigated. The conversion of food wastes in value-added products is becoming a more economically viable and interesting practice. Food waste, collected in the distribution sector and citrus industries, was characterised for its potential as a raw material to use in fermentation processes. In this study, the production of single-cell protein (SCP) using food waste as a substrate was investigated. The purpose of this study has been to produce SCP from mixtures of food waste using Saccharomyces cerevisiae. The main fermentation test was carried out using a 25 l bioreactor. The utilisation of food waste can allow us to not only to reduce environmental pollution, but also to obtain value-added products such as protein supply for animal feed.

Calculation of accurate small angle X-ray scattering curves from coarse-grained protein models

Directory of Open Access Journals (Sweden)

Stovgaard Kasper

2010-08-01

Full Text Available Abstract Background Genome sequencing projects have expanded the gap between the amount of known protein sequences and structures. The limitations of current high resolution structure determination methods make it unlikely that this gap will disappear in the near future. Small angle X-ray scattering (SAXS is an established low resolution method for routinely determining the structure of proteins in solution. The purpose of this study is to develop a method for the efficient calculation of accurate SAXS curves from coarse-grained protein models. Such a method can for example be used to construct a likelihood function, which is paramount for structure determination based on statistical inference. Results We present a method for the efficient calculation of accurate SAXS curves based on the Debye formula and a set of scattering form factors for dummy atom representations of amino acids. Such a method avoids the computationally costly iteration over all atoms. We estimated the form factors using generated data from a set of high quality protein structures. No ad hoc scaling or correction factors are applied in the calculation of the curves. Two coarse-grained representations of protein structure were investigated; two scattering bodies per amino acid led to significantly better results than a single scattering body. Conclusion We show that the obtained point estimates allow the calculation of accurate SAXS curves from coarse-grained protein models. The resulting curves are on par with the current state-of-the-art program CRYSOL, which requires full atomic detail. Our method was also comparable to CRYSOL in recognizing native structures among native-like decoys. As a proof-of-concept, we combined the coarse-grained Debye calculation with a previously described probabilistic model of protein structure, TorusDBN. This resulted in a significant improvement in the decoy recognition performance. In conclusion, the presented method shows great promise for

Assigning Significance in Label-Free Quantitative Proteomics to Include Single-Peptide-Hit Proteins with Low Replicates

OpenAIRE

Li, Qingbo

2010-01-01

When sample replicates are limited in a label-free proteomics experiment, selecting differentially regulated proteins with an assignment of statistical significance remains difficult for proteins with a single-peptide hit or a small fold-change. This paper aims to address this issue. An important component of the approach employed here is to utilize the rule of Minimum number of Permuted Significant Pairings (MPSP) to reduce false positives. The MPSP rule generates permuted sample pairings fr...

Exploring structural variability in X-ray crystallographic models using protein local optimization by torsion-angle sampling

International Nuclear Information System (INIS)

Knight, Jennifer L.; Zhou, Zhiyong; Gallicchio, Emilio; Himmel, Daniel M.; Friesner, Richard A.; Arnold, Eddy; Levy, Ronald M.

2008-01-01

Torsion-angle sampling, as implemented in the Protein Local Optimization Program (PLOP), is used to generate multiple structurally variable single-conformer models which are in good agreement with X-ray data. An ensemble-refinement approach to differentiate between positional uncertainty and conformational heterogeneity is proposed. Modeling structural variability is critical for understanding protein function and for modeling reliable targets for in silico docking experiments. Because of the time-intensive nature of manual X-ray crystallographic refinement, automated refinement methods that thoroughly explore conformational space are essential for the systematic construction of structurally variable models. Using five proteins spanning resolutions of 1.0–2.8 Å, it is demonstrated how torsion-angle sampling of backbone and side-chain libraries with filtering against both the chemical energy, using a modern effective potential, and the electron density, coupled with minimization of a reciprocal-space X-ray target function, can generate multiple structurally variable models which fit the X-ray data well. Torsion-angle sampling as implemented in the Protein Local Optimization Program (PLOP) has been used in this work. Models with the lowest R free values are obtained when electrostatic and implicit solvation terms are included in the effective potential. HIV-1 protease, calmodulin and SUMO-conjugating enzyme illustrate how variability in the ensemble of structures captures structural variability that is observed across multiple crystal structures and is linked to functional flexibility at hinge regions and binding interfaces. An ensemble-refinement procedure is proposed to differentiate between variability that is a consequence of physical conformational heterogeneity and that which reflects uncertainty in the atomic coordinates

Protein Folding: Search for Basic Physical Models

Directory of Open Access Journals (Sweden)

Ivan Y. Torshin

2003-01-01

Full Text Available How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context of in vivo protein folding (which has been studied only for a few proteins, the roles of the fundamental physical forces in the in vitro folding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces. Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

Single Molecule Science for Personalized Nanomedicine: Atomic Force Microscopy of Biopolymer-Protein Interactions

Science.gov (United States)

Hsueh, Carlin

Nanotechnology has a unique and relatively untapped utility in the fields of medicine and dentistry at the level of single-biopolymer and -molecule diagnostics. In recent years atomic force microscopy (AFM) has garnered much interest due to its ability to obtain atomic-resolution of molecular structures and probe biophysical behaviors of biopolymers and proteins in a variety of biologically significant environments. The work presented in this thesis focuses on the nanoscale manipulation and observation of biopolymers to develop an innovative technology for personalized medicine while understanding complex biological systems. These studies described here primarily use AFM to observe biopolymer interactions with proteins and its surroundings with unprecedented resolution, providing a better understanding of these systems and interactions at the nanoscale. Transcriptional profiling, the measure of messenger RNA (mRNA) abundance in a single cell, is a powerful technique that detects "behavior" or "symptoms" at the tissue and cellular level. We have sought to develop an alternative approach, using our expertise in AFM and single molecule nanotechnology, to achieve a cost-effective high throughput method for sensitive detection and profiling of subtle changes in transcript abundance. The technique does not require amplification of the mRNA sample because the AFM provides three-dimensional views of molecules with unprecedented resolution, requires minimal sample preparation, and utilizes a simple tagging chemistry on cDNA molecules. AFM images showed collagen polymers in teeth and of Drebrin-A remodeling of filamentous actin structure and mechanics. AFM was used to image collagen on exposed dentine tubules and confirmed tubule occlusion with a desensitizing prophylaxis paste by Colgate-Palmolive. The AFM also superseded other microscopy tools in resolving F-actin helix remodeling and possible cooperative binding by a neuronal actin binding protein---Drebrin-A, an

Models of crk adaptor proteins in cancer.

Science.gov (United States)

Bell, Emily S; Park, Morag

2012-05-01

The Crk family of adaptor proteins (CrkI, CrkII, and CrkL), originally discovered as the oncogene fusion product, v-Crk, of the CT10 chicken retrovirus, lacks catalytic activity but engages with multiple signaling pathways through their SH2 and SH3 domains. Crk proteins link upstream tyrosine kinase and integrin-dependent signals to downstream effectors, acting as adaptors in diverse signaling pathways and cellular processes. Crk proteins are now recognized to play a role in the malignancy of many human cancers, stimulating renewed interest in their mechanism of action in cancer progression. The contribution of Crk signaling to malignancy has been predominantly studied in fibroblasts and in hematopoietic models and more recently in epithelial models. A mechanistic understanding of Crk proteins in cancer progression in vivo is still poorly understood in part due to the highly pleiotropic nature of Crk signaling. Recent advances in the structural organization of Crk domains, new roles in kinase regulation, and increased knowledge of the mechanisms and frequency of Crk overexpression in human cancers have provided an incentive for further study in in vivo models. An understanding of the mechanisms through which Crk proteins act as oncogenic drivers could have important implications in therapeutic targeting.

Modelling Protein Dynamics on the Microsecond Time Scale

DEFF Research Database (Denmark)

Siuda, Iwona Anna

Recent years have shown an increase in coarse-grained (CG) molecular dynamics simulations, providing structural and dynamic details of large proteins and enabling studies of self-assembly of biological materials. It is not easy to acquire such data experimentally, and access is also still limited...... in atomistic simulations. During her PhD studies, Iwona Siuda used MARTINI CG models to study the dynamics of different globular and membrane proteins. In several cases, the MARTINI model was sufficient to study conformational changes of small, purely alpha-helical proteins. However, in studies of larger......ELNEDIN was therefore proposed as part of the work. Iwona Siuda’s results from the CG simulations had biological implications that provide insights into possible mechanisms of the periplasmic leucine-binding protein, the sarco(endo)plasmic reticulum calcium pump, and several proteins from the saposin-like proteins...

Introducing a Clustering Step in a Consensus Approach for the Scoring of Protein-Protein Docking Models

KAUST Repository

Chermak, Edrisse; De Donato, Renato; Lensink, Marc F.; Petta, Andrea; Serra, Luigi; Scarano, Vittorio; Cavallo, Luigi; Oliva, Romina

2016-01-01

Correctly scoring protein-protein docking models to single out native-like ones is an open challenge. It is also an object of assessment in CAPRI (Critical Assessment of PRedicted Interactions), the community-wide blind docking experiment. We introduced in the field the first pure consensus method, CONSRANK, which ranks models based on their ability to match the most conserved contacts in the ensemble they belong to. In CAPRI, scorers are asked to evaluate a set of available models and select the top ten ones, based on their own scoring approach. Scorers' performance is ranked based on the number of targets/interfaces for which they could provide at least one correct solution. In such terms, blind testing in CAPRI Round 30 (a joint prediction round with CASP11) has shown that critical cases for CONSRANK are represented by targets showing multiple interfaces or for which only a very small number of correct solutions are available. To address these challenging cases, CONSRANK has now been modified to include a contact-based clustering of the models as a preliminary step of the scoring process. We used an agglomerative hierarchical clustering based on the number of common inter-residue contacts within the models. Two criteria, with different thresholds, were explored in the cluster generation, setting either the number of common contacts or of total clusters. For each clustering approach, after selecting the top (most populated) ten clusters, CONSRANK was run on these clusters and the top-ranked model for each cluster was selected, in the limit of 10 models per target. We have applied our modified scoring approach, Clust-CONSRANK, to SCORE_SET, a set of CAPRI scoring models made recently available by CAPRI assessors, and to the subset of homodimeric targets in CAPRI Round 30 for which CONSRANK failed to include a correct solution within the ten selected models. Results show that, for the challenging cases, the clustering step typically enriches the ten top ranked

Introducing a Clustering Step in a Consensus Approach for the Scoring of Protein-Protein Docking Models

KAUST Repository

Chermak, Edrisse

2016-11-15

Correctly scoring protein-protein docking models to single out native-like ones is an open challenge. It is also an object of assessment in CAPRI (Critical Assessment of PRedicted Interactions), the community-wide blind docking experiment. We introduced in the field the first pure consensus method, CONSRANK, which ranks models based on their ability to match the most conserved contacts in the ensemble they belong to. In CAPRI, scorers are asked to evaluate a set of available models and select the top ten ones, based on their own scoring approach. Scorers\\' performance is ranked based on the number of targets/interfaces for which they could provide at least one correct solution. In such terms, blind testing in CAPRI Round 30 (a joint prediction round with CASP11) has shown that critical cases for CONSRANK are represented by targets showing multiple interfaces or for which only a very small number of correct solutions are available. To address these challenging cases, CONSRANK has now been modified to include a contact-based clustering of the models as a preliminary step of the scoring process. We used an agglomerative hierarchical clustering based on the number of common inter-residue contacts within the models. Two criteria, with different thresholds, were explored in the cluster generation, setting either the number of common contacts or of total clusters. For each clustering approach, after selecting the top (most populated) ten clusters, CONSRANK was run on these clusters and the top-ranked model for each cluster was selected, in the limit of 10 models per target. We have applied our modified scoring approach, Clust-CONSRANK, to SCORE_SET, a set of CAPRI scoring models made recently available by CAPRI assessors, and to the subset of homodimeric targets in CAPRI Round 30 for which CONSRANK failed to include a correct solution within the ten selected models. Results show that, for the challenging cases, the clustering step typically enriches the ten top ranked

Mixture of Regression Models with Single-Index

OpenAIRE

Xiang, Sijia; Yao, Weixin

2016-01-01

In this article, we propose a class of semiparametric mixture regression models with single-index. We argue that many recently proposed semiparametric/nonparametric mixture regression models can be considered special cases of the proposed model. However, unlike existing semiparametric mixture regression models, the new pro- posed model can easily incorporate multivariate predictors into the nonparametric components. Backfitting estimates and the corresponding algorithms have been proposed for...

A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation

Directory of Open Access Journals (Sweden)

Patrizia Marinelli

2018-04-01

Full Text Available Oxidatively modified forms of proteins accumulate during aging. Oxidized protein conformers might act as intermediates in the formation of amyloids in age-related disorders. However, it is not known whether this amyloidogenic conversion requires an extensive protein oxidative damage or it can be promoted just by a discrete, localized post-translational modification of certain residues. Here, we demonstrate that the irreversible oxidation of a single free Cys suffices to severely perturb the folding energy landscape of a stable globular protein, compromise its kinetic stability, and lead to the formation of amyloids under physiological conditions. Experiments and simulations converge to indicate that this specific oxidation-promoted protein aggregation requires only local unfolding. Indeed, a large scale analysis indicates that many cellular proteins are at risk of undergoing this kind of deleterious transition; explaining how oxidative stress can impact cell proteostasis and subsequently lead to the onset of pathological states. Keywords: Protein oxidation, Protein misfolding, Protein aggregation, Oxidative stress, Post-translational modification

Comparison of Model Reliabilities from Single-Step and Bivariate Blending Methods

DEFF Research Database (Denmark)

Taskinen, Matti; Mäntysaari, Esa; Lidauer, Martin

2013-01-01

Model based reliabilities in genetic evaluation are compared between three methods: animal model BLUP, single-step BLUP, and bivariate blending after genomic BLUP. The original bivariate blending is revised in this work to better account animal models. The study data is extracted from...... be calculated. Model reliabilities by the single-step and the bivariate blending methods were higher than by animal model due to genomic information. Compared to the single-step method, the bivariate blending method reliability estimates were, in general, lower. Computationally bivariate blending method was......, on the other hand, lighter than the single-step method....

Immunogenicity of therapeutic proteins: the use of animal models.

Science.gov (United States)

Brinks, Vera; Jiskoot, Wim; Schellekens, Huub

2011-10-01

Immunogenicity of therapeutic proteins lowers patient well-being and drastically increases therapeutic costs. Preventing immunogenicity is an important issue to consider when developing novel therapeutic proteins and applying them in the clinic. Animal models are increasingly used to study immunogenicity of therapeutic proteins. They are employed as predictive tools to assess different aspects of immunogenicity during drug development and have become vital in studying the mechanisms underlying immunogenicity of therapeutic proteins. However, the use of animal models needs critical evaluation. Because of species differences, predictive value of such models is limited, and mechanistic studies can be restricted. This review addresses the suitability of animal models for immunogenicity prediction and summarizes the insights in immunogenicity that they have given so far.

Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

Science.gov (United States)

Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

2015-11-01

Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

Multiple graph regularized protein domain ranking.

Science.gov (United States)

Wang, Jim Jing-Yan; Bensmail, Halima; Gao, Xin

2012-11-19

Protein domain ranking is a fundamental task in structural biology. Most protein domain ranking methods rely on the pairwise comparison of protein domains while neglecting the global manifold structure of the protein domain database. Recently, graph regularized ranking that exploits the global structure of the graph defined by the pairwise similarities has been proposed. However, the existing graph regularized ranking methods are very sensitive to the choice of the graph model and parameters, and this remains a difficult problem for most of the protein domain ranking methods. To tackle this problem, we have developed the Multiple Graph regularized Ranking algorithm, MultiG-Rank. Instead of using a single graph to regularize the ranking scores, MultiG-Rank approximates the intrinsic manifold of protein domain distribution by combining multiple initial graphs for the regularization. Graph weights are learned with ranking scores jointly and automatically, by alternately minimizing an objective function in an iterative algorithm. Experimental results on a subset of the ASTRAL SCOP protein domain database demonstrate that MultiG-Rank achieves a better ranking performance than single graph regularized ranking methods and pairwise similarity based ranking methods. The problem of graph model and parameter selection in graph regularized protein domain ranking can be solved effectively by combining multiple graphs. This aspect of generalization introduces a new frontier in applying multiple graphs to solving protein domain ranking applications.

Co-cultivation of Green Microalgae and Methanotrophic Bacteria for Single Cell Protein Production from Wastewater

DEFF Research Database (Denmark)

Rasouli, Zahra; Valverde Pérez, Borja; D'Este, Martina

2017-01-01

microalgae – as a means to recover nutrients from industrial wastewater and upcycle them to feed grade single cell protein. Results demonstrated that both algae and bacteria could remove or assimilate most of the organic carbon present in the wastewater. However, their growth stopped before nutrients...

Scoring predictive models using a reduced representation of proteins: model and energy definition.

Science.gov (United States)

Fogolari, Federico; Pieri, Lidia; Dovier, Agostino; Bortolussi, Luca; Giugliarelli, Gilberto; Corazza, Alessandra; Esposito, Gennaro; Viglino, Paolo

2007-03-23

Reduced representations of proteins have been playing a keyrole in the study of protein folding. Many such models are available, with different representation detail. Although the usefulness of many such models for structural bioinformatics applications has been demonstrated in recent years, there are few intermediate resolution models endowed with an energy model capable, for instance, of detecting native or native-like structures among decoy sets. The aim of the present work is to provide a discrete empirical potential for a reduced protein model termed here PC2CA, because it employs a PseudoCovalent structure with only 2 Centers of interactions per Amino acid, suitable for protein model quality assessment. All protein structures in the set top500H have been converted in reduced form. The distribution of pseudobonds, pseudoangle, pseudodihedrals and distances between centers of interactions have been converted into potentials of mean force. A suitable reference distribution has been defined for non-bonded interactions which takes into account excluded volume effects and protein finite size. The correlation between adjacent main chain pseudodihedrals has been converted in an additional energetic term which is able to account for cooperative effects in secondary structure elements. Local energy surface exploration is performed in order to increase the robustness of the energy function. The model and the energy definition proposed have been tested on all the multiple decoys' sets in the Decoys'R'us database. The energetic model is able to recognize, for almost all sets, native-like structures (RMSD less than 2.0 A). These results and those obtained in the blind CASP7 quality assessment experiment suggest that the model compares well with scoring potentials with finer granularity and could be useful for fast exploration of conformational space. Parameters are available at the url: http://www.dstb.uniud.it/~ffogolari/download/.

Mixture models for single-cell assays with applications to vaccine studies

OpenAIRE

Finak, Greg; McDavid, Andrew; Chattopadhyay, Pratip; Dominguez, Maria; De Rosa, Steve; Roederer, Mario; Gottardo, Raphael

2013-01-01

Blood and tissue are composed of many functionally distinct cell subsets. In immunological studies, these can be measured accurately only using single-cell assays. The characterization of these small cell subsets is crucial to decipher system-level biological changes. For this reason, an increasing number of studies rely on assays that provide single-cell measurements of multiple genes and proteins from bulk cell samples. A common problem in the analysis of such data is to identify biomarkers...

Predicting nucleic acid binding interfaces from structural models of proteins.

Science.gov (United States)

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2012-02-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

Energy Technology Data Exchange (ETDEWEB)

Tsuji, Toshikazu; Kawai-Noma, Shigeko [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Pack, Chan-Gi [Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198 (Japan); Terajima, Hideki [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Yajima, Junichiro; Nishizaka, Takayuki [Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588 (Japan); Kinjo, Masataka [Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021 (Japan); Taguchi, Hideki, E-mail: taguchi@bio.titech.ac.jp [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)

2011-02-25

Research highlights: {yields} We develop a method to track a quantum dot-conjugated protein in yeast cells. {yields} We incorporate the conjugated quantum dot proteins into yeast spheroplasts. {yields} We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

International Nuclear Information System (INIS)

Tsuji, Toshikazu; Kawai-Noma, Shigeko; Pack, Chan-Gi; Terajima, Hideki; Yajima, Junichiro; Nishizaka, Takayuki; Kinjo, Masataka; Taguchi, Hideki

2011-01-01

Research highlights: → We develop a method to track a quantum dot-conjugated protein in yeast cells. → We incorporate the conjugated quantum dot proteins into yeast spheroplasts. → We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

Quantitative occupational risk model: Single hazard

International Nuclear Information System (INIS)

Papazoglou, I.A.; Aneziris, O.N.; Bellamy, L.J.; Ale, B.J.M.; Oh, J.

2017-01-01

A model for the quantification of occupational risk of a worker exposed to a single hazard is presented. The model connects the working conditions and worker behaviour to the probability of an accident resulting into one of three types of consequence: recoverable injury, permanent injury and death. Working conditions and safety barriers in place to reduce the likelihood of an accident are included. Logical connections are modelled through an influence diagram. Quantification of the model is based on two sources of information: a) number of accidents observed over a period of time and b) assessment of exposure data of activities and working conditions over the same period of time and the same working population. Effectiveness of risk reducing measures affecting the working conditions, worker behaviour and/or safety barriers can be quantified through the effect of these measures on occupational risk. - Highlights: • Quantification of occupational risk from a single hazard. • Influence diagram connects working conditions, worker behaviour and safety barriers. • Necessary data include the number of accidents and the total exposure of worker • Effectiveness of risk reducing measures is quantified through the impact on the risk • An example illustrates the methodology.

Highly sensitive immunoassay of protein molecules based on single nanoparticle fluorescence detection in a nanowell

Science.gov (United States)

Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

2011-03-01

A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

Descriptor Data Bank (DDB): A Cloud Platform for Multiperspective Modeling of Protein-Ligand Interactions.

Science.gov (United States)

Ashtawy, Hossam M; Mahapatra, Nihar R

2018-01-22

Protein-ligand (PL) interactions play a key role in many life processes such as molecular recognition, molecular binding, signal transmission, and cell metabolism. Examples of interaction forces include hydrogen bonding, hydrophobic effects, steric clashes, electrostatic contacts, and van der Waals attractions. Currently, a large number of hypotheses and perspectives to model these interaction forces are scattered throughout the literature and largely forgotten. Instead, had they been assembled and utilized collectively, they would have substantially improved the accuracy of predicting binding affinity of protein-ligand complexes. In this work, we present Descriptor Data Bank (DDB), a data-driven platform on the cloud for facilitating multiperspective modeling of PL interactions. DDB is an open-access hub for depositing, hosting, executing, and sharing descriptor extraction tools and data for a large number of interaction modeling hypotheses. The platform also implements a machine-learning (ML) toolbox for automatic descriptor filtering and analysis and scoring function (SF) fitting and prediction. The descriptor filtering module is used to filter out irrelevant and/or noisy descriptors and to produce a compact subset from all available features. We seed DDB with 16 diverse descriptor extraction tools developed in-house and collected from the literature. The tools altogether generate over 2700 descriptors that characterize (i) proteins, (ii) ligands, and (iii) protein-ligand complexes. The in-house descriptors we extract are protein-specific which are based on pairwise primary and tertiary alignment of protein structures followed by clustering and trilateration. We built and used DDB's ML library to fit SFs to the in-house descriptors and those collected from the literature. We then evaluated them on several data sets that were constructed to reflect real-world drug screening scenarios. We found that multiperspective SFs that were constructed using a large number

Fast loop modeling for protein structures

Science.gov (United States)

Zhang, Jiong; Nguyen, Son; Shang, Yi; Xu, Dong; Kosztin, Ioan

2015-03-01

X-ray crystallography is the main method for determining 3D protein structures. In many cases, however, flexible loop regions of proteins cannot be resolved by this approach. This leads to incomplete structures in the protein data bank, preventing further computational study and analysis of these proteins. For instance, all-atom molecular dynamics (MD) simulation studies of structure-function relationship require complete protein structures. To address this shortcoming, we have developed and implemented an efficient computational method for building missing protein loops. The method is database driven and uses deep learning and multi-dimensional scaling algorithms. We have implemented the method as a simple stand-alone program, which can also be used as a plugin in existing molecular modeling software, e.g., VMD. The quality and stability of the generated structures are assessed and tested via energy scoring functions and by equilibrium MD simulations. The proposed method can also be used in template-based protein structure prediction. Work supported by the National Institutes of Health [R01 GM100701]. Computer time was provided by the University of Missouri Bioinformatics Consortium.

Bioinformatic Analysis of Deleterious Non-Synonymous Single Nucleotide Polymorphisms (nsSNPs in the Coding Regions of Human Prion Protein Gene (PRNP

Directory of Open Access Journals (Sweden)

Kourosh Bamdad

2016-12-01

Full Text Available Background & Objective: Single nucleotide polymorphisms are the cause of genetic variation to living organisms. Single nucleotide polymorphisms alter residues in the protein sequence. In this investigation, the relationship between prion protein gene polymorphisms and its relevance to pathogenicity was studied. Material & Method: Amino acid sequence of the main isoform from the human prion protein gene (PRNP was extracted from UniProt database and evaluated by FoldAmyloid and AmylPred servers. All non-synonymous single nucleotide polymorphisms (nsSNPs from SNP database (dbSNP were further analyzed by bioinformatics servers including SIFT, PolyPhen-2, I-Mutant-3.0, PANTHER, SNPs & GO, PHD-SNP, Meta-SNP, and MutPred to determine the most damaging nsSNPs. Results: The results of the first structure analyses by FoldAmyloid and AmylPerd servers implied that regions including 5-15, 174-178, 180-184, 211-217, and 240-252 were the most sensitive parts of the protein sequence to amyloidosis. Screening all nsSNPs of the main protein isoform using bioinformatic servers revealed that substitution of Aspartic acid with Valine at position 178 (ID code: rs11538766 was the most deleterious nsSNP in the protein structure. Conclusion:  Substitution of the Aspartic acid with Valine at position 178 (D178V was the most pathogenic mutation in the human prion protein gene. Analyses from the MutPred server also showed that beta-sheets’ increment in the secondary structure was the main reason behind the molecular mechanism of the prion protein aggregation.

Modeling protein structures: construction and their applications.

Science.gov (United States)

Ring, C S; Cohen, F E

1993-06-01

Although no general solution to the protein folding problem exists, the three-dimensional structures of proteins are being successfully predicted when experimentally derived constraints are used in conjunction with heuristic methods. In the case of interleukin-4, mutagenesis data and CD spectroscopy were instrumental in the accurate assignment of secondary structure. In addition, the tertiary structure was highly constrained by six cysteines separated by many residues that formed three disulfide bridges. Although the correct structure was a member of a short list of plausible structures, the "best" structure was the topological enantiomer of the experimentally determined conformation. For many proteases, other experimentally derived structures can be used as templates to identify the secondary structure elements. In a procedure called modeling by homology, the structure of a known protein is used as a scaffold to predict the structure of another related protein. This method has been used to model a serine and a cysteine protease that are important in the schistosome and malarial life cycles, respectively. The model structures were then used to identify putative small molecule enzyme inhibitors computationally. Experiments confirm that some of these nonpeptidic compounds are active at concentrations of less than 10 microM.

The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins

DEFF Research Database (Denmark)

Farver, O; Skov, L K; van de Kamp, M

1992-01-01

-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine...... the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism....

A computational model of the LGI1 protein suggests a common binding site for ADAM proteins.

Directory of Open Access Journals (Sweden)

Emanuela Leonardi

Full Text Available Mutations of human leucine-rich glioma inactivated (LGI1 gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE, a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.

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

Bridging scales through multiscale modeling: A case study on Protein Kinase A

Directory of Open Access Journals (Sweden)

Sophia P Hirakis

2015-09-01

Full Text Available The goal of multiscale modeling in biology is to use structurally based physico-chemical models to integrate across temporal and spatial scales of biology and thereby improve mechanistic understanding of, for example, how a single mutation can alter organism-scale phenotypes. This approach may also inform therapeutic strategies or identify candidate drug targets that might otherwise have been overlooked. However, in many cases, it remains unclear how best to synthesize information obtained from various scales and analysis approaches, such as atomistic molecular models, Markov state models (MSM, subcellular network models, and whole cell models. In this paper, we use protein kinase A (PKA activation as a case study to explore how computational methods that model different physical scales can complement each other and integrate into an improved multiscale representation of the biological mechanisms. Using measured crystal structures, we show how molecular dynamics (MD simulations coupled with atomic-scale MSMs can provide conformations for Brownian dynamics (BD simulations to feed transitional states and kinetic parameters into protein-scale MSMs. We discuss how milestoning can give reaction probabilities and forward-rate constants of cAMP association events by seamlessly integrating MD and BD simulation scales. These rate constants coupled with MSMs provide a robust representation of the free energy landscape, enabling access to kinetic and thermodynamic parameters unavailable from current experimental data. These approaches have helped to illuminate the cooperative nature of PKA activation in response to distinct cAMP binding events. Collectively, this approach exemplifies a general strategy for multiscale model development that is applicable to a wide range of biological problems.

Scalable rule-based modelling of allosteric proteins and biochemical networks.

Directory of Open Access Journals (Sweden)

Julien F Ollivier

2010-11-01

Full Text Available Much of the complexity of biochemical networks comes from the information-processing abilities of allosteric proteins, be they receptors, ion-channels, signalling molecules or transcription factors. An allosteric protein can be uniquely regulated by each combination of input molecules that it binds. This "regulatory complexity" causes a combinatorial increase in the number of parameters required to fit experimental data as the number of protein interactions increases. It therefore challenges the creation, updating, and re-use of biochemical models. Here, we propose a rule-based modelling framework that exploits the intrinsic modularity of protein structure to address regulatory complexity. Rather than treating proteins as "black boxes", we model their hierarchical structure and, as conformational changes, internal dynamics. By modelling the regulation of allosteric proteins through these conformational changes, we often decrease the number of parameters required to fit data, and so reduce over-fitting and improve the predictive power of a model. Our method is thermodynamically grounded, imposes detailed balance, and also includes molecular cross-talk and the background activity of enzymes. We use our Allosteric Network Compiler to examine how allostery can facilitate macromolecular assembly and how competitive ligands can change the observed cooperativity of an allosteric protein. We also develop a parsimonious model of G protein-coupled receptors that explains functional selectivity and can predict the rank order of potency of agonists acting through a receptor. Our methodology should provide a basis for scalable, modular and executable modelling of biochemical networks in systems and synthetic biology.

What precision-protein-tuning and nano-resolved single molecule sciences can do for each other.

Science.gov (United States)

Milles, Sigrid; Lemke, Edward A

2013-01-01

While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how the impact of modern technologies is influenced not only by further hard- and software developments, but also by the availability and suitability of protein-engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints. Copyright © 2013 WILEY Periodicals, Inc.

Beta-Poisson model for single-cell RNA-seq data analyses.

Science.gov (United States)

Vu, Trung Nghia; Wills, Quin F; Kalari, Krishna R; Niu, Nifang; Wang, Liewei; Rantalainen, Mattias; Pawitan, Yudi

2016-07-15

Single-cell RNA-sequencing technology allows detection of gene expression at the single-cell level. One typical feature of the data is a bimodality in the cellular distribution even for highly expressed genes, primarily caused by a proportion of non-expressing cells. The standard and the over-dispersed gamma-Poisson models that are commonly used in bulk-cell RNA-sequencing are not able to capture this property. We introduce a beta-Poisson mixture model that can capture the bimodality of the single-cell gene expression distribution. We further integrate the model into the generalized linear model framework in order to perform differential expression analyses. The whole analytical procedure is called BPSC. The results from several real single-cell RNA-seq datasets indicate that ∼90% of the transcripts are well characterized by the beta-Poisson model; the model-fit from BPSC is better than the fit of the standard gamma-Poisson model in > 80% of the transcripts. Moreover, in differential expression analyses of simulated and real datasets, BPSC performs well against edgeR, a conventional method widely used in bulk-cell RNA-sequencing data, and against scde and MAST, two recent methods specifically designed for single-cell RNA-seq data. An R package BPSC for model fitting and differential expression analyses of single-cell RNA-seq data is available under GPL-3 license at https://github.com/nghiavtr/BPSC CONTACT: yudi.pawitan@ki.se or mattias.rantalainen@ki.se Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Tests of the single-pion exchange model

International Nuclear Information System (INIS)

Treiman, S.B.; Yang, C.N.

1983-01-01

The single-pion exchange model (SPEM) of high-energy particle reactions provides an attractively simple picture of seemingly complex processes and has accordingly been much discussed in recent times. The purpose of this note is to call attention to the possibility of subjecting the model to certain tests precisely in the domain where the model stands the best chance of making sense

Noninvasive imaging of protein metabolic labeling in single human cells using stable isotopes and Raman microscopy

NARCIS (Netherlands)

van Manen, H.J.; Lenferink, Aufrid T.M.; Otto, Cornelis

2008-01-01

We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C−D

Tackling Bet v 1 and associated food allergies with a single hybrid protein.

Science.gov (United States)

Hofer, Heidi; Asam, Claudia; Hauser, Michael; Nagl, Birgit; Laimer, Josef; Himly, Martin; Briza, Peter; Ebner, Christof; Lang, Roland; Hawranek, Thomas; Bohle, Barbara; Lackner, Peter; Ferreira, Fátima; Wallner, Michael

2017-08-01

Allergy vaccines should be easily applicable, safe, and efficacious. For Bet v 1-mediated birch pollen and associated food allergies, a single wild-type allergen does not provide a complete solution. We aimed to combine immunologically relevant epitopes of Bet v 1 and the 2 clinically most important related food allergens from apple and hazelnut to a single hybrid protein, termed MBC4. After identification of T cell epitope-containing parts on each of the 3 parental allergens, the hybrid molecule was designed to cover relevant epitopes and evaluated in silico. Thereby a mutation was introduced into the hybrid sequence, which should alter the secondary structure without compromising the immunogenic properties of the molecule. MBC4 and the parental allergens were purified to homogeneity. Analyses of secondary structure elements revealed substantial changes rendering the hybrid de facto nonreactive with patients' serum IgE. Nevertheless, the protein was monomeric in solution. MBC4 was able to activate T-cell lines from donors with birch pollen allergy and from mice immunized with the parental allergens. Moreover, on immunization of mice and rabbits, MBC4 induced cross-reactive IgG antibodies, which were able to block the binding of human serum IgE. Directed epitope rearrangements combined with a knowledge-based structural modification resulted in a protein unable to bind IgE from allergic patients. Still, properties to activate specific T cells or induce blocking antibodies were conserved. This suggests that MBC4 is a suitable vaccine candidate for the simultaneous treatment of Bet v 1 and associated food allergies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Trade-off between positive and negative design of protein stability: from lattice models to real proteins.

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Orly Noivirt-Brik

2009-12-01

Full Text Available Two different strategies for stabilizing proteins are (i positive design in which the native state is stabilized and (ii negative design in which competing non-native conformations are destabilized. Here, the circumstances under which one strategy might be favored over the other are explored in the case of lattice models of proteins and then generalized and discussed with regard to real proteins. The balance between positive and negative design of proteins is found to be determined by their average "contact-frequency", a property that corresponds to the fraction of states in the conformational ensemble of the sequence in which a pair of residues is in contact. Lattice model proteins with a high average contact-frequency are found to use negative design more than model proteins with a low average contact-frequency. A mathematical derivation of this result indicates that it is general and likely to hold also for real proteins. Comparison of the results of correlated mutation analysis for real proteins with typical contact-frequencies to those of proteins likely to have high contact-frequencies (such as disordered proteins and proteins that are dependent on chaperonins for their folding indicates that the latter tend to have stronger interactions between residues that are not in contact in their native conformation. Hence, our work indicates that negative design is employed when insufficient stabilization is achieved via positive design owing to high contact-frequencies.

Roles of beta-turns in protein folding: from peptide models to protein engineering.

Science.gov (United States)

Marcelino, Anna Marie C; Gierasch, Lila M

2008-05-01

Reverse turns are a major class of protein secondary structure; they represent sites of chain reversal and thus sites where the globular character of a protein is created. It has been speculated for many years that turns may nucleate the formation of structure in protein folding, as their propensity to occur will favor the approximation of their flanking regions and their general tendency to be hydrophilic will favor their disposition at the solvent-accessible surface. Reverse turns are local features, and it is therefore not surprising that their structural properties have been extensively studied using peptide models. In this article, we review research on peptide models of turns to test the hypothesis that the propensities of turns to form in short peptides will relate to the roles of corresponding sequences in protein folding. Turns with significant stability as isolated entities should actively promote the folding of a protein, and by contrast, turn sequences that merely allow the chain to adopt conformations required for chain reversal are predicted to be passive in the folding mechanism. We discuss results of protein engineering studies of the roles of turn residues in folding mechanisms. Factors that correlate with the importance of turns in folding indeed include their intrinsic stability, as well as their topological context and their participation in hydrophobic networks within the protein's structure.

An Integrated Framework Advancing Membrane Protein Modeling and Design.

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Rebecca F Alford

2015-09-01

Full Text Available Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1 prediction of free energy changes upon mutation; (2 high-resolution structural refinement; (3 protein-protein docking; and (4 assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design.

Multiple graph regularized protein domain ranking

KAUST Repository

Wang, Jim Jing-Yan

2012-11-19

Background: Protein domain ranking is a fundamental task in structural biology. Most protein domain ranking methods rely on the pairwise comparison of protein domains while neglecting the global manifold structure of the protein domain database. Recently, graph regularized ranking that exploits the global structure of the graph defined by the pairwise similarities has been proposed. However, the existing graph regularized ranking methods are very sensitive to the choice of the graph model and parameters, and this remains a difficult problem for most of the protein domain ranking methods.Results: To tackle this problem, we have developed the Multiple Graph regularized Ranking algorithm, MultiG-Rank. Instead of using a single graph to regularize the ranking scores, MultiG-Rank approximates the intrinsic manifold of protein domain distribution by combining multiple initial graphs for the regularization. Graph weights are learned with ranking scores jointly and automatically, by alternately minimizing an objective function in an iterative algorithm. Experimental results on a subset of the ASTRAL SCOP protein domain database demonstrate that MultiG-Rank achieves a better ranking performance than single graph regularized ranking methods and pairwise similarity based ranking methods.Conclusion: The problem of graph model and parameter selection in graph regularized protein domain ranking can be solved effectively by combining multiple graphs. This aspect of generalization introduces a new frontier in applying multiple graphs to solving protein domain ranking applications. 2012 Wang et al; licensee BioMed Central Ltd.

Multiple graph regularized protein domain ranking

KAUST Repository

Wang, Jim Jing-Yan; Bensmail, Halima; Gao, Xin

2012-01-01

Background: Protein domain ranking is a fundamental task in structural biology. Most protein domain ranking methods rely on the pairwise comparison of protein domains while neglecting the global manifold structure of the protein domain database. Recently, graph regularized ranking that exploits the global structure of the graph defined by the pairwise similarities has been proposed. However, the existing graph regularized ranking methods are very sensitive to the choice of the graph model and parameters, and this remains a difficult problem for most of the protein domain ranking methods.Results: To tackle this problem, we have developed the Multiple Graph regularized Ranking algorithm, MultiG-Rank. Instead of using a single graph to regularize the ranking scores, MultiG-Rank approximates the intrinsic manifold of protein domain distribution by combining multiple initial graphs for the regularization. Graph weights are learned with ranking scores jointly and automatically, by alternately minimizing an objective function in an iterative algorithm. Experimental results on a subset of the ASTRAL SCOP protein domain database demonstrate that MultiG-Rank achieves a better ranking performance than single graph regularized ranking methods and pairwise similarity based ranking methods.Conclusion: The problem of graph model and parameter selection in graph regularized protein domain ranking can be solved effectively by combining multiple graphs. This aspect of generalization introduces a new frontier in applying multiple graphs to solving protein domain ranking applications. 2012 Wang et al; licensee BioMed Central Ltd.

Multiple graph regularized protein domain ranking

Directory of Open Access Journals (Sweden)

Wang Jim

2012-11-01

Full Text Available Abstract Background Protein domain ranking is a fundamental task in structural biology. Most protein domain ranking methods rely on the pairwise comparison of protein domains while neglecting the global manifold structure of the protein domain database. Recently, graph regularized ranking that exploits the global structure of the graph defined by the pairwise similarities has been proposed. However, the existing graph regularized ranking methods are very sensitive to the choice of the graph model and parameters, and this remains a difficult problem for most of the protein domain ranking methods. Results To tackle this problem, we have developed the Multiple Graph regularized Ranking algorithm, MultiG-Rank. Instead of using a single graph to regularize the ranking scores, MultiG-Rank approximates the intrinsic manifold of protein domain distribution by combining multiple initial graphs for the regularization. Graph weights are learned with ranking scores jointly and automatically, by alternately minimizing an objective function in an iterative algorithm. Experimental results on a subset of the ASTRAL SCOP protein domain database demonstrate that MultiG-Rank achieves a better ranking performance than single graph regularized ranking methods and pairwise similarity based ranking methods. Conclusion The problem of graph model and parameter selection in graph regularized protein domain ranking can be solved effectively by combining multiple graphs. This aspect of generalization introduces a new frontier in applying multiple graphs to solving protein domain ranking applications.

Studying repair of a single protein-bound nick in vivo using the Flp-nick system

DEFF Research Database (Denmark)

Nielsen, Ida; Andersen, Anni Hangaard; Bjergbæk, Lotte

2012-01-01

The Flp-nick system is a simple in vivo system developed for studying the cellular responses to a protein-bound nick at a single genomic site in the budding yeast Saccharomyces cerevisiae. The Flp-nick system takes advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp ...

Interactions of Histone Acetyltransferase p300 with the Nuclear Proteins Histone and HMGB1, As Revealed by Single Molecule Atomic Force Spectroscopy.

Science.gov (United States)

Banerjee, S; Rakshit, T; Sett, S; Mukhopadhyay, R

2015-10-22

One of the important properties of the transcriptional coactivator p300 is histone acetyltransferase (HAT) activity that enables p300 to influence chromatin action via histone modulation. p300 can exert its HAT action upon the other nuclear proteins too--one notable example being the transcription-factor-like protein HMGB1, which functions also as a cytokine, and whose accumulation in the cytoplasm, as a response to tissue damage, is triggered by its acetylation. Hitherto, no information on the structure and stability of the complexes between full-length p300 (p300FL) (300 kDa) and the histone/HMGB1 proteins are available, probably due to the presence of unstructured regions within p300FL that makes it difficult to be crystallized. Herein, we have adopted the high-resolution atomic force microscopy (AFM) approach, which allows molecularly resolved three-dimensional contour mapping of a protein molecule of any size and structure. From the off-rate and activation barrier values, obtained using single molecule dynamic force spectroscopy, the biochemical proposition of preferential binding of p300FL to histone H3, compared to the octameric histone, can be validated. Importantly, from the energy landscape of the dissociation events, a model for the p300-histone and the p300-HMGB1 dynamic complexes that HAT forms, can be proposed. The lower unbinding forces of the complexes observed in acetylating conditions, compared to those observed in non-acetylating conditions, indicate that upon acetylation, p300 tends to weakly associate, probably as an outcome of charge alterations on the histone/HMGB1 surface and/or acetylation-induced conformational changes. To our knowledge, for the first time, a single molecule level treatment of the interactions of HAT, where the full-length protein is considered, is being reported.

Modeling of calcination of single kaolinitic clay particle

DEFF Research Database (Denmark)

Gebremariam, Abraham Teklay; Yin, Chungen; Rosendahl, Lasse

The present work aims at modeling of the calcination (dehydroxylation) process of clay particles, specifically kaolinite, and its thermal transformation. For such purpose, 1D single particle calcination model was developed based on the concept of shrinking core model to assess the dehydroxylation...

Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

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Huang Jian-dong

2011-04-01

Full Text Available Abstract Background SXT is an integrating conjugative element (ICE originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo and single strand annealing protein (S065, SXT-Bet encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively. Results SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn2+ or Mg2+ ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb. When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo. Conclusions The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V

Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein*

Science.gov (United States)

Rahman, M. Azizur; Oomori, Tamotsu; Wörheide, Gert

2011-01-01

Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called “calcite and aragonite seas.” Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that –OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature. PMID:21768106

Predicted cycloartenol synthase protein from Kandelia obovata and Rhizophora stylosa using online software of Phyre2 and Swiss-model

Science.gov (United States)

Basyuni, M.; Sulistiyono, N.; Wati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.

2018-03-01

Cloning of Kandelia obovata KcCAS gene (previously known as Kandelia candel) and Rhizophora stylosa RsCAS have already have been reported and encoded cycloartenol synthases. In this study, the predicted KcCAS and RsCAS protein were analyzed using online software of Phyre2 and Swiss-model. The protein modelling for KcCAS and RsCAS cycloartenol synthases was determined using Pyre2 had similar results with slightly different in sequence identity. By contrast, the Swiss-model for KcCAS slightly had higher sequence identity (47.31%) and Qmean (0.70) compared to RsCAS. No difference of ligands binding site which is considered as modulators for both cycloartenol synthases. The range of predicted protein derived from 91-757 amino acid residues with coverage sequence similarities 0.86, respectively from template model of lanosterol synthase from the human. Homology modelling revealed that 706 residues (93% of the amino acid sequence) had been modelled with 100.0% confidence by the single highest scoring template for both KcCAS and RsCAS using Phyre2. This coverage was more elevated than swiss-model predicted (86%). The present study suggested that both genes are responsible for the genesis of cycloartenol in these mangrove plants.

Multiple protonation equilibria in electrostatics of protein-protein binding.

Science.gov (United States)

Piłat, Zofia; Antosiewicz, Jan M

2008-11-27

All proteins contain groups capable of exchanging protons with their environment. We present here an approach, based on a rigorous thermodynamic cycle and the partition functions for energy levels characterizing protonation states of the associating proteins and their complex, to compute the electrostatic pH-dependent contribution to the free energy of protein-protein binding. The computed electrostatic binding free energies include the pH of the solution as the variable of state, mutual "polarization" of associating proteins reflected as changes in the distribution of their protonation states upon binding and fluctuations between available protonation states. The only fixed property of both proteins is the conformation; the structure of the monomers is kept in the same conformation as they have in the complex structure. As a reference, we use the electrostatic binding free energies obtained from the traditional Poisson-Boltzmann model, computed for a single macromolecular conformation fixed in a given protonation state, appropriate for given solution conditions. The new approach was tested for 12 protein-protein complexes. It is shown that explicit inclusion of protonation degrees of freedom might lead to a substantially different estimation of the electrostatic contribution to the binding free energy than that based on the traditional Poisson-Boltzmann model. This has important implications for the balancing of different contributions to the energetics of protein-protein binding and other related problems, for example, the choice of protein models for Brownian dynamics simulations of their association. Our procedure can be generalized to include conformational degrees of freedom by combining it with molecular dynamics simulations at constant pH. Unfortunately, in practice, a prohibitive factor is an enormous requirement for computer time and power. However, there may be some hope for solving this problem by combining existing constant pH molecular dynamics

Mechanical Modeling and Computer Simulation of Protein Folding

Science.gov (United States)

Prigozhin, Maxim B.; Scott, Gregory E.; Denos, Sharlene

2014-01-01

In this activity, science education and modern technology are bridged to teach students at the high school and undergraduate levels about protein folding and to strengthen their model building skills. Students are guided from a textbook picture of a protein as a rigid crystal structure to a more realistic view: proteins are highly dynamic…

Spectrally resolved single-molecule electrometry

Science.gov (United States)

Ruggeri, F.; Krishnan, M.

2018-03-01

Escape-time electrometry is a recently developed experimental technique that offers the ability to measure the effective electrical charge of a single biomolecule in solution with sub-elementary charge precision. The approach relies on measuring the average escape-time of a single charged macromolecule or molecular species transiently confined in an electrostatic fluidic trap. Comparing the experiments with the predictions of a mean-field model of molecular electrostatics, we have found that the measured effective charge even reports on molecular conformation, e.g., folded or disordered state, and non-uniform charge distribution in disordered proteins or polyelectrolytes. Here we demonstrate the ability to use the spectral dimension to distinguish minute differences in electrical charge between individual molecules or molecular species in a single simultaneous measurement, under identical experimental conditions. Using one spectral channel for referenced measurement, this kind of photophysical distinguishability essentially eliminates the need for accurate knowledge of key experimental parameters, otherwise obtained through intensive characterization of the experimental setup. As examples, we demonstrate the ability to detect small differences (˜5%) in the length of double-stranded DNA fragments as well as single amino acid exchange in an intrinsically disordered protein, prothymosin α.

Determination of selenium in BCR single cell protein via destructive neutron activation analysis

International Nuclear Information System (INIS)

Goeij, J.J.M. de; Zegers, C.

1978-10-01

The amount of selenium in single cell protein (SCP), a product of BP Research Centre at Sunbury-at-Thames, England, was determined by neutron activation analysis. The SCP-samples were irradiated in the reactor of the Interuniversity Reactor Institute at Delft, in a neutron flux of 1.0 x 10 13 n/cm 2 s for 24 hours. After chemical destruction of the samples the amount of selenium was determined by measuring the γ-peaks of selenium-75

Molecular modeling of protein materials: case study of elastin

International Nuclear Information System (INIS)

Tarakanova, Anna; Buehler, Markus J

2013-01-01

Molecular modeling of protein materials is a quickly growing area of research that has produced numerous contributions in fields ranging from structural engineering to medicine and biology. We review here the history and methods commonly employed in molecular modeling of protein materials, emphasizing the advantages for using modeling as a complement to experimental work. We then consider a case study of the protein elastin, a critically important ‘mechanical protein’ to exemplify the approach in an area where molecular modeling has made a significant impact. We outline the progression of computational modeling studies that have considerably enhanced our understanding of this important protein which endows elasticity and recoil to the tissues it is found in, including the skin, lungs, arteries and the heart. A vast collection of literature has been directed at studying the structure and function of this protein for over half a century, the first molecular dynamics study of elastin being reported in the 1980s. We review the pivotal computational works that have considerably enhanced our fundamental understanding of elastin's atomistic structure and its extraordinary qualities—focusing on two in particular: elastin's superb elasticity and the inverse temperature transition—the remarkable ability of elastin to take on a more structured conformation at higher temperatures, suggesting its effectiveness as a biomolecular switch. Our hope is to showcase these methods as both complementary and enriching to experimental approaches that have thus far dominated the study of most protein-based materials. (topical review)

Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

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Stormo Gary D

2005-07-01

Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

Nutrient recovery from industrial wastewater as single cell protein by a co-culture of green microalgae and methanotrophs

DEFF Research Database (Denmark)

Rasouli, Zahra; Valverde Pérez, Borja; D'Este, Martina

2018-01-01

wastewater and upcycle them to feed grade single cell protein. Results demonstrated that both algae and bacteria could remove or assimilate most of the organic carbon present in the wastewater (~95% removal for monocultures and 91% for the algal-bacterial consortium). However, their growth stopped before......, for all cultures the protein content (45% of dry weight, DW, for methanotrophs; 52.5% of DW for algae; and 27.6% of DW for consortium) and amino acid profile was suitable for substitution of conventional protein sources. Further research should focus on increasing productivity of biomass grown...

In silico modelling and validation of differential expressed proteins in lung cancer

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Bhagavathi S

2012-05-01

Full Text Available Objective: The present study aims predict the three dimensional structure of three major proteins responsible for causing Lung cancer. Methods: These are the differentially expressed proteins in lung cancer dataset. Initially, the structural template for these proteins is identified from structural database using homology search and perform homology modelling approach to predict its native 3D structure. Three-dimensional model obtained was validated using Ramachandran plot analysis to find the reliability of the model. Results: Four proteins were differentially expressed and were significant proteins in causing lung cancer. Among the four proteins, Matrixmetallo proteinase (P39900 had a known 3D structure and hence was not considered for modelling. The remaining proteins Polo like kinase I Q58A51, Trophinin B1AKF1, Thrombomodulin P07204 were modelled and validated. Conclusions: The three dimensional structure of proteins provides insights about the functional aspect and regulatory aspect of the protein. Thus, this study will be a breakthrough for further lung cancer related studies.

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

Science.gov (United States)

Lee, Young Kwang; Low-Nam, Shalini T; Chung, Jean K; Hansen, Scott D; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T

2017-04-28

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

A single-vendor and a single-buyer integrated inventory model with ordering cost reduction dependent on lead time

Science.gov (United States)

Vijayashree, M.; Uthayakumar, R.

2017-09-01

Lead time is one of the major limits that affect planning at every stage of the supply chain system. In this paper, we study a continuous review inventory model. This paper investigates the ordering cost reductions are dependent on lead time. This study addressed two-echelon supply chain problem consisting of a single vendor and a single buyer. The main contribution of this study is that the integrated total cost of the single vendor and the single buyer integrated system is analyzed by adopting two different (linear and logarithmic) types ordering cost reductions act dependent on lead time. In both cases, we develop effective solution procedures for finding the optimal solution and then illustrative numerical examples are given to illustrate the results. The solution procedure is to determine the optimal solutions of order quantity, ordering cost, lead time and the number of deliveries from the single vendor and the single buyer in one production run, so that the integrated total cost incurred has the minimum value. Ordering cost reduction is the main aspect of the proposed model. A numerical example is given to validate the model. Numerical example solved by using Matlab software. The mathematical model is solved analytically by minimizing the integrated total cost. Furthermore, the sensitivity analysis is included and the numerical examples are given to illustrate the results. The results obtained in this paper are illustrated with the help of numerical examples. The sensitivity of the proposed model has been checked with respect to the various major parameters of the system. Results reveal that the proposed integrated inventory model is more applicable for the supply chain manufacturing system. For each case, an algorithm procedure of finding the optimal solution is developed. Finally, the graphical representation is presented to illustrate the proposed model and also include the computer flowchart in each model.

Visualization of protein folding funnels in lattice models.

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Antonio B Oliveira

Full Text Available Protein folding occurs in a very high dimensional phase space with an exponentially large number of states, and according to the energy landscape theory it exhibits a topology resembling a funnel. In this statistical approach, the folding mechanism is unveiled by describing the local minima in an effective one-dimensional representation. Other approaches based on potential energy landscapes address the hierarchical structure of local energy minima through disconnectivity graphs. In this paper, we introduce a metric to describe the distance between any two conformations, which also allows us to go beyond the one-dimensional representation and visualize the folding funnel in 2D and 3D. In this way it is possible to assess the folding process in detail, e.g., by identifying the connectivity between conformations and establishing the paths to reach the native state, in addition to regions where trapping may occur. Unlike the disconnectivity maps method, which is based on the kinetic connections between states, our methodology is based on structural similarities inferred from the new metric. The method was developed in a 27-mer protein lattice model, folded into a 3×3×3 cube. Five sequences were studied and distinct funnels were generated in an analysis restricted to conformations from the transition-state to the native configuration. Consistent with the expected results from the energy landscape theory, folding routes can be visualized to probe different regions of the phase space, as well as determine the difficulty in folding of the distinct sequences. Changes in the landscape due to mutations were visualized, with the comparison between wild and mutated local minima in a single map, which serves to identify different trapping regions. The extension of this approach to more realistic models and its use in combination with other approaches are discussed.

Simulation studies of protein-induced bilayer deformations, and lipid-induced protein tilting, on a mesoscopic model for lipid bilayers with embedded proteins

DEFF Research Database (Denmark)

Venturoli, M.; Smit, B.; Sperotto, Maria Maddalena

2005-01-01

membranes. Here we present a mesoscopic model for lipid bilayers with embedded proteins, which we have studied with the help of the dissipative particle dynamics simulation technique. Because hydrophobic matching is believed to be one of the main physical mechanisms regulating lipid-protein interactions......-induced protein tilt, with the hydrophobic mismatch ( positive and negative) between the protein hydrophobic length and the pure lipid bilayer hydrophobic thickness. The protein-induced bilayer perturbation was quantified in terms of a coherence length, xi(P), of the lipid bilayer hydrophobic thickness pro. le...... for positive values of mismatch; a dependence on the protein size appears as well. In the case of large model proteins experiencing extreme mismatch conditions, in the region next to the so-called lipid annulus, there appears an undershooting ( or overshooting) region where the bilayer hydrophobic thickness...

A single grain approach applied to modelling recrystallization kinetics in a single-phase metal

NARCIS (Netherlands)

Chen, S.P.; Zwaag, van der S.

2004-01-01

A comprehensive model for the recrystallization kinetics is proposed which incorporates both microstructure and the textural components in the deformed state. The model is based on the single-grain approach proposed previously. The influence of the as-deformed grain orientation, which affects the

SynechoNET: integrated protein-protein interaction database of a model cyanobacterium Synechocystis sp. PCC 6803

OpenAIRE

Kim, Woo-Yeon; Kang, Sungsoo; Kim, Byoung-Chul; Oh, Jeehyun; Cho, Seongwoong; Bhak, Jong; Choi, Jong-Soon

2008-01-01

Background Cyanobacteria are model organisms for studying photosynthesis, carbon and nitrogen assimilation, evolution of plant plastids, and adaptability to environmental stresses. Despite many studies on cyanobacteria, there is no web-based database of their regulatory and signaling protein-protein interaction networks to date. Description We report a database and website SynechoNET that provides predicted protein-protein interactions. SynechoNET shows cyanobacterial domain-domain interactio...

A nonadaptive origin of a beneficial trait: in silico selection for free energy of folding leads to the neutral emergence of mutational robustness in single domain proteins.

Science.gov (United States)

Pagan, Rafael F; Massey, Steven E

2014-02-01

Proteins are regarded as being robust to the deleterious effects of mutations. Here, the neutral emergence of mutational robustness in a population of single domain proteins is explored using computer simulations. A pairwise contact model was used to calculate the ΔG of folding (ΔG folding) using the three dimensional protein structure of leech eglin C. A random amino acid sequence with low mutational robustness, defined as the average ΔΔG resulting from a point mutation (ΔΔG average), was threaded onto the structure. A population of 1,000 threaded sequences was evolved under selection for stability, using an upper and lower energy threshold. Under these conditions, mutational robustness increased over time in the most common sequence in the population. In contrast, when the wild type sequence was used it did not show an increase in robustness. This implies that the emergence of mutational robustness is sequence specific and that wild type sequences may be close to maximal robustness. In addition, an inverse relationship between ∆∆G average and protein stability is shown, resulting partly from a larger average effect of point mutations in more stable proteins. The emergence of mutational robustness was also observed in the Escherichia coli colE1 Rop and human CD59 proteins, implying that the property may be common in single domain proteins under certain simulation conditions. The results indicate that at least a portion of mutational robustness in small globular proteins might have arisen by a process of neutral emergence, and could be an example of a beneficial trait that has not been directly selected for, termed a "pseudaptation."

Electron-helium S-wave model benchmark calculations. I. Single ionization and single excitation

Science.gov (United States)

Bartlett, Philip L.; Stelbovics, Andris T.

2010-02-01

A full four-body implementation of the propagating exterior complex scaling (PECS) method [J. Phys. B 37, L69 (2004)] is developed and applied to the electron-impact of helium in an S-wave model. Time-independent solutions to the Schrödinger equation are found numerically in coordinate space over a wide range of energies and used to evaluate total and differential cross sections for a complete set of three- and four-body processes with benchmark precision. With this model we demonstrate the suitability of the PECS method for the complete solution of the full electron-helium system. Here we detail the theoretical and computational development of the four-body PECS method and present results for three-body channels: single excitation and single ionization. Four-body cross sections are presented in the sequel to this article [Phys. Rev. A 81, 022716 (2010)]. The calculations reveal structure in the total and energy-differential single-ionization cross sections for excited-state targets that is due to interference from autoionization channels and is evident over a wide range of incident electron energies.

Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer.

Science.gov (United States)

Wollman, Adam J M; Miller, Helen; Zhou, Zhaokun; Leake, Mark C

2015-04-01

DNA-interacting proteins have roles in multiple processes, many operating as molecular machines which undergo dynamic meta-stable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner, fast enough to sample the molecular transitions across the free-energy landscape. Progress has been made over the past decade in utilizing cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection signal-to-noise of molecular signatures and transition events. In the present paper, we discuss a range of techniques we have developed to monitor DNA-protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our laboratory made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new super-resolution fluorescence microscope.

Comparing single- and dual-process models of memory development.

Science.gov (United States)

Hayes, Brett K; Dunn, John C; Joubert, Amy; Taylor, Robert

2017-11-01

This experiment examined single-process and dual-process accounts of the development of visual recognition memory. The participants, 6-7-year-olds, 9-10-year-olds and adults, were presented with a list of pictures which they encoded under shallow or deep conditions. They then made recognition and confidence judgments about a list containing old and new items. We replicated the main trends reported by Ghetti and Angelini () in that recognition hit rates increased from 6 to 9 years of age, with larger age changes following deep than shallow encoding. Formal versions of the dual-process high threshold signal detection model and several single-process models (equal variance signal detection, unequal variance signal detection, mixture signal detection) were fit to the developmental data. The unequal variance and mixture signal detection models gave a better account of the data than either of the other models. A state-trace analysis found evidence for only one underlying memory process across the age range tested. These results suggest that single-process memory models based on memory strength are a viable alternative to dual-process models for explaining memory development. © 2016 John Wiley & Sons Ltd.

General model of phospholipid bilayers in fluid phase within the single chain mean field theory

Energy Technology Data Exchange (ETDEWEB)

Guo, Yachong; Baulin, Vladimir A. [Departament d’Enginyeria Química, Universitat Rovira i Virgili, Av. dels Paisos Catalans 26, 43007 Tarragona (Spain); Pogodin, Sergey [Institute of Chemical Research of Catalonia, ICIQ, Av. Paisos Catalans 16, 43007 Tarragona (Spain)

2014-05-07

Coarse-grained model for saturated phospholipids: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and unsaturated phospholipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) is introduced within the single chain mean field theory. A single set of parameters adjusted for DMPC bilayers gives an adequate description of equilibrium and mechanical properties of a range of saturated lipid molecules that differ only in length of their hydrophobic tails and unsaturated (POPC, DOPC) phospholipids which have double bonds in the tails. A double bond is modeled with a fixed angle of 120°, while the rest of the parameters are kept the same as saturated lipids. The thickness of the bilayer and its hydrophobic core, the compressibility, and the equilibrium area per lipid correspond to experimentally measured values for each lipid, changing linearly with the length of the tail. The model for unsaturated phospholipids also fetches main thermodynamical properties of the bilayers. This model is used for an accurate estimation of the free energies of the compressed or stretched bilayers in stacks or multilayers and gives reasonable estimates for free energies. The proposed model may further be used for studies of mixtures of lipids, small molecule inclusions, interactions of bilayers with embedded proteins.

Development of nondestructive screening methods for single kernel characterization of wheat

DEFF Research Database (Denmark)

Nielsen, J.P.; Pedersen, D.K.; Munck, L.

2003-01-01

predictability. However, by applying an averaging approach, in which single seed replicate measurements are mathematically simulated, a very good NIT prediction model was achieved. This suggests that the single seed NIT spectra contain hardness information, but that a single seed hardness method with higher......The development of nondestructive screening methods for single seed protein, vitreousness, density, and hardness index has been studied for single kernels of European wheat. A single kernel procedure was applied involving, image analysis, near-infrared transmittance (NIT) spectroscopy, laboratory...

Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

Science.gov (United States)

Hackenberg, Dieter; McKain, Michael R; Lee, Soon Goo; Roy Choudhury, Swarup; McCann, Tyler; Schreier, Spencer; Harkess, Alex; Pires, J Chris; Wong, Gane Ka-Shu; Jez, Joseph M; Kellogg, Elizabeth A; Pandey, Sona

2017-10-01

Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Hydration dynamics near a model protein surface

International Nuclear Information System (INIS)

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

2003-01-01

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

BiP clustering facilitates protein folding in the endoplasmic reticulum.

Directory of Open Access Journals (Sweden)

Marc Griesemer

2014-07-01

Full Text Available The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER: translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as 'clusters'. Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.

Statistical-mechanical lattice models for protein-DNA binding in chromatin

International Nuclear Information System (INIS)

Teif, Vladimir B; Rippe, Karsten

2010-01-01

Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibria measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical-mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quantitative models for the regulation of gene expression.

SAAFEC: Predicting the Effect of Single Point Mutations on Protein Folding Free Energy Using a Knowledge-Modified MM/PBSA Approach.

Science.gov (United States)

Getov, Ivan; Petukh, Marharyta; Alexov, Emil

2016-04-07

Folding free energy is an important biophysical characteristic of proteins that reflects the overall stability of the 3D structure of macromolecules. Changes in the amino acid sequence, naturally occurring or made in vitro, may affect the stability of the corresponding protein and thus could be associated with disease. Several approaches that predict the changes of the folding free energy caused by mutations have been proposed, but there is no method that is clearly superior to the others. The optimal goal is not only to accurately predict the folding free energy changes, but also to characterize the structural changes induced by mutations and the physical nature of the predicted folding free energy changes. Here we report a new method to predict the Single Amino Acid Folding free Energy Changes (SAAFEC) based on a knowledge-modified Molecular Mechanics Poisson-Boltzmann (MM/PBSA) approach. The method is comprised of two main components: a MM/PBSA component and a set of knowledge based terms delivered from a statistical study of the biophysical characteristics of proteins. The predictor utilizes a multiple linear regression model with weighted coefficients of various terms optimized against a set of experimental data. The aforementioned approach yields a correlation coefficient of 0.65 when benchmarked against 983 cases from 42 proteins in the ProTherm database. the webserver can be accessed via http://compbio.clemson.edu/SAAFEC/.

Exploiting conformational ensembles in modeling protein-protein interactions on the proteome scale

Science.gov (United States)

Kuzu, Guray; Gursoy, Attila; Nussinov, Ruth; Keskin, Ozlem

2013-01-01

Cellular functions are performed through protein-protein interactions; therefore, identification of these interactions is crucial for understanding biological processes. Recent studies suggest that knowledge-based approaches are more useful than ‘blind’ docking for modeling at large scales. However, a caveat of knowledge-based approaches is that they treat molecules as rigid structures. The Protein Data Bank (PDB) offers a wealth of conformations. Here, we exploited ensemble of the conformations in predictions by a knowledge-based method, PRISM. We tested ‘difficult’ cases in a docking-benchmark dataset, where the unbound and bound protein forms are structurally different. Considering alternative conformations for each protein, the percentage of successfully predicted interactions increased from ~26% to 66%, and 57% of the interactions were successfully predicted in an ‘unbiased’ scenario, in which data related to the bound forms were not utilized. If the appropriate conformation, or relevant template interface, is unavailable in the PDB, PRISM could not predict the interaction successfully. The pace of the growth of the PDB promises a rapid increase of ensemble conformations emphasizing the merit of such knowledge-based ensemble strategies for higher success rates in protein-protein interaction predictions on an interactome-scale. We constructed the structural network of ERK interacting proteins as a case study. PMID:23590674

Investigation of sliding DNA clamp dynamics by single-molecule fluorescence, mass spectrometry and structure-based modeling

Science.gov (United States)

Gadkari, Varun V; Harvey, Sophie R; Raper, Austin T; Chu, Wen-Ting; Wang, Jin; Wysocki, Vicki H; Suo, Zucai

2018-01-01

Abstract Proliferating cell nuclear antigen (PCNA) is a trimeric ring-shaped clamp protein that encircles DNA and interacts with many proteins involved in DNA replication and repair. Despite extensive structural work to characterize the monomeric, dimeric, and trimeric forms of PCNA alone and in complex with interacting proteins, no structure of PCNA in a ring-open conformation has been published. Here, we use a multidisciplinary approach, including single-molecule Förster resonance energy transfer (smFRET), native ion mobility-mass spectrometry (IM-MS), and structure-based computational modeling, to explore the conformational dynamics of a model PCNA from Sulfolobus solfataricus (Sso), an archaeon. We found that Sso PCNA samples ring-open and ring-closed conformations even in the absence of its clamp loader complex, replication factor C, and transition to the ring-open conformation is modulated by the ionic strength of the solution. The IM-MS results corroborate the smFRET findings suggesting that PCNA dynamics are maintained in the gas phase and further establishing IM-MS as a reliable strategy to investigate macromolecular motions. Our molecular dynamic simulations agree with the experimental data and reveal that ring-open PCNA often adopts an out-of-plane left-hand geometry. Collectively, these results implore future studies to define the roles of PCNA dynamics in DNA loading and other PCNA-mediated interactions. PMID:29529283

CONFOLD2: improved contact-driven ab initio protein structure modeling.

Science.gov (United States)

Adhikari, Badri; Cheng, Jianlin

2018-01-25

Contact-guided protein structure prediction methods are becoming more and more successful because of the latest advances in residue-residue contact prediction. To support contact-driven structure prediction, effective tools that can quickly build tertiary structural models of good quality from predicted contacts need to be developed. We develop an improved contact-driven protein modelling method, CONFOLD2, and study how it may be effectively used for ab initio protein structure prediction with predicted contacts as input. It builds models using various subsets of input contacts to explore the fold space under the guidance of a soft square energy function, and then clusters the models to obtain the top five models. CONFOLD2 obtains an average reconstruction accuracy of 0.57 TM-score for the 150 proteins in the PSICOV contact prediction dataset. When benchmarked on the CASP11 contacts predicted using CONSIP2 and CASP12 contacts predicted using Raptor-X, CONFOLD2 achieves a mean TM-score of 0.41 on both datasets. CONFOLD2 allows to quickly generate top five structural models for a protein sequence when its secondary structures and contacts predictions at hand. The source code of CONFOLD2 is publicly available at https://github.com/multicom-toolbox/CONFOLD2/ .

On the Predictiveness of Single-Field Inflationary Models

CERN Document Server

Burgess, C.P.; Trott, Michael

2014-01-01

We re-examine the predictiveness of single-field inflationary models and discuss how an unknown UV completion can complicate determining inflationary model parameters from observations, even from precision measurements. Besides the usual naturalness issues associated with having a shallow inflationary potential, we describe another issue for inflation, namely, unknown UV physics modifies the running of Standard Model (SM) parameters and thereby introduces uncertainty into the potential inflationary predictions. We illustrate this point using the minimal Higgs Inflationary scenario, which is arguably the most predictive single-field model on the market, because its predictions for $A_s$, $r$ and $n_s$ are made using only one new free parameter beyond those measured in particle physics experiments, and run up to the inflationary regime. We find that this issue can already have observable effects. At the same time, this UV-parameter dependence in the Renormalization Group allows Higgs Inflation to occur (in prin...

Metabolism of whole body protein in pregnant and non-pregnant gilts using 15N-glycine single-dose end-product method

International Nuclear Information System (INIS)

Wu De; Liu Huifang; Zhou Anguo; Wang Kangning; Yang Feng

2007-01-01

The metabolism of whole-body protein for pregnant and non-pregnant gilts was investigated using single-dose of 15 N-glycine end-product method. The results showed that there were no differences (P>0.05) in protein dynamic metabolism, amino acids utilization rate between pregnant and non-pregnant gilts at breeding. However, N flux, protein turnover rate, protein synthesis rate and breakdown rate of pregnant gilts were lower (P<0.05) than those of non-pregnant gilts at 30days after breeding, but the protein aggradiation's rate increased by 25% (P<0.05). During late gestation, N flux, protein turnover rate, protein synthesis rate and breakdown rate of pregnant sows were significantly increased (P<0.01), and protein aggradation's rate increased by 71.1%, compared with that of non-pregnant gilts. (authors)

Are animal models predictive for human postmortem muscle protein degradation?

Science.gov (United States)

Ehrenfellner, Bianca; Zissler, Angela; Steinbacher, Peter; Monticelli, Fabio C; Pittner, Stefan

2017-11-01

A most precise determination of the postmortem interval (PMI) is a crucial aspect in forensic casework. Although there are diverse approaches available to date, the high heterogeneity of cases together with the respective postmortal changes often limit the validity and sufficiency of many methods. Recently, a novel approach for time since death estimation by the analysis of postmortal changes of muscle proteins was proposed. It is however necessary to improve the reliability and accuracy, especially by analysis of possible influencing factors on protein degradation. This is ideally investigated on standardized animal models that, however, require legitimization by a comparison of human and animal tissue, and in this specific case of protein degradation profiles. Only if protein degradation events occur in comparable fashion within different species, respective findings can sufficiently be transferred from the animal model to application in humans. Therefor samples from two frequently used animal models (mouse and pig), as well as forensic cases with representative protein profiles of highly differing PMIs were analyzed. Despite physical and physiological differences between species, western blot analysis revealed similar patterns in most of the investigated proteins. Even most degradation events occurred in comparable fashion. In some other aspects, however, human and animal profiles depicted distinct differences. The results of this experimental series clearly indicate the huge importance of comparative studies, whenever animal models are considered. Although animal models could be shown to reflect the basic principles of protein degradation processes in humans, we also gained insight in the difficulties and limitations of the applicability of the developed methodology in different mammalian species regarding protein specificity and methodic functionality.

Hidden Markov model-derived structural alphabet for proteins: the learning of protein local shapes captures sequence specificity.

Science.gov (United States)

Camproux, A C; Tufféry, P

2005-08-05

Understanding and predicting protein structures depend on the complexity and the accuracy of the models used to represent them. We have recently set up a Hidden Markov Model to optimally compress protein three-dimensional conformations into a one-dimensional series of letters of a structural alphabet. Such a model learns simultaneously the shape of representative structural letters describing the local conformation and the logic of their connections, i.e. the transition matrix between the letters. Here, we move one step further and report some evidence that such a model of protein local architecture also captures some accurate amino acid features. All the letters have specific and distinct amino acid distributions. Moreover, we show that words of amino acids can have significant propensities for some letters. Perspectives point towards the prediction of the series of letters describing the structure of a protein from its amino acid sequence.

SNP2Structure: A Public and Versatile Resource for Mapping and Three-Dimensional Modeling of Missense SNPs on Human Protein Structures

Directory of Open Access Journals (Sweden)

Difei Wang

2015-01-01

Full Text Available One of the long-standing challenges in biology is to understand how non-synonymous single nucleotide polymorphisms (nsSNPs change protein structure and further affect their function. While it is impractical to solve all the mutated protein structures experimentally, it is quite feasible to model the mutated structures in silico. Toward this goal, we built a publicly available structure database resource (SNP2Structure, https://apps.icbi.georgetown.edu/snp2structure focusing on missense mutations, msSNP. Compared with web portals with similar aims, SNP2Structure has the following major advantages. First, our portal offers direct comparison of two related 3D structures. Second, the protein models include all interacting molecules in the original PDB structures, so users are able to determine regions of potential interaction changes when a protein mutation occurs. Third, the mutated structures are available to download locally for further structural and functional analysis. Fourth, we used Jsmol package to display the protein structure that has no system compatibility issue. SNP2Structure provides reliable, high quality mapping of nsSNPs to 3D protein structures enabling researchers to explore the likely functional impact of human disease-causing mutations.

Hidden markov model for the prediction of transmembrane proteins using MATLAB.

Science.gov (United States)

Chaturvedi, Navaneet; Shanker, Sudhanshu; Singh, Vinay Kumar; Sinha, Dhiraj; Pandey, Paras Nath

2011-01-01

Since membranous proteins play a key role in drug targeting therefore transmembrane proteins prediction is active and challenging area of biological sciences. Location based prediction of transmembrane proteins are significant for functional annotation of protein sequences. Hidden markov model based method was widely applied for transmembrane topology prediction. Here we have presented a revised and a better understanding model than an existing one for transmembrane protein prediction. Scripting on MATLAB was built and compiled for parameter estimation of model and applied this model on amino acid sequence to know the transmembrane and its adjacent locations. Estimated model of transmembrane topology was based on TMHMM model architecture. Only 7 super states are defined in the given dataset, which were converted to 96 states on the basis of their length in sequence. Accuracy of the prediction of model was observed about 74 %, is a good enough in the area of transmembrane topology prediction. Therefore we have concluded the hidden markov model plays crucial role in transmembrane helices prediction on MATLAB platform and it could also be useful for drug discovery strategy. The database is available for free at bioinfonavneet@gmail.comvinaysingh@bhu.ac.in.

Experiments and modeling of single plastic particle conversion in suspension

DEFF Research Database (Denmark)

Nakhaei, Mohammadhadi; Wu, Hao; Grévain, Damien

2018-01-01

Conversion of single high density polyethylene (PE) particles has been studied by experiments and modeling. The experiments were carried out in a single particle combustor for five different shapes and masses of particles at temperature conditions of 900 and 1100°C. Each experiment was recorded...... against the experiments as well as literature data. Furthermore, a simplified isothermal model appropriate for CFD applications was developed, in order to model the combustion of plastic particles in cement calciners. By comparing predictions with the isothermal and the non–isothermal models under typical...

Latent Membrane Protein 1 as a molecular adjuvant for single-cycle lentiviral vaccines

Directory of Open Access Journals (Sweden)

Rahmberg Andrew R

2011-05-01

Full Text Available Abstract Background Molecular adjuvants are a promising method to enhance virus-specific immune responses and protect against HIV-1 infection. Immune activation by ligands for receptors such as CD40 can induce dendritic cell activation and maturation. Here we explore the incorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or an LMP1-CD40 chimera, into a strain of SIV that was engineered to be limited to a single cycle of infection. Results Full length LMP1 or the chimeric protein LMP1-CD40 was cloned into the nef-locus of single-cycle SIV. Human and Macaque monocyte derived macrophages and DC were infected with these viruses. Infected cells were analyzed for activation surface markers by flow cytometry. Cells were also analyzed for secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12p70 and TNF by cytometric bead array. Conclusions Overall, single-cycle SIV expressing LMP1 and LMP1-CD40 produced a broad and potent TH1-biased immune response in human as well as rhesus macaque macrophages and DC when compared with control virus. Single-cycle SIV-LMP1 also enhanced antigen presentation by lentiviral vector vaccines, suggesting that LMP1-mediated immune activation may enhance lentiviral vector vaccines against HIV-1.

Interaction of amidated single-walled carbon nanotubes with protein by multiple spectroscopic methods

Energy Technology Data Exchange (ETDEWEB)

Li, Lili [China Pharmaceutical University, Nanjing 210009 (China); The Nursing College of Pingdingshan University, Pingdingshan 467000 (China); Lin, Rui [Yancheng Health Vocational and Technical College, Yancheng 224005 (China); He, Hua, E-mail: dochehua@163.com [China Pharmaceutical University, Nanjing 210009 (China); Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, Nanjing 210009 (China); Sun, Meiling, E-mail: sml-nir@sohu.com [China Pharmaceutical University, Nanjing 210009 (China); Jiang, Li; Gao, Mengmeng [China Pharmaceutical University, Nanjing 210009 (China)

2014-01-15

The aim of this work was to investigate the detailed interaction between BSA and amidated single walled carbon nanotubes (e-SWNTs) in vitro. Ethylenediamine (EDA) was successfully linked on the surface of single-walled carbon nanotubes (SWNTs) via acylation to improve their dispersion and to introduce active groups. Bovine serum albumin (BSA) was selected as the template protein to inspect the interaction of e-SWNTs with protein. Decreases in fluorescence intensity of BSA induced by e-SWNTs demonstrated the occurrence of interaction between BSA and e-SWNTs. Quenching parameters and different absorption spectra for e-SWNTs–BSA show that the quenching effect of e-SWNTs was static quenching. Hydrophobic force had a leading contribution to the binding roles of BSA on e-SWNTs, which was confirmed by positive enthalpy change and entropy change. The interference of Na{sup +} with the quenching effect of e-SWNTs authenticated that electrostatic force existed in the interactive process simultaneously. The hydrophobicity of amino acid residues markedly increased with the addition of e-SWNTs viewed from UV spectra of BSA. The content of α-helix structure in BSA decreased by 6.8% due to the addition of e-SWNTs, indicating that e-SWNTs have an effect on the secondary conformation of BSA. -- Highlights: • The interaction between e-SWNTs and BSA was investigated by multiple spectroscopic methods. • Quenching mechanism was static quenching. • Changes in structure of BSA were inspected by synchronous fluorescence, UV–vis and CD spectrum.

Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins.

Science.gov (United States)

Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J

2017-07-25

Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.

A model in which heat shock protein 90 targets protein-folding clefts: rationale for a new approach to neuroprotective treatment of protein folding diseases.

Science.gov (United States)

Pratt, William B; Morishima, Yoshihiro; Gestwicki, Jason E; Lieberman, Andrew P; Osawa, Yoichi

2014-11-01

In an EBM Minireview published in 2010, we proposed that the heat shock protein (Hsp)90/Hsp70-based chaperone machinery played a major role in determining the selection of proteins that have undergone oxidative or other toxic damage for ubiquitination and proteasomal degradation. The proposal was based on a model in which the Hsp90 chaperone machinery regulates signaling by modulating ligand-binding clefts. The model provides a framework for thinking about the development of neuroprotective therapies for protein-folding diseases like Alzheimer's disease (AD), Parkinson's disease (PD), and the polyglutamine expansion disorders, such as Huntington's disease (HD) and spinal and bulbar muscular atrophy (SBMA). Major aberrant proteins that misfold and accumulate in these diseases are "client" proteins of the abundant and ubiquitous stress chaperone Hsp90. These Hsp90 client proteins include tau (AD), α-synuclein (PD), huntingtin (HD), and the expanded glutamine androgen receptor (polyQ AR) (SBMA). In this Minireview, we update our model in which Hsp90 acts on protein-folding clefts and show how it forms a rational basis for developing drugs that promote the targeted elimination of these aberrant proteins. © 2014 by the Society for Experimental Biology and Medicine.

Rapid concentration and dialysis of proteins with single hollow fibers: possible applications in analysis of protein secretion by isolated cells and steroid radioimmunoassays

International Nuclear Information System (INIS)

Rommerts, F.F.G.; Clotscher, W.F.; Van der Molen, H.J.

1977-01-01

Single hollow fibers were used in specially made cells for fast concentration and dialysis of solutions containing macromolecules. Volumes on the order of 5 ml of diluted protein solutions could be concentrated to 50--100 μl or less within 7 min with a protein recovery of 60--80%. More than 99% of the molecules with a molecular weight less than 500 could be removed in less than 1 hr. A possible application of the rapid dialysis method for the mechanization of radioimmunoassays is indicated. It was shown that in the radioimmunoassay of steriods the unbound steroids could be removed after incubation with antiserum, within 10 min and without a change in volume

Models of protein and amino acid requirements for cattle

Directory of Open Access Journals (Sweden)

Luis Orlindo Tedeschi

2015-03-01

Full Text Available Protein supply and requirements by ruminants have been studied for more than a century. These studies led to the accumulation of lots of scientific information about digestion and metabolism of protein by ruminants as well as the characterization of the dietary protein in order to maximize animal performance. During the 1980s and 1990s, when computers became more accessible and powerful, scientists began to conceptualize and develop mathematical nutrition models, and to program them into computers to assist with ration balancing and formulation for domesticated ruminants, specifically dairy and beef cattle. The most commonly known nutrition models developed during this period were the National Research Council (NRC in the United States, Agricultural Research Council (ARC in the United Kingdom, Institut National de la Recherche Agronomique (INRA in France, and the Commonwealth Scientific and Industrial Research Organization (CSIRO in Australia. Others were derivative works from these models with different degrees of modifications in the supply or requirement calculations, and the modeling nature (e.g., static or dynamic, mechanistic, or deterministic. Circa 1990s, most models adopted the metabolizable protein (MP system over the crude protein (CP and digestible CP systems to estimate supply of MP and the factorial system to calculate MP required by the animal. The MP system included two portions of protein (i.e., the rumen-undegraded dietary CP - RUP - and the contributions of microbial CP - MCP as the main sources of MP for the animal. Some models would explicitly account for the impact of dry matter intake (DMI on the MP required for maintenance (MPm; e.g., Cornell Net Carbohydrate and Protein System - CNCPS, the Dutch system - DVE/OEB, while others would simply account for scurf, urinary, metabolic fecal, and endogenous contributions independently of DMI. All models included milk yield and its components in estimating MP required for lactation

Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Levin, Johannes [Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany); German Center for Neurodegenerative Diseases – DZNE, Site Munich, Feodor-Lynen-Str. 17, 81377 Munich (Germany); Hillmer, Andreas S. [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany); Högen, Tobias [Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany); McLean, Pamela J. [Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224 (United States); Giese, Armin, E-mail: armin.giese@med.uni-muenchen.de [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany)

2016-08-12

Synucleinopathies such as dementia with Lewy bodies or Parkinson’s disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation. - Highlights: • Single particle spectroscopy detects intracellular formed α-synuclein aggregates. • Fusion proteins allow detection of protein

A resource for benchmarking the usefulness of protein structure models.

Science.gov (United States)

Carbajo, Daniel; Tramontano, Anna

2012-08-02

Increasingly, biologists and biochemists use computational tools to design experiments to probe the function of proteins and/or to engineer them for a variety of different purposes. The most effective strategies rely on the knowledge of the three-dimensional structure of the protein of interest. However it is often the case that an experimental structure is not available and that models of different quality are used instead. On the other hand, the relationship between the quality of a model and its appropriate use is not easy to derive in general, and so far it has been analyzed in detail only for specific application. This paper describes a database and related software tools that allow testing of a given structure based method on models of a protein representing different levels of accuracy. The comparison of the results of a computational experiment on the experimental structure and on a set of its decoy models will allow developers and users to assess which is the specific threshold of accuracy required to perform the task effectively. The ModelDB server automatically builds decoy models of different accuracy for a given protein of known structure and provides a set of useful tools for their analysis. Pre-computed data for a non-redundant set of deposited protein structures are available for analysis and download in the ModelDB database. IMPLEMENTATION, AVAILABILITY AND REQUIREMENTS: Project name: A resource for benchmarking the usefulness of protein structure models. Project home page: http://bl210.caspur.it/MODEL-DB/MODEL-DB_web/MODindex.php.Operating system(s): Platform independent. Programming language: Perl-BioPerl (program); mySQL, Perl DBI and DBD modules (database); php, JavaScript, Jmol scripting (web server). Other requirements: Java Runtime Environment v1.4 or later, Perl, BioPerl, CPAN modules, HHsearch, Modeller, LGA, NCBI Blast package, DSSP, Speedfill (Surfnet) and PSAIA. License: Free. Any restrictions to use by non-academics: No.

A resource for benchmarking the usefulness of protein structure models

Directory of Open Access Journals (Sweden)

Carbajo Daniel

2012-08-01

Full Text Available Abstract Background Increasingly, biologists and biochemists use computational tools to design experiments to probe the function of proteins and/or to engineer them for a variety of different purposes. The most effective strategies rely on the knowledge of the three-dimensional structure of the protein of interest. However it is often the case that an experimental structure is not available and that models of different quality are used instead. On the other hand, the relationship between the quality of a model and its appropriate use is not easy to derive in general, and so far it has been analyzed in detail only for specific application. Results This paper describes a database and related software tools that allow testing of a given structure based method on models of a protein representing different levels of accuracy. The comparison of the results of a computational experiment on the experimental structure and on a set of its decoy models will allow developers and users to assess which is the specific threshold of accuracy required to perform the task effectively. Conclusions The ModelDB server automatically builds decoy models of different accuracy for a given protein of known structure and provides a set of useful tools for their analysis. Pre-computed data for a non-redundant set of deposited protein structures are available for analysis and download in the ModelDB database. Implementation, availability and requirements Project name: A resource for benchmarking the usefulness of protein structure models. Project home page: http://bl210.caspur.it/MODEL-DB/MODEL-DB_web/MODindex.php. Operating system(s: Platform independent. Programming language: Perl-BioPerl (program; mySQL, Perl DBI and DBD modules (database; php, JavaScript, Jmol scripting (web server. Other requirements: Java Runtime Environment v1.4 or later, Perl, BioPerl, CPAN modules, HHsearch, Modeller, LGA, NCBI Blast package, DSSP, Speedfill (Surfnet and PSAIA. License: Free. Any

A resource for benchmarking the usefulness of protein structure models.

KAUST Repository

Carbajo, Daniel

2012-08-02

BACKGROUND: Increasingly, biologists and biochemists use computational tools to design experiments to probe the function of proteins and/or to engineer them for a variety of different purposes. The most effective strategies rely on the knowledge of the three-dimensional structure of the protein of interest. However it is often the case that an experimental structure is not available and that models of different quality are used instead. On the other hand, the relationship between the quality of a model and its appropriate use is not easy to derive in general, and so far it has been analyzed in detail only for specific application. RESULTS: This paper describes a database and related software tools that allow testing of a given structure based method on models of a protein representing different levels of accuracy. The comparison of the results of a computational experiment on the experimental structure and on a set of its decoy models will allow developers and users to assess which is the specific threshold of accuracy required to perform the task effectively. CONCLUSIONS: The ModelDB server automatically builds decoy models of different accuracy for a given protein of known structure and provides a set of useful tools for their analysis. Pre-computed data for a non-redundant set of deposited protein structures are available for analysis and download in the ModelDB database. IMPLEMENTATION, AVAILABILITY AND REQUIREMENTS: Project name: A resource for benchmarking the usefulness of protein structure models. Project home page: http://bl210.caspur.it/MODEL-DB/MODEL-DB_web/MODindex.php.Operating system(s): Platform independent. Programming language: Perl-BioPerl (program); mySQL, Perl DBI and DBD modules (database); php, JavaScript, Jmol scripting (web server). Other requirements: Java Runtime Environment v1.4 or later, Perl, BioPerl, CPAN modules, HHsearch, Modeller, LGA, NCBI Blast package, DSSP, Speedfill (Surfnet) and PSAIA. License: Free. Any restrictions to use by

A resource for benchmarking the usefulness of protein structure models.

KAUST Repository

Carbajo, Daniel; Tramontano, Anna

2012-01-01

BACKGROUND: Increasingly, biologists and biochemists use computational tools to design experiments to probe the function of proteins and/or to engineer them for a variety of different purposes. The most effective strategies rely on the knowledge of the three-dimensional structure of the protein of interest. However it is often the case that an experimental structure is not available and that models of different quality are used instead. On the other hand, the relationship between the quality of a model and its appropriate use is not easy to derive in general, and so far it has been analyzed in detail only for specific application. RESULTS: This paper describes a database and related software tools that allow testing of a given structure based method on models of a protein representing different levels of accuracy. The comparison of the results of a computational experiment on the experimental structure and on a set of its decoy models will allow developers and users to assess which is the specific threshold of accuracy required to perform the task effectively. CONCLUSIONS: The ModelDB server automatically builds decoy models of different accuracy for a given protein of known structure and provides a set of useful tools for their analysis. Pre-computed data for a non-redundant set of deposited protein structures are available for analysis and download in the ModelDB database. IMPLEMENTATION, AVAILABILITY AND REQUIREMENTS: Project name: A resource for benchmarking the usefulness of protein structure models. Project home page: http://bl210.caspur.it/MODEL-DB/MODEL-DB_web/MODindex.php.Operating system(s): Platform independent. Programming language: Perl-BioPerl (program); mySQL, Perl DBI and DBD modules (database); php, JavaScript, Jmol scripting (web server). Other requirements: Java Runtime Environment v1.4 or later, Perl, BioPerl, CPAN modules, HHsearch, Modeller, LGA, NCBI Blast package, DSSP, Speedfill (Surfnet) and PSAIA. License: Free. Any restrictions to use by

Bacillus subtilis single-stranded DNA-binding protein SsbA is phosphorylated at threonine 38 by the serine/threonine kinase YabT

DEFF Research Database (Denmark)

Derouiche, Abderahmane; Petranovic, Dina; Macek, Boris

2016-01-01

Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA......-binding proteins have previously been found to be phosphorylated on tyrosine and arginine residues. While tyrosine phosphorylation was shown to enhance the DNA-binding properties of SsbA, arginine phosphorylation was not functionally characterized.Materials and methods: We used mass spectrometry analysis to detect...... phosphorylation of SsbA purified from B. subtilis cells. The detected phosphorylation site was assessed for its influence on DNA-binding in vitro, using electrophoretic mobility shift assays. The ability of B. subtilis serine/threonine kinases to phosphorylate SsbA was assessed using in vitro phosphorylation...

Exploring Transduction Mechanisms of Protein Transduction Domains (PTDs in Living Cells Utilizing Single-Quantum Dot Tracking (SQT Technology

Directory of Open Access Journals (Sweden)

Yasuhiro Suzuki

2012-01-01

Full Text Available Specific protein domains known as protein transduction domains (PTDs can permeate cell membranes and deliver proteins or bioactive materials into living cells. Various approaches have been applied for improving their transduction efficacy. It is, therefore, crucial to clarify the entry mechanisms and to identify the rate-limiting steps. Because of technical limitations for imaging PTD behavior on cells with conventional fluorescent-dyes, how PTDs enter the cells has been a topic of much debate. Utilizing quantum dots (QDs, we recently tracked the behavior of PTD that was derived from HIV-1 Tat (TatP in living cells at the single-molecule level with 7-nm special precision. In this review article, we initially summarize the controversy on TatP entry mechanisms; thereafter, we will focus on our recent findings on single-TatP-QD tracking (SQT, to identify the major sequential steps of intracellular delivery in living cells and to discuss how SQT can easily provide direct information on TatP entry mechanisms. As a primer for SQT study, we also discuss the latest findings on single particle tracking of various molecules on the plasma membrane. Finally, we discuss the problems of QDs and the challenges for the future in utilizing currently available QD probes for SQT. In conclusion, direct identification of the rate-limiting steps of PTD entry with SQT should dramatically improve the methods for enhancing transduction efficiency.

Topology and weights in a protein domain interaction network--a novel way to predict protein interactions.

Science.gov (United States)

Wuchty, Stefan

2006-05-23

While the analysis of unweighted biological webs as diverse as genetic, protein and metabolic networks allowed spectacular insights in the inner workings of a cell, biological networks are not only determined by their static grid of links. In fact, we expect that the heterogeneity in the utilization of connections has a major impact on the organization of cellular activities as well. We consider a web of interactions between protein domains of the Protein Family database (PFAM), which are weighted by a probability score. We apply metrics that combine the static layout and the weights of the underlying interactions. We observe that unweighted measures as well as their weighted counterparts largely share the same trends in the underlying domain interaction network. However, we only find weak signals that weights and the static grid of interactions are connected entities. Therefore assuming that a protein interaction is governed by a single domain interaction, we observe strong and significant correlations of the highest scoring domain interaction and the confidence of protein interactions in the underlying interactions of yeast and fly. Modeling an interaction between proteins if we find a high scoring protein domain interaction we obtain 1, 428 protein interactions among 361 proteins in the human malaria parasite Plasmodium falciparum. Assessing their quality by a logistic regression method we observe that increasing confidence of predicted interactions is accompanied by high scoring domain interactions and elevated levels of functional similarity and evolutionary conservation. Our results indicate that probability scores are randomly distributed, allowing to treat static grid and weights of domain interactions as separate entities. In particular, these finding confirms earlier observations that a protein interaction is a matter of a single interaction event on domain level. As an immediate application, we show a simple way to predict potential protein interactions

Preclinical models used for immunogenicity prediction of therapeutic proteins.

Science.gov (United States)

Brinks, Vera; Weinbuch, Daniel; Baker, Matthew; Dean, Yann; Stas, Philippe; Kostense, Stefan; Rup, Bonita; Jiskoot, Wim

2013-07-01

All therapeutic proteins are potentially immunogenic. Antibodies formed against these drugs can decrease efficacy, leading to drastically increased therapeutic costs and in rare cases to serious and sometimes life threatening side-effects. Many efforts are therefore undertaken to develop therapeutic proteins with minimal immunogenicity. For this, immunogenicity prediction of candidate drugs during early drug development is essential. Several in silico, in vitro and in vivo models are used to predict immunogenicity of drug leads, to modify potentially immunogenic properties and to continue development of drug candidates with expected low immunogenicity. Despite the extensive use of these predictive models, their actual predictive value varies. Important reasons for this uncertainty are the limited/insufficient knowledge on the immune mechanisms underlying immunogenicity of therapeutic proteins, the fact that different predictive models explore different components of the immune system and the lack of an integrated clinical validation. In this review, we discuss the predictive models in use, summarize aspects of immunogenicity that these models predict and explore the merits and the limitations of each of the models.

Model test on the relationship feed energy and protein ratio to the production and quality of milk protein

Science.gov (United States)

Hartanto, R.; Jantra, M. A. C.; Santosa, S. A. B.; Purnomoadi, A.

2018-01-01

The purpose of this research was to find an appropriate relationship model between the feed energy and protein ratio with the amount of production and quality of milk proteins. This research was conducted at Getasan Sub-district, Semarang Regency, Central Java Province, Indonesia using 40 samples (Holstein Friesian cattle, lactation period II-III and lactation month 3-4). Data were analyzed using linear and quadratic regressions, to predict the production and quality of milk protein from feed energy and protein ratio that describe the diet. The significance of model was tested using analysis of variance. Coefficient of determination (R2), residual variance (RV) and root mean square prediction error (RMSPE) were reported for the developed equations as an indicator of the goodness of model fit. The results showed no relationship in milk protein (kg), milk casein (%), milk casein (kg) and milk urea N (mg/dl) as function of CP/TDN. The significant relationship was observed in milk production (L or kg) and milk protein (%) as function of CP/TDN, both in linear and quadratic models. In addition, a quadratic change in milk production (L) (P = 0.003), milk production (kg) (P = 0.003) and milk protein concentration (%) (P = 0.026) were observed with increase of CP/TDN. It can be concluded that quadratic equation was the good fitting model for this research, because quadratic equation has larger R2, smaller RV and smaller RMSPE than those of linear equation.

Synchrotron radiation-based Fourier-transform infrared spectromicroscopy for characterization of the protein/peptide distribution in single microspheres

Directory of Open Access Journals (Sweden)

Manli Wang

2015-05-01

Full Text Available The present study establishes a visualization method for the measurement of the distribution and localization of protein/peptide constituents within a single poly-lactide-co-glycolide (PLGA microsphere using synchrotron radiation–based Fourier-transform infrared spectromicroscopy (SR-FTIR. The representative infrared wavenumbers specific for protein/peptide (Exenatide and excipient (PLGA were identified and chemical maps at the single microsphere level were generated by measuring and plotting the intensity of these specific bands. For quantitative analysis of the distribution within microspheres, Matlab software was used to transform the map file into a 3D matrix and the matrix values specific for the drug and excipient were extracted. Comparison of the normalized SR-FTIR maps of PLGA and Exenatide indicated that PLGA was uniformly distributed, while Exenatide was relatively non-uniformly distributed in the microspheres. In conclusion, SR-FTIR is a rapid, nondestructive and sensitive detection technology to provide the distribution of chemical constituents and functional groups in microparticles and microspheres.

Protein structure analysis using the resonant recognition model and wavelet transforms

International Nuclear Information System (INIS)

Fang, Q.; Cosic, I.

1998-01-01

An approach based on the resonant recognition model and the discrete wavelet transform is introduced here for characterising proteins' biological function. The protein sequence is converted into a numerical series by assigning the electron-ion interaction potential to each amino acid from N-terminal to C-terminal. A set of peaks is found after performing a wavelet transform onto a numerical series representing a group of homologous proteins. These peaks are related to protein structural and functional properties and named characteristic vector of that protein group. Further more, the amino acids contributing mostly to a protein's biological functions, the so-called 'hot spots' amino acids, are predicted by the continuous wavelet transform. It is found that the hot spots are clustered around the protein's cleft structure. The wavelets approach provides a novel methods for amino acid sequence analysis as well as an expansion for the newly established macromolecular interaction model: the resonant recognition model. Copyright (1998) Australasian Physical and Engineering Sciences in Medicine

Fis protein induced λF-DNA bending observed by single-pair fluorescence resonance energy transfer

Science.gov (United States)

Chi-Cheng, Fu; Wunshain, Fann; Yuan Hanna, S.

2006-03-01

Fis, a site-specific DNA binding protein, regulates many biological processes including recombination, transcription, and replication in E.coli. Fis induced DNA bending plays an important role in regulating these functions and bending angle range from ˜50 to 95 dependent on the DNA sequence. For instance, the average bending angle of λF-DNA (26 bp, 8.8nm long, contained λF binding site on the center) measured by gel mobility shift assays was ˜ 94 . But the traditional method cannot provide information about the dynamics and the angle distribution. In this study, λF-DNA was labeled with donor (Alexa Fluor 546) and acceptor (Alexa Fluor 647) dyes on its two 5' ends and the donor-acceptor distances were measured using single-pair fluorescence resonance energy transfer (sp-FRET) with and without the present of Fis protein. Combing with structure information of Fis-DNA complex, the sp-FRET results are used to estimate the protein induced DNA bending angle distribution and dynamics.

Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein

International Nuclear Information System (INIS)

Yazaki, Paul J.; Kassa, Thewodros; Cheung, Chia-wei; Crow, Desiree M.; Sherman, Mark A.; Bading, James R.; Anderson, Anne-Line J.; Colcher, David; Raubitschek, Andrew

2008-01-01

Albumin fusion proteins have demonstrated the ability to prolong the in vivo half-life of small therapeutic proteins/peptides in the circulation and thereby potentially increase their therapeutic efficacy. To evaluate if this format can be employed for antibody-based imaging, an anticarcinoembryonic antigen (CEA) single-chain antibody(scFv)-albumin fusion protein was designed, expressed and radiolabeled for biodistribution and imaging studies in athymic mice bearing human colorectal carcinoma LS-174T xenografts. The [ 125 I]-T84.66 fusion protein demonstrated rapid tumor uptake of 12.3% injected dose per gram (ID/g) at 4 h that reached a plateau of 22.7% ID/g by 18 h. This was a dramatic increase in tumor uptake compared to 4.9% ID/g for the scFv alone. The radiometal [ 111 In]-labeled version resulted in higher tumor uptake, 37.2% ID/g at 18 h, which persisted at the tumor site with tumor: blood ratios reaching 18:1 and with normal tissues showing limited uptake. Based on these favorable imaging properties, a pilot [ 64 Cu]-positron emission tomography imaging study was performed with promising results. The anti-CEA T84.66 scFv-albumin fusion protein demonstrates highly specific tumor uptake that is comparable to cognate recombinant antibody fragments. The radiometal-labeled version, which shows lower normal tissue accumulation than these recombinant antibodies, provides a promising and novel platform for antibody-based imaging agents

Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.

Science.gov (United States)

Arcand, Mathieu; Roby, Philippe; Bossé, Roger; Lipari, Francesco; Padrós, Jaime; Beaudet, Lucille; Marcil, Alexandre; Dahan, Sophie

2010-04-20

We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.

A generative, probabilistic model of local protein structure

DEFF Research Database (Denmark)

Boomsma, Wouter; Mardia, Kanti V.; Taylor, Charles C.

2008-01-01

Despite significant progress in recent years, protein structure prediction maintains its status as one of the prime unsolved problems in computational biology. One of the key remaining challenges is an efficient probabilistic exploration of the structural space that correctly reflects the relative...... conformational stabilities. Here, we present a fully probabilistic, continuous model of local protein structure in atomic detail. The generative model makes efficient conformational sampling possible and provides a framework for the rigorous analysis of local sequence-structure correlations in the native state...

Modeling and Stability Assessment of Single-Phase Grid Synchronization Techniques

DEFF Research Database (Denmark)

Golestan, Saeed; Guerrero, Josep M.; Vasquez, Juan

2018-01-01

(GSTs) is of vital importance. This task is most often based on obtaining a linear time-invariant (LTI) model for the GST and applying standard stability tests to it. Another option is modeling and dynamics/stability assessment of GSTs in the linear time-periodic (LTP) framework, which has received...... a very little attention. In this letter, the procedure of deriving the LTP model for single-phase GSTs is first demonstrated. The accuracy of the LTP model in predicting the GST dynamic behavior and stability is then evaluated and compared with that of the LTI one. Two well-known single-phase GSTs, i...

Plasmonic welded single walled carbon nanotubes on monolayer graphene for sensing target protein

Energy Technology Data Exchange (ETDEWEB)

Kim, Jangheon; Kim, Soohyun [Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, 373-1 Guseong, Yuseong, Daejeon 305-806 (Korea, Republic of); Kim, Gi Gyu; Jung, Wonsuk, E-mail: wonsuk81@wku.ac.kr [Department of Mechanical and Automotive Engineering, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of)

2016-05-16

We developed plasmonic welded single walled carbon nanotubes (SWCNTs) on monolayer graphene as a biosensor to detect target antigen molecules, fc fusion protein without any treatment to generate binder groups for linker and antibody. This plasmonic welding induces atomic networks between SWCNTs as junctions containing carboxylic groups and improves the electrical sensitivity of a SWCNTs and the graphene membrane to detect target protein. We investigated generation of the atomic networks between SWCNTs by field-emission scanning electron microscopy and atomic force microscopy after plasmonic welding process. We compared the intensity ratios of D to G peaks from the Raman spectra and electrical sheet resistance of welded SWCNTs with the results of normal SWCNTs, which decreased from 0.115 to 0.086 and from 10.5 to 4.12, respectively. Additionally, we measured the drain current via source/drain voltage after binding of the antigen to the antibody molecules. This electrical sensitivity of the welded SWCNTs was 1.55 times larger than normal SWCNTs.

THE BERTRAND MODEL OF THE SINGLE MARKET

Directory of Open Access Journals (Sweden)

Vadasan Ioana

2010-12-01

Full Text Available Starting with the signification of the rationality hypothesis when the agent’s contentment is directly affected by the other agents’ decisions, the theory of games defines solutions for solving different situations of conflict. The economic actors have different behaviours of the Single Market. Oligopoly strategic behaviours were analysed by the Bertrand model. The two types revealed in the work show that strategic interactions are sensitive to the companies’ features, products and markets. Regarding the situation when we have an oligopoly competition, the companies make interdependent decisions in the environment affected by risk and uncertainty of the Single Market. For this reason it is an opportunity to study the structure of oligopoly type of of the Single Market with the aid of non – cooperative games.

Protein and oil composition predictions of single soybeans by transmission Raman spectroscopy.

Science.gov (United States)

Schulmerich, Matthew V; Walsh, Michael J; Gelber, Matthew K; Kong, Rong; Kole, Matthew R; Harrison, Sandra K; McKinney, John; Thompson, Dennis; Kull, Linda S; Bhargava, Rohit

2012-08-22

The soybean industry requires rapid, accurate, and precise technologies for the analyses of seed/grain constituents. While the current gold standard for nondestructive quantification of economically and nutritionally important soybean components is near-infrared spectroscopy (NIRS), emerging technology may provide viable alternatives and lead to next generation instrumentation for grain compositional analysis. In principle, Raman spectroscopy provides the necessary chemical information to generate models for predicting the concentration of soybean constituents. In this communication, we explore the use of transmission Raman spectroscopy (TRS) for nondestructive soybean measurements. We show that TRS uses the light scattering properties of soybeans to effectively homogenize the heterogeneous bulk of a soybean for representative sampling. Working with over 1000 individual intact soybean seeds, we developed a simple partial least-squares model for predicting oil and protein content nondestructively. We find TRS to have a root-mean-standard error of prediction (RMSEP) of 0.89% for oil measurements and 0.92% for protein measurements. In both calibration and validation sets, the predicative capabilities of the model were similar to the error in the reference methods.

Selection of single grain seeds by 14N(n,γ)15N nuclear reaction for protein improvement

International Nuclear Information System (INIS)

Andras, L.; Csoke, A.; Nagy, A.Z.; Balint, A.

1979-01-01

A new non-destructive screening technique was developed for determining the protein (total nitrogen) content of single grain seeds. Here, our first experiment is described where in the case of maize samples, 300 s was used to perform one measurement on a seed with a semiautomatic device. (author)

Conformational Sampling in Template-Free Protein Loop Structure Modeling: An Overview

OpenAIRE

Li, Yaohang

2013-01-01

Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a “mini protein folding problem” under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increas...

Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale

Science.gov (United States)

Yu, Xiong; Egelman, Edward H.

2010-01-01

Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single stranded DNA with ∼ 9 subunits per turn in contrast to the filaments formed on double stranded DNA with ∼ 6.4 subunits per turn, and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy (STEM) that the Dmc1 filament formed on single stranded DNA has a mass per unit length expected from ∼ 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions, and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or STEM to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated. PMID:20600108

Single step purification of recombinant proteins using the metal ion-inducible autocleavage (MIIA) domain as linker for tag removal.

Science.gov (United States)

Ibe, Susan; Schirrmeister, Jana; Zehner, Susanne

2015-08-20

For fast and easy purification, proteins are typically fused with an affinity tag, which often needs to be removed after purification. Here, we present a method for the removal of the affinity tag from the target protein in a single step protocol. The protein VIC_001052 of the coral pathogen Vibrio coralliilyticus ATCC BAA-450 contains a metal ion-inducible autocatalytic cleavage (MIIA) domain. Its coding sequence was inserted into an expression vector for the production of recombinant fusion proteins. Following, the target proteins MalE and mCherry were produced as MIIA-Strep fusion proteins in Escherichia coli. The target proteins could be separated from the MIIA-Strep part simply by the addition of calcium or manganese(II) ions within minutes. The cleavage is not affected in the pH range from 5.0 to 9.0 or at low temperatures (6°C). Autocleavage was also observed with immobilized protein on an affinity column. The protein yield was similar to that achieved with a conventional purification protocol. Copyright © 2015 Elsevier B.V. All rights reserved.

A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis.

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Leslie Goo

2017-02-01

Full Text Available The structural flexibility or 'breathing' of the envelope (E protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F of the West Nile virus (WNV E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV, but not Zika virus (ZIKV, E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing.

Protein Structure Classification and Loop Modeling Using Multiple Ramachandran Distributions

KAUST Repository

Najibi, Seyed Morteza; Maadooliat, Mehdi; Zhou, Lan; Huang, Jianhua Z.; Gao, Xin

2017-01-01

Recently, the study of protein structures using angular representations has attracted much attention among structural biologists. The main challenge is how to efficiently model the continuous conformational space of the protein structures based on the differences and similarities between different Ramachandran plots. Despite the presence of statistical methods for modeling angular data of proteins, there is still a substantial need for more sophisticated and faster statistical tools to model the large-scale circular datasets. To address this need, we have developed a nonparametric method for collective estimation of multiple bivariate density functions for a collection of populations of protein backbone angles. The proposed method takes into account the circular nature of the angular data using trigonometric spline which is more efficient compared to existing methods. This collective density estimation approach is widely applicable when there is a need to estimate multiple density functions from different populations with common features. Moreover, the coefficients of adaptive basis expansion for the fitted densities provide a low-dimensional representation that is useful for visualization, clustering, and classification of the densities. The proposed method provides a novel and unique perspective to two important and challenging problems in protein structure research: structure-based protein classification and angular-sampling-based protein loop structure prediction.

Protein Structure Classification and Loop Modeling Using Multiple Ramachandran Distributions

KAUST Repository

Najibi, Seyed Morteza

2017-02-08

Recently, the study of protein structures using angular representations has attracted much attention among structural biologists. The main challenge is how to efficiently model the continuous conformational space of the protein structures based on the differences and similarities between different Ramachandran plots. Despite the presence of statistical methods for modeling angular data of proteins, there is still a substantial need for more sophisticated and faster statistical tools to model the large-scale circular datasets. To address this need, we have developed a nonparametric method for collective estimation of multiple bivariate density functions for a collection of populations of protein backbone angles. The proposed method takes into account the circular nature of the angular data using trigonometric spline which is more efficient compared to existing methods. This collective density estimation approach is widely applicable when there is a need to estimate multiple density functions from different populations with common features. Moreover, the coefficients of adaptive basis expansion for the fitted densities provide a low-dimensional representation that is useful for visualization, clustering, and classification of the densities. The proposed method provides a novel and unique perspective to two important and challenging problems in protein structure research: structure-based protein classification and angular-sampling-based protein loop structure prediction.

Nucleation phenomena in protein folding: the modulating role of protein sequence

International Nuclear Information System (INIS)

Travasso, Rui D M; FaIsca, Patricia F N; Gama, Margarida M Telo da

2007-01-01

For the vast majority of naturally occurring, small, single-domain proteins, folding is often described as a two-state process that lacks detectable intermediates. This observation has often been rationalized on the basis of a nucleation mechanism for protein folding whose basic premise is the idea that, after completion of a specific set of contacts forming the so-called folding nucleus, the native state is achieved promptly. Here we propose a methodology to identify folding nuclei in small lattice polymers and apply it to the study of protein molecules with a chain length of N = 48. To investigate the extent to which protein topology is a robust determinant of the nucleation mechanism, we compare the nucleation scenario of a native-centric model with that of a sequence-specific model sharing the same native fold. To evaluate the impact of the sequence's finer details in the nucleation mechanism, we consider the folding of two non-homologous sequences. We conclude that, in a sequence-specific model, the folding nucleus is, to some extent, formed by the most stable contacts in the protein and that the less stable linkages in the folding nucleus are solely determined by the fold's topology. We have also found that, independently of the protein sequence, the folding nucleus performs the same 'topological' function. This unifying feature of the nucleation mechanism results from the residues forming the folding nucleus being distributed along the protein chain in a similar and well-defined manner that is determined by the fold's topological features

A single product perishing inventory model with demand interaction

African Journals Online (AJOL)

The paper describes a single perishing product inventory model in which ... continuous review inventory models have been studied recently by Yadavalli et al ...... stochastic inventory system with lost sales, Stochastic Analysis and Applications ...

Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy

KAUST Repository

Reymer, A.

2009-07-08

To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a filament structure of a human Rad51 protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleo-protein complex. An inhomogeneously stretched double-stranded DNA fitted into the filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-protein filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning protein monomers along the DNA strand upon filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.

A transdisciplinary approach to the initial validation of a single cell protein as an alternative protein source for use in aquafeeds

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Michael Tlusty

2017-04-01

Full Text Available The human population is growing and, globally, we must meet the challenge of increased protein needs required to feed this population. Single cell proteins (SCP, when coupled to aquaculture production, offer a means to ensure future protein needs can be met without direct competition with food for people. To demonstrate a given type of SCP has potential as a protein source for use in aquaculture feed, a number of steps need to be validated including demonstrating that the SCP is accepted by the species in question, leads to equivalent survival and growth, does not result in illness or other maladies, is palatable to the consumer, is cost effective to produce and can easily be incorporated into diets using existing technology. Here we examine white shrimp (Litopenaeus vannamei growth and consumer taste preference, smallmouth grunt (Haemulon chrysargyreum growth, survival, health and gut microbiota, and Atlantic salmon (Salmo salar digestibility when fed diets that substitute the bacterium Methylobacterium extorquens at a level of 30% (grunts, 100% (shrimp, or 55% (salmon of the fishmeal in a compound feed. In each of these tests, animals performed equivalently when fed diets containing M. extorquens as when fed a standard aquaculture diet. This transdisciplinary approach is a first validation of this bacterium as a potential SCP protein substitute in aquafeeds. Given the ease to produce this SCP through an aerobic fermentation process, the broad applicability for use in aquaculture indicates the promise of M. extorquens in leading toward greater food security in the future.

Conformational Dynamics of the Receptor Protein Galactose/Glucose Binding Protein

Science.gov (United States)

Messina, Troy; Talaga, David

2006-03-01

We have performed time-correlated single photon counting (TCSPC) anisotropy and Stokes Shift measurements on bulk solutions of galactose/glucose binding protein. Site-directed mutagenesis was used to provide a single cysteine amino acid near the sugar-binding center of the protein (glutamine 26 to cysteine -- Q26C). The cysteine was covalently labeled with the environmentally-sensitive fluorophore acrylodan, and a long-lived ruthenium complex was covalently attached to the N-terminus to provide a fluorescent reference. The TCSPC data were analyzed using global convolute-and-compare fitting routines over the entire glucose titration and temperature range to provide minimal reduced chi-squared values and the highest time resolution possible. Using a standard ligand-binding model, the resulting distributions show that the closed (ligand-bound) conformation exists even at zero glucose concentration. At 20^oC, the relative abundance of this conformation is as high as 40%. The temperature dependence of this conformational study will be discussed and related to the ligand-binding free energy surface.

Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

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Françoise Paquet

Full Text Available In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1 from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

Topology and weights in a protein domain interaction network – a novel way to predict protein interactions

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Wuchty Stefan

2006-05-01

Full Text Available Abstract Background While the analysis of unweighted biological webs as diverse as genetic, protein and metabolic networks allowed spectacular insights in the inner workings of a cell, biological networks are not only determined by their static grid of links. In fact, we expect that the heterogeneity in the utilization of connections has a major impact on the organization of cellular activities as well. Results We consider a web of interactions between protein domains of the Protein Family database (PFAM, which are weighted by a probability score. We apply metrics that combine the static layout and the weights of the underlying interactions. We observe that unweighted measures as well as their weighted counterparts largely share the same trends in the underlying domain interaction network. However, we only find weak signals that weights and the static grid of interactions are connected entities. Therefore assuming that a protein interaction is governed by a single domain interaction, we observe strong and significant correlations of the highest scoring domain interaction and the confidence of protein interactions in the underlying interactions of yeast and fly. Modeling an interaction between proteins if we find a high scoring protein domain interaction we obtain 1, 428 protein interactions among 361 proteins in the human malaria parasite Plasmodium falciparum. Assessing their quality by a logistic regression method we observe that increasing confidence of predicted interactions is accompanied by high scoring domain interactions and elevated levels of functional similarity and evolutionary conservation. Conclusion Our results indicate that probability scores are randomly distributed, allowing to treat static grid and weights of domain interactions as separate entities. In particular, these finding confirms earlier observations that a protein interaction is a matter of a single interaction event on domain level. As an immediate application, we

Protein homology model refinement by large-scale energy optimization.

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Park, Hahnbeom; Ovchinnikov, Sergey; Kim, David E; DiMaio, Frank; Baker, David

2018-03-20

Proteins fold to their lowest free-energy structures, and hence the most straightforward way to increase the accuracy of a partially incorrect protein structure model is to search for the lowest-energy nearby structure. This direct approach has met with little success for two reasons: first, energy function inaccuracies can lead to false energy minima, resulting in model degradation rather than improvement; and second, even with an accurate energy function, the search problem is formidable because the energy only drops considerably in the immediate vicinity of the global minimum, and there are a very large number of degrees of freedom. Here we describe a large-scale energy optimization-based refinement method that incorporates advances in both search and energy function accuracy that can substantially improve the accuracy of low-resolution homology models. The method refined low-resolution homology models into correct folds for 50 of 84 diverse protein families and generated improved models in recent blind structure prediction experiments. Analyses of the basis for these improvements reveal contributions from both the improvements in conformational sampling techniques and the energy function.

Interaction of single and multi-layer graphene oxide with fetal bovine serum: assessing the protein corona formation

Energy Technology Data Exchange (ETDEWEB)

Franqui, Lidiane Silva; Farias, Marcelo Alexandre de; Portugal, Rodrigo Villares; Costa, Carlos Alberto; Leme, Adriana Franco Paes; Martinez, Diego Stefani Teodoro, E-mail: lidiane.franqui@pos.ft.unicamp.br [Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP (Brazil); Coluci, Vitor Rafael [Universidade Estadual de Campinas (UNICAMP), SP (Brazil)

2016-07-01

Full text: When in contact with biological systems, nanomaterials surface adsorbs biomolecules present in the biological medium, mainly proteins, yielding a molecular coating 'protein corona' which affects the biological response and toxicity of the nanomaterials. Several factors can influence the protein corona formation, such as nanomaterial physicochemical properties and the nature of biological medium. In this work, we have performed a comparative study between the single and multi-layer graphene oxide nanomaterials (SL-GO and ML-GO, respectively) after their interaction with DMEM cell culture medium containing fetal bovine serum (FBS). Bare GOs and FBS protein corona-coated GOs were characterized using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), cryogenic transmission electron microscopy (Cryo-TEM) and X-ray photoelectron spectroscopy (XPS). The protein corona composition was characterized by gel electrophoresis (SDS-PAGE) and mass spectrometry (LC-MS/MS). Our results showed that, after interaction with FBS, GO particle size increased due to the protein corona formation. Besides, the presence of proteins also has significantly increased the dispersion stability of SLGO and ML-GO over time. Whereas the main proteins have been identified in both SL-GO and ML-GO, SL-GO has adsorbed larger amounts of proteins than ML-GO. Finally, the number of GO layers influences its interactions with FBS proteins and dispersion stability in DMEM medium. These results point out implications for in vitro cytotoxicity assessment and biomedical applications of these promising carbon nanomaterials. (author)

Interaction of single and multi-layer graphene oxide with fetal bovine serum: assessing the protein corona formation

International Nuclear Information System (INIS)

Franqui, Lidiane Silva; Farias, Marcelo Alexandre de; Portugal, Rodrigo Villares; Costa, Carlos Alberto; Leme, Adriana Franco Paes; Martinez, Diego Stefani Teodoro; Coluci, Vitor Rafael

2016-01-01

Full text: When in contact with biological systems, nanomaterials surface adsorbs biomolecules present in the biological medium, mainly proteins, yielding a molecular coating 'protein corona' which affects the biological response and toxicity of the nanomaterials. Several factors can influence the protein corona formation, such as nanomaterial physicochemical properties and the nature of biological medium. In this work, we have performed a comparative study between the single and multi-layer graphene oxide nanomaterials (SL-GO and ML-GO, respectively) after their interaction with DMEM cell culture medium containing fetal bovine serum (FBS). Bare GOs and FBS protein corona-coated GOs were characterized using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), cryogenic transmission electron microscopy (Cryo-TEM) and X-ray photoelectron spectroscopy (XPS). The protein corona composition was characterized by gel electrophoresis (SDS-PAGE) and mass spectrometry (LC-MS/MS). Our results showed that, after interaction with FBS, GO particle size increased due to the protein corona formation. Besides, the presence of proteins also has significantly increased the dispersion stability of SLGO and ML-GO over time. Whereas the main proteins have been identified in both SL-GO and ML-GO, SL-GO has adsorbed larger amounts of proteins than ML-GO. Finally, the number of GO layers influences its interactions with FBS proteins and dispersion stability in DMEM medium. These results point out implications for in vitro cytotoxicity assessment and biomedical applications of these promising carbon nanomaterials. (author)

A single WAP domain (SWD)-containing protein with antiviral activity from Pacific white shrimp Litopenaeus vannamei.

Science.gov (United States)

Yang, Linwei; Niu, Shengwen; Gao, Jiefeng; Zuo, Hongliang; Yuan, Jia; Weng, Shaoping; He, Jianguo; Xu, Xiaopeng

2018-02-01

The single whey acidic protein (WAP) domain (SWD)-containing proteins, also called type III crustins, are a group of antimicrobial peptides (AMPs) in crustaceans. At present, a number of SWDs have been identified in shrimp, which showed essential antibacterial activities. However, the roles of SWDs in antiviral immune responses have not been reported up to now. In this study, a novel SWD (LvSWD3) was identified from Pacific white shrimp, Litopenaeus vannamei, which contained a typical single WAP domain homologous to those of other crustacean SWDs. Although lacking the pro and arg-rich region between the signal peptide and the WAP domain, LvSWD3 was closely clustered with other shrimp SWDs in the phylogenetic tree. Similar to many shrimp SWDs, the highest expression of LvSWD3 was detected in hemocytes. The LvSWD3 expression exhibited only limited changes after challenges with Vibrio parahaemolyticus, Poly (I:C) and lipopolysaccharide, but was significantly up-regulated after white spot syndrome virus (WSSV) infection. Silencing of LvSWDs significantly accelerated the death of the WSSV-infected but not the V. parahaemolyticus-infected shrimp. The recombinant LvSWD3 protein did not show proteinase inhibitory and antibacterial activities but could significantly postpone the death of WSSV-infected shrimp and reduce the viral load in tissues. These suggested that LvSWD3 was a novel SWD with antiviral activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

The research methods and model of protein turnover in animal

International Nuclear Information System (INIS)

Wu Xilin; Yang Feng

2002-01-01

The author discussed the concept and research methods of protein turnover in animal body. The existing problems and the research results of animal protein turnover in recent years were presented. Meanwhile, the measures to improve the models of animal protein turnover were analyzed

Cultivation and utilization of Jerusalem artichoke for ethanol, single cell protein, and high-fructose syrup production

Energy Technology Data Exchange (ETDEWEB)

Bajpai, P.K.; Bajpai, Pratima (Thapar Corporate Research and Development Center, Patiala (IN). Div. of Chemical and Biochemical Engineering)

1991-04-01

Jerusalem artichoke has one of the highest carbohydrate yields of the known agricultural crops and has many distinct advantages over traditional crops. This brief review presents data on the yield and composition of Jerusalem artichoke, techniques of carbohydrate extraction and its utilization for the production of ethanol, single cell protein (SCP), and high-fructose syrup, along with economic considerations. (author).

A simple quantitative model of macromolecular crowding effects on protein folding: Application to the murine prion protein(121-231)

Science.gov (United States)

Bergasa-Caceres, Fernando; Rabitz, Herschel A.

2013-06-01

A model of protein folding kinetics is applied to study the effects of macromolecular crowding on protein folding rate and stability. Macromolecular crowding is found to promote a decrease of the entropic cost of folding of proteins that produces an increase of both the stability and the folding rate. The acceleration of the folding rate due to macromolecular crowding is shown to be a topology-dependent effect. The model is applied to the folding dynamics of the murine prion protein (121-231). The differential effect of macromolecular crowding as a function of protein topology suffices to make non-native configurations relatively more accessible.

Single daily dosing of antibiotics: importance of in vitro killing rate, serum half-life, and protein binding.

Science.gov (United States)

Potel, G; Chau, N P; Pangon, B; Fantin, B; Vallois, J M; Faurisson, F; Carbon, C

1991-10-01

The relative importance of pharmacokinetic and pharmacodynamic parameters for the feasibility of a single daily dose (SDD) of antibiotics remains to be established. Therefore, we studied the relationship between in vitro bacteriological parameters (MIC, MBC, and killing rate [KR], defined as the reduction in the inoculum within 3 h), pharmacokinetic parameters (t1/2 and protein binding [PB], and in vivo antibacterial effect of a single antibiotic dose in an experimental rabbit model of Escherichia coli endocarditis. Nine antibiotics were investigated: two aminoglycosides, two quinolones, and five beta-lactams. For each drug, the minimal effective dose (MED) (in milligrams per kilogram) was defined as the lowest dose able to achieve a significant difference (P less than 0.05) of CFU in the vegetations in comparison with controls 24 h after a single intravenous injection. Aminoglycosides and quinolones had the lowest MEDs, followed by beta-lactams. Univariate regression analysis showed that KR was the major determinant of MED. A stepwise regression analysis showed that t1/2 significantly improved the predictive value of KR, while PB, MIC, and MBC did not. The final equation was MED = 1,586-238 KR-297 t1/2 (r = 0.90, P = 0.01). We concluded that the pharmacodynamic parameters (especially the high KR) of aminoglycosides and quinolones explained their low MEDs and might allow SDD. In contrast, the low KR of beta-lactams emphasized the critical importance of a long t1/2, as for ceftriaxone, allowing the use of this beta-lactam alone in SDD.

Physics of protein folding

Science.gov (United States)

Finkelstein, A. V.; Galzitskaya, O. V.

2004-04-01

Protein physics is grounded on three fundamental experimental facts: protein, this long heteropolymer, has a well defined compact three-dimensional structure; this structure can spontaneously arise from the unfolded protein chain in appropriate environment; and this structure is separated from the unfolded state of the chain by the “all-or-none” phase transition, which ensures robustness of protein structure and therefore of its action. The aim of this review is to consider modern understanding of physical principles of self-organization of protein structures and to overview such important features of this process, as finding out the unique protein structure among zillions alternatives, nucleation of the folding process and metastable folding intermediates. Towards this end we will consider the main experimental facts and simple, mostly phenomenological theoretical models. We will concentrate on relatively small (single-domain) water-soluble globular proteins (whose structure and especially folding are much better studied and understood than those of large or membrane and fibrous proteins) and consider kinetic and structural aspects of transition of initially unfolded protein chains into their final solid (“native”) 3D structures.

Possibilities of microscopic detection of isolated porcine proteins in model meat products

Directory of Open Access Journals (Sweden)

Michaela Petrášová

2016-05-01

Full Text Available In recent years, various protein additives intended for manufacture of meat products have increasing importance in the food industry. These ingredients include both, plant-origin as well as animal-origin proteins. Among animal proteins, blood plasma, milk protein or collagen are used most commonly. Collagen is obtained from pork, beef, and poultry or fish skin. Collagen does not contain all the essential amino acids, thus it is not a full protein in terms of essential amino acids supply for one's organism. However, it is rather rich in amino acids of glycine, hydroxyproline and proline which are almost absent in other proteins and their synthesis is very energy intensive. Collagen, which is added to the soft and small meat products in the form of isolated porcine protein, significantly affects the organoleptic properties of these products. This work focused on detection of isolated porcine protein in model meat products where detection of isolated porcine protein was verified by histological staining and light microscopy. Seven model meat products from poultry meat and 7 model meat products from beef and pork in the ratio of 1:1, which contained 2.5% concentration of various commercially produced isolated porcine proteins, were examined. These model meat products were histologically processed by means of cryosections and stained with hematoxylin-eosin staining, toluidine blue staining and Calleja. For the validation phase, Calleja was utilized. To determine the sensitivity and specificity, five model meat products containing the addition of isolated porcine protein and five model meat products free of it were used. The sensitivity was determined for isolated porcine protein at 1.00 and specificity was determined at 1.00. The detection limit of the method was at the level of 0.001% addition. Repeatability of the method was carried out using products with addition as well as without addition of isolated porcine protein and detection was repeated

A single molecule approach for measuring the transport properties and energetics of membrane proteins in heterogeneous planar bio-mimetic assemblies

Science.gov (United States)

Poudel, Kumud Raj

The significance of transmembrane protein research is well documented. Numerous studies have clearly established the biological, biophysical and pharmaceutical importance that these membrane components serve. Communications through receptors regulate countless body functions and they also provide structural support to the cell. However, a lack of high-resolution structure data has limited our understanding of these proteins that make it necessary to study them in in-vitro platforms or 'bio-mimetic' assemblies. Albeit that an assortment of platforms have been suggested for in-vitro studies, the issues, however, remain the same. The lack of mobility of the proteins in artificial environments, the question of functionality that arises with mobility and the search in general for the best assembly, is still a work in progress. In this work, we have taken some of the most accepted platforms in the field and characterized them through the lens of single molecule spectroscopy. We have addressed the question of mobility by reducing it down to a single molecule and comparing it with the bulk. By utilizing the Serotonin Receptor 5HT3A we have shown that techniques such as passivation of the substrates in the assemblies by Bovine Serum Albumin has a significant effect at the molecular level. The larger size of the intracellular domain for the 5HT3A served as a great probe to understand and evaluate the interaction of a surface passivator with the integrated membrane protein. We have also taken this a step further by developing a novel, single cushion 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) assembly and added another degree of complexity- through a phase transition. We have utilized phase transition to get an insight into the local protein environment, activation energies, heterogeneity and diffusion characteristics by using Annexin V as our probe. The work presented here studies two completely different biological platforms using two entirely different transmembrane

A linear programming model for protein inference problem in shotgun proteomics.

Science.gov (United States)

Huang, Ting; He, Zengyou

2012-11-15

Assembling peptides identified from tandem mass spectra into a list of proteins, referred to as protein inference, is an important issue in shotgun proteomics. The objective of protein inference is to find a subset of proteins that are truly present in the sample. Although many methods have been proposed for protein inference, several issues such as peptide degeneracy still remain unsolved. In this article, we present a linear programming model for protein inference. In this model, we use a transformation of the joint probability that each peptide/protein pair is present in the sample as the variable. Then, both the peptide probability and protein probability can be expressed as a formula in terms of the linear combination of these variables. Based on this simple fact, the protein inference problem is formulated as an optimization problem: minimize the number of proteins with non-zero probabilities under the constraint that the difference between the calculated peptide probability and the peptide probability generated from peptide identification algorithms should be less than some threshold. This model addresses the peptide degeneracy issue by forcing some joint probability variables involving degenerate peptides to be zero in a rigorous manner. The corresponding inference algorithm is named as ProteinLP. We test the performance of ProteinLP on six datasets. Experimental results show that our method is competitive with the state-of-the-art protein inference algorithms. The source code of our algorithm is available at: https://sourceforge.net/projects/prolp/. zyhe@dlut.edu.cn. Supplementary data are available at Bioinformatics Online.

Structure of a second crystal form of Bence-Jones protein Loc: Strikingly different domain associations in two crystal forms of a single protein

International Nuclear Information System (INIS)

Schiffer, M.; Ainsworth, C.; Xu, Z.B.; Carperos, W.; Olsen, K.; Solomon, A.; Stevens, F.J.; Chang, C.H.

1989-01-01

The authors have determined the structure of the immunoglobulin light-chain dimer Loc in a second crystal form that was grown from distilled water. The crystal structure was determined to 2.8-angstrom resolution; the R factor is 0.22. The two variable domains are related by local 2-fold axes and form an antigen binding pocket. The variable domain-variable domain interaction observed in this crystal form differs from the one exhibited by the protein when crystallized from ammonium sulfate in which the two variable domains formed a protrusion. The structure attained in the distilled water crystals is similar to, but not identical with, the one observed for the Mcg light-chain dimer in crystals grown from ammonium sulfate. Thus, two strikingly different structures were attained by this multisubunit protein in crystals grown under two different, commonly used, crystallization techniques. The quaternary interactions exhibited by the protein in the two crystal forms are sufficiently different to suggest fundamentally different interpretations of the structural basis for the function of this protein. This observation may have general implications regarding the use of single crystallographic determinations for detailed identification of structural and functional relationships. On the other hand, proteins whose structures can be altered by manipulation of crystallization conditions may provide useful systems for study of fundamental structural chemistry

Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.

Science.gov (United States)

Yamniuk, Aaron P; Newitt, John A; Doyle, Michael L; Arisaka, Fumio; Giannetti, Anthony M; Hensley, Preston; Myszka, David G; Schwarz, Fred P; Thomson, James A; Eisenstein, Edward

2015-12-01

A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.

Identify High-Quality Protein Structural Models by Enhanced K-Means.

Science.gov (United States)

Wu, Hongjie; Li, Haiou; Jiang, Min; Chen, Cheng; Lv, Qiang; Wu, Chuang

2017-01-01

Background. One critical issue in protein three-dimensional structure prediction using either ab initio or comparative modeling involves identification of high-quality protein structural models from generated decoys. Currently, clustering algorithms are widely used to identify near-native models; however, their performance is dependent upon different conformational decoys, and, for some algorithms, the accuracy declines when the decoy population increases. Results. Here, we proposed two enhanced K -means clustering algorithms capable of robustly identifying high-quality protein structural models. The first one employs the clustering algorithm SPICKER to determine the initial centroids for basic K -means clustering ( SK -means), whereas the other employs squared distance to optimize the initial centroids ( K -means++). Our results showed that SK -means and K -means++ were more robust as compared with SPICKER alone, detecting 33 (59%) and 42 (75%) of 56 targets, respectively, with template modeling scores better than or equal to those of SPICKER. Conclusions. We observed that the classic K -means algorithm showed a similar performance to that of SPICKER, which is a widely used algorithm for protein-structure identification. Both SK -means and K -means++ demonstrated substantial improvements relative to results from SPICKER and classical K -means.

Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes.

Science.gov (United States)

Rackiewicz, Michal; Große-Hovest, Ludger; Alpert, Andrew J; Zarei, Mostafa; Dengjel, Jörn

2017-06-02

Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

Polymorphism of myofibrillar proteins of rabbit skeletal-muscle fibres. An electrophoretic study of single fibres.

OpenAIRE

Salviati, G; Betto, R; Danieli Betto, D

1982-01-01

Rabbit predominantly fast-twitch-fibre and predominantly slow-twitch-fibre skeletal muscles of the hind limbs, the psoas, the diaphragm and the masseter muscles were fibre-typed by one-dimensional polyacrylamide-gel electrophoresis of the myofibrillar proteins of chemically skinned single fibres. Investigation of the distribution of fast-twitch-fibre and slow-twitch-fibre isoforms of myosin light chains and the type of myosin heavy chains, based on peptide 'maps' published in Cleveland. Fisch...

NASCENT: an automatic protein interaction network generation tool for non-model organisms.

Science.gov (United States)

Banky, Daniel; Ordog, Rafael; Grolmusz, Vince

2009-04-24

Large quantity of reliable protein interaction data are available for model organisms in public depositories (e.g., MINT, DIP, HPRD, INTERACT). Most data correspond to experiments with the proteins of Saccharomyces cerevisiae, Drosophila melanogaster, Homo sapiens, Caenorhabditis elegans, Escherichia coli and Mus musculus. For other important organisms the data availability is poor or non-existent. Here we present NASCENT, a completely automatic web-based tool and also a downloadable Java program, capable of modeling and generating protein interaction networks even for non-model organisms. The tool performs protein interaction network modeling through gene-name mapping, and outputs the resulting network in graphical form and also in computer-readable graph-forms, directly applicable by popular network modeling software. http://nascent.pitgroup.org.

Combining NMR ensembles and molecular dynamics simulations provides more realistic models of protein structures in solution and leads to better chemical shift prediction

International Nuclear Information System (INIS)

Lehtivarjo, Juuso; Tuppurainen, Kari; Hassinen, Tommi; Laatikainen, Reino; Peräkylä, Mikael

2012-01-01

While chemical shifts are invaluable for obtaining structural information from proteins, they also offer one of the rare ways to obtain information about protein dynamics. A necessary tool in transforming chemical shifts into structural and dynamic information is chemical shift prediction. In our previous work we developed a method for 4D prediction of protein 1 H chemical shifts in which molecular motions, the 4th dimension, were modeled using molecular dynamics (MD) simulations. Although the approach clearly improved the prediction, the X-ray structures and single NMR conformers used in the model cannot be considered fully realistic models of protein in solution. In this work, NMR ensembles (NMRE) were used to expand the conformational space of proteins (e.g. side chains, flexible loops, termini), followed by MD simulations for each conformer to map the local fluctuations. Compared with the non-dynamic model, the NMRE+MD model gave 6–17% lower root-mean-square (RMS) errors for different backbone nuclei. The improved prediction indicates that NMR ensembles with MD simulations can be used to obtain a more realistic picture of protein structures in solutions and moreover underlines the importance of short and long time-scale dynamics for the prediction. The RMS errors of the NMRE+MD model were 0.24, 0.43, 0.98, 1.03, 1.16 and 2.39 ppm for 1 Hα, 1 HN, 13 Cα, 13 Cβ, 13 CO and backbone 15 N chemical shifts, respectively. The model is implemented in the prediction program 4DSPOT, available at http://www.uef.fi/4dspothttp://www.uef.fi/4dspot.