Enhanced Stereoselectivity of a Cu(II) Complex Chiral Auxiliary in the Synthesis of Fmoc-L-γ-carboxyglutamic Acid | Center for Cancer Research

Science.gov (United States)

Bridging bioinorganic chemistry with asymmetric synthesis: a naturally occurring metalloprotein is used for the structure-based evolution of chiral auxiliaries that prove to be effective in the synthesis of Fmoc-L-γ-carboxyglutamic acid.

Investigation of an optimal cell lysis method for the study of the zinc metalloproteome of Histoplasma capsulatum.

Science.gov (United States)

Donnell, Anna M; Lewis, Stephanie; Abraham, Sami; Subramanian, Kavitha; Figueroa, Julio Landero; Deepe, George S; Vonderheide, Anne P

2017-10-01

This work sought to assess optimal extraction conditions in the study of the metalloproteome of the dimorphic fungus Histoplasma capsulatum. One of the body's responses to H. capsulatum infection is sequestration of zinc within host macrophage (MØ), as reported by Vignesh et al. (Immunity 39:697-710, 2013) and Vignesh et al. (PLOS Pathog 9:E1003815, 2013). Thus, metalloproteins containing zinc were of greatest interest as it plays a critical role in survival of the fungus. One challenge in metalloproteomics is the preservation of the native structure of proteins to retain non-covalently bound metals. Many of the conventional cell lysis, separation, and identification techniques in proteomics are carried out under conditions that could lead to protein denaturation. Various cell lysis techniques were investigated in an effort to both maintain the metalloproteins during lysis and subsequent analysis while, at the same time, serving to be strong enough to break the cell wall, allowing access to cytosolic metalloproteins. The addition of 1% Triton x-100, a non-ionic detergent, to the lysis buffer was also studied. Seven lysis methods were considered and these included: Glass Homogenizer (H), Bead Beater (BB), Sonication Probe (SP), Vortex with 1% Triton x-100 (V, T), Vortex with no Triton x-100 (V, NT), Sonication Bath, Vortex, and 1% Triton x-100 (SB, V, T) and Sonication Bath, Vortex, and no Triton x-100 (SB, V, NT). A Qubit® Assay was used to compare total protein concentration and inductively coupled plasma-mass spectrometry (ICP-MS) was utilized for total metal analysis of cell lysates. Size exclusion chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS) was used for separation of the metalloproteins in the cell lysate and the concentration of Zn over a wide molecular weight range was examined. Additional factors such as potential contamination sources were also considered. A cell lysis method involving vortexing H. capsulatum yeast cells with 500 μm glass beads

Surface Immobilized His-tagged Azurin as a Model Interface for the Investigation of Vectorial Electron Transfer in Biological Systems

International Nuclear Information System (INIS)

Casalini, Stefano; Berto, Marcello; Kovtun, Alessandro; Operamolla, Alessandra; Di Rocco, Giulia; Facci, Paolo; Liscio, Andrea; Farinola, Gianluca M.; Borsari, Marco; Bortolotti, Carlo A.

2015-01-01

A model system for the electrochemical investigation of vectorial electron transfer in biological systems was designed, assembled and characterized. Gold electrodes, functionalized with a -OCH_3 terminated, aromatic self-assembled monolayer, were used as a substrate for the adsorption of variants of copper-containing, redox metalloprotein azurin. The engineered azurin bears a polyhistidine tag at its C-terminus. Thanks to the presence of the solvent exposed tag, which chelates Cu"2"+ ions in solution, we introduced an exogenous redox centre. The different reduction potentials of the two redox centres and their positioning with respect to the surface are such that electron transfer from the exogenous copper centre and the electrode is mediated by the native azurin active site, closely paralleling electron transfer processes in naturally occurring multicentre metalloproteins.

The Role of Copper in Neurodegenerative Disease

Science.gov (United States)

Rose, Francis M.

My research concerns the fundamental atomistic mechanisms of neurodegenerative diseases and the methodologies by which they may be discerned. This thesis consists of three primary parts. The introductory material is the raison d'etre for this work and a critical overview of the specific physics, mathematics and algorithms used in this research. The methods are presented along with specific details in order to facilitate future replication and enhancement. With the groundwork of mechanisms and methods out of the way, we then explore a nouveau atomistic mechanism describing the onset of Parkinson's disease, a disease that has been closely linked to misfolded metalloproteins. Further exploration of neurodegeneration takes place in the following chapter, where a remedial approach to Alzheimer's disease via a simulated chelation of a metalloprotein is undertaken. Altogether, the methods and techniques applied here allow for simulated exploration of both the atomistic mechanisms of neurodegeneration and their potential remediation strategies. The beginning portion of the research efforts explore protein misfolding dynamics in the presence a copper ion. Misfolding of the human alpha-synuclein (aS) protein has been implicated as a central constituent in neurodegenerative disease. In Parkinson's disease (PD) in particular, aS is thought to be the causative participant when found concentrated into neuritic plaques. Here we propose a scenario involving the metal ion Cu2+ as the protein misfolding initiator of fibrillized aS, the chief component of neuritic plaques. From experimental results we know these misfolded proteins have a rich beta--sheet signature, a marker that we reproduce with our simulated model. This model identifies a process of structural modifications to a natively unfolded alpha-synuclein resulting in a partially folded intermediate with a well defined nucleation site. It serves as a precursor to the fully misfolded protein. Understanding the nucleation

Characterization of biological macromolecules by electrophoresis and neutron activation

International Nuclear Information System (INIS)

Stone, S.F.; Hancock, D.; Zeisler, R.

1987-01-01

A procedure combining polyacrylamide gel electrophoresis (PAGE) with INAA and autoradiography was developed to study biological macromolecules and their associated trace elements. Results from the application of this method to several metalloproteins are presented. (author)

The Au-S bond in biomolecular adsorption and electrochemical electron transfer

DEFF Research Database (Denmark)

Ford, M. J.; Hush, N. S.; Marcuccio, S.

Interfacial electrochemical electron transfer (ET) of redox metalloproteins is long established. For the proteins to retain full ET or enzyme activity, modification of the electrode surfaces, such as goldsurfaces by self-assembled molecular monolayers (SAMs), is nearly always required, where pure...

Plasma Ang2 and ADAM17 levels are elevated during clinical malaria; Ang2 level correlates with severity and expression of EPCR-binding PfEMP1

DEFF Research Database (Denmark)

Petersen, Jens E V; Mkumbaye, Sixbert I; Vaaben, Anna V

2016-01-01

The pathogenesis of Plasmodium falciparum malaria involves a complex interplay between parasite adhesion and inflammatory response that includes release of cytokines and activation of the endothelium with accompanying release of Angiopoitin 2 (Ang2) to the plasma. A-disintegrin and metalloprotein...

Redox Potentials and Electronic States of Iron Porphyrin IX Adsorbed on Single Crystal Gold Electrode Surfaces

DEFF Research Database (Denmark)

Zhang, Ling; Kepp, Kasper P.; Ulstrup, Jens

2018-01-01

Metalloporphyrins are active sites in metalloproteins and synthetic catalysts. They have also been studied extensively by electrochemistry as well as being prominent targets in electrochemical scanning tunneling microscopy (STM). Previous studies of FePPIX adsorbed on graphite and alkylthiol modi...

Sarkar, Prof. Sabyasachi

Indian Academy of Sciences (India)

Home; Fellowship. Fellow Profile. Elected: 1997 Section: Chemistry. Sarkar, Prof. Sabyasachi Ph.D. (Gorakhpur). Date of birth: 17 May 1947. Specialization: Graphene & Carbon Quantum Dots, Drug Delivery, Bio-geoinorganic Chemistry, Structure-functional analogues of metallo-proteins, Carbon Quantum Dots, Bio- ...

Molecular Assembly of Hemin on Single-Crystal Au(111)-electrode Surfaces

DEFF Research Database (Denmark)

Zhang, Ling; Ulstrup, Jens; Zhang, Jingdong

Iron porphyrin, hemin, is the active core in cytochromes, haemoglobin and myoglobin, andenzymes such as the peroxidases. These metalloproteins are engaged in respiratory electron transfer, oxygen transport and storage, and enzyme catalysis in the biosynthesis of a range of metabolites. Hemin itse...

Novel Chiroptical Analysis of Hemoglobin by Surface Enhanced Resonance Raman Optical Activity Spectroscopy

DEFF Research Database (Denmark)

Brazhe, Nadezda; Brazhe, Alexey; Sosnovtseva, Olga

2010-01-01

The metalloprotein hemoglobin (Hb) was studied using surface enhanced resonance Raman spectroscopy (SERRS) and surface enhanced resonance Raman optical activity (SERROA). The SERROA results are analyzed and compared with the SERRS, and the later to the resonance Raman (RRS) performed on Hb...

Development and evaluation of an indirect ELISA for detection of exfoliative toxin ExhA, ExhB or ExhC produced by Staphylococcus hyicus

DEFF Research Database (Denmark)

Andresen, Lars Ole

1999-01-01

Immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) confirmed previous reports that the Staphylococcus hyicus exfoliative toxins ExhA and ExhB are metalloproteins, and further indicated that ExhC is also a metalloprotein. An indirect ELISA. was developed for the detection of toxigenic...... strains as an alternative method to the use of phage typing for selection of S. hyicus isolates to be used in autogenous vaccine against exudative epidermitis in pigs. The indirect ELISA was evaluated by investigating the presence of toxin among a total of 655 S. hyicus isolates from 69 pig skin samples......, one from each of the 69 pig herds with outbreak of exudative epidermitis. Toxigenic S. hyicus were detected in 74% of the cases by ELISA. From each of the five cases, in which initially no toxigenic S. hyicus were found, a further 40 S. hyicus-like colonies were tested in ELISA. Testing of this number...

Ion mobility spectrometry focusing on speciation analysis of metals/metalloids bound to carbonic anhydrase.

Science.gov (United States)

Pessôa, Gustavo de Souza; Pilau, Eduardo Jorge; Gozzo, Fábio Cesar; Arruda, Marco Aurélio Zezzi

2013-09-01

In the present work, traveling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS) was applied to speciation analysis of metalloproteins. The influence of pH on complexation conditions between some metals and bovine carbonic anhydrase was evaluated from pH 6 to 9, as well as the time involved in their complexation (0-24 h). Employing TWIMS-MS, two conformational states of bovine carbonic anhydrase were observed with charge states of +12 and +11; these configurations being evaluated in terms of the folded state of the apo form and this protein (at charge state +11) being linked to barium, lead, copper, and zinc in their divalent forms. Metalloprotein speciation analysis was carried out for copper (Cu(+) and Cu(2+)), lead (Pb(2+) and Pb(4+)), and selenium (Se(4+) and Se(6+)) species complexed with bovine carbonic anhydrase. Mobilities of all complexed species were compared, also considering the apo form of this protein.

Electrochemistry and bioelectrochemistry towards the single-molecule level: Theoretical notions and systems

International Nuclear Information System (INIS)

Zhang Jingdong; Chi Qijin; Albrecht, Tim; Kuznetsov, Alexander M.; Grubb, Mikala; Hansen, Allan G.; Wackerbarth, Hainer; Welinder, Anne C.; Ulstrup, Jens

2005-01-01

Surface structures controlled at the nanometer and single-molecule levels, with functions crucially determined by interfacial electron transfer (ET) are broadly reported in recent years, with different kinds of electrochemically controlled nanoscale/single molecule systems. One is the broad class of metallic and semiconductor-based nanoparticles, nano-arrays, nanotubes, and nanopits. Others are based on self-assembled molecular monolayers. The latter extend to bioelectrochemical systems with redox metalloproteins and DNA-based molecules as targets. We overview here some recent achievements in areas of interfacial electrochemical ET systems, mapped to the nanoscale and single-molecule levels. Focus is on both experimental and theoretical studies in our group. Systems addressed are organized monolayers of redox active transition metal complexes, and metalloproteins and metalloenzymes on single-crystal Au(1 1 1)-electrode surfaces. These systems have been investigated by voltammetry, spectroscopy, microcantilever technology, and scanning probe microscopy. A class of Os-complexes has shown suitable as targets for electrochemical in situ scanning tunnelling microscopy (STM), with close to single-molecule scanning tunnelling spectroscopic (STS) features. Mapping of redox metalloproteins from the three major classes, i.e. blue copper proteins, heme proteins, and iron-sulfur proteins, at the monolayer and single-molecule levels have also been achieved. In situ STM and spectroscopy of redox molecules and biomolecules have been supported by new theoretical frames, which extend established theory of interfacial electrochemical ET. The electrochemical nanoscale and single-molecule systems discussed are compared with other recent nanoscale and single-molecule systems with conspicuous device-like properties, particularly unimolecular rectifiers and single-molecule transistors. Both of these show analogies to electrochemical in situ STM features of redox molecules and

Journal of Biosciences | Indian Academy of Sciences

Indian Academy of Sciences (India)

Metallothioneins (MTs), a low-mass class of metalloproteins, are characterized by a high thiolate sulphur and metal content. MTs are involved in metal homeostasis and heavy metal detoxification, and are efficient scavengers of free radicals. This article describes zinc release from human MT-1 and modification of its amino ...

Solid state protein monolayers: Morphological, conformational, and functional properties

Science.gov (United States)

Pompa, P. P.; Biasco, A.; Frascerra, V.; Calabi, F.; Cingolani, R.; Rinaldi, R.; Verbeet, M. Ph.; de Waal, E.; Canters, G. W.

2004-12-01

We have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely atomic force microscopy, intrinsic fluorescence spectroscopy, and scanning tunneling microscopy. We demonstrate that a "solid state protein film" maintains its nativelike conformation and ET function, even after removal of the aqueous solvent.

Expanding the biological periodic table.

Science.gov (United States)

Seravalli, Javier; Ragsdale, Stephen W

2010-08-27

Metal ions play an indispensable role in biology, enabling enzymes to perform their functions and lending support to the structures of numerous macromolecules. Despite their prevalence and importance, the metalloproteome is still relatively unexplored. Cvetkovic et al. (2010) now describe an approach to identify metalloproteins on a genome-wide scale. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

METALLOPROTEINS DURING DEVELOPMENT OF WALKER-256 CARCINOSARCOMA RESISTANT PHENOTYPE.

Science.gov (United States)

Chekhun, V F; Lozovska, Yu V; Burlaka, A P; Ganusevich, I I; Shvets, Yu V; Lukianova, N Yu; Todor, I M; Demash, D V; Pavlova, A A; Naleskina, L A

2015-01-01

The study was focused on the detection of changes in serum and tumor metal-containing proteins in animals during development ofdoxorubicin-resistant phenotype in malignant cells after 12 courses of chemotherapy. We found that on every stage of resistance development there was a significant increase in content of ferritin and transferrin proteins (which take part in iron traffick and storage) in Walker-256 carc'inosarcoma tissue. We observed decreased serumferritin levels at the beginning stage of the resistance development and significant elevation of this protein levels in the cases withfully developed resistance phenotype. Transferrin content showed changes opposite to that offerritin. During the development of resistance phenotype the tumor tissue also exhibited increased 'free iron' concentration that putatively correlate with elevation of ROS generation and levels of MMP-2 and MMP-9 active forms. The tumor non-protein thiol content increases gradually as well. The serum of animals with early stages of resistance phenotype development showed high ceruloplasmin activity and its significant reduction after loss of tumor sensitivity to doxorubicin. Therefore, the development of resistance phenotype in Walker-256 carcinosarcoma is accompanied by both the deregulation of metal-containing proteins in serum and tumor tissue and by the changes in activity of antioxidant defense system. Thus, the results of this study allow us to determine the spectrum of metal-containing proteins that are involved in the development of resistant tumor phenotype and that may be targeted for methods for doxorubicin sensitivity correction therapy.

Metalloproteins during development of Walker-256 carcinosarcoma resistant phenotype

Directory of Open Access Journals (Sweden)

V. F. Chekhun

2015-04-01

Full Text Available The study was focused on the detection of changes in serum and tumor metal-containing proteins in animals during development of doxorubicin-resistant phenotype in malignant cells after 12 courses of chemotherapy. We found that on every stage of resistance development there was a significant increase in content of ferritin and transferrin proteins (which take part in iron traffick and storage in Walker-256 carcinosarcoma tissue. We observed decreased serum ferritin levels at the beginning stage of the resistance development and significant elevation of this protein levels in the cases with fully developed resistance phenotype. Transferrin content showed changes opposite to that of ferritin. During the development of resistance phenotype the tumor tissue also exhibited increased ‘free iron’ concentration that putatively correlate with elevation of ROS generation and levels of MMP-2 and MMP-9 active forms. The tumor non-protein thiol content increases gradually as well. The serum of animals with early stages of resistance phenotype development showed high ceruloplasmin activity and its significant reduction after loss of tumor sensitivity to doxorubicin. Therefore, the development of resistance phenotype in Walker-256 carcinosarcoma is accompanied by both the deregulation of metal-containing proteins in serum and tumor tissue and by the changes in activity of antioxidant defense system. Thus, the results of this study allow us to determine the spectrum of metal-containing proteins that are involved in the development of resistant tumor phenotype and that may be targeted for methods for doxorubicin sensitivity correction therapy.

Hydrolytic catalysis and structural stabilization in a designed metalloprotein

Science.gov (United States)

Zastrow, Melissa L.; Peacock, Anna F. A.; Stuckey, Jeanne A.; Pecoraro, Vincent L.

2011-01-01

Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions – a Zn(II) ion which is important for catalytic activity and a Hg(II) ion which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate hydrolysis (pNPA) to within ~100-fold of the efficiency of human carbonic anhydrase (CA)II and is at least 550-fold better than comparable synthetic complexes. Similarly, CO2 hydration occurs with an efficiency within ~500-fold of CAII. While histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO2 hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme uncovers necessary design features for future metalloenzymes containing one or more metals. PMID:22270627

Specific Labeling of Zinc Finger Proteins using Non-canonical Amino Acids and Copper-free Click Chemistry

Science.gov (United States)

Kim, Younghoon; Kim, Sung Hoon; Ferracane, Dean; Katzenellenbogen, John A.

2012-01-01

Zinc finger proteins (ZFPs) play a key role in transcriptional regulation and serve as invaluable tools for gene modification and genetic engineering. Development of efficient strategies for labeling metalloproteins such as ZFPs is essential for understanding and controlling biological processes. In this work, we engineered ZFPs containing cysteine-histidine (Cys2-His2) motifs by metabolic incorporation of the unnatural amino acid azidohomoalanine (AHA), followed by specific protein labeling via click chemistry. We show that cyclooctyne promoted [3 + 2] dipolar cycloaddition with azides, known as copper-free click chemistry, provides rapid and specific labeling of ZFPs at high yields as determined by mass spectrometry analysis. We observe that the DNA-binding activity of ZFPs labeled by conventional copper-mediated click chemistry was completely abolished, whereas ZFPs labeled by copper-free click chemistry retain their sequence-specific DNA-binding activity under native conditions, as determined by electrophoretic mobility shift assays, protein microarrays and kinetic binding assays based on Förster resonance energy transfer (FRET). Our work provides a general framework to label metalloproteins such as ZFPs by metabolic incorporation of unnatural amino acids followed by copper-free click chemistry. PMID:22871171

Speciation of protein-bound trace elements by gel electrophoresis and atomic spectrometry.

Science.gov (United States)

Ma, Renli; McLeod, Cameron W; Tomlinson, Kerry; Poole, Robert K

2004-08-01

The metabolism of trace elements, in particular their binding to proteins in biological systems is of great importance in biochemical, toxicological, and pharmacological studies. As a result there has been a sustained interest over the last two decades in the speciation of protein-bound metals. Various analytical approaches have been employed, combining efficient separation of metalloproteins by liquid chromatography or electrophoresis with high-sensitivity elemental detection. Slab-gel electrophoresis (GE) is a key platform for high-resolution protein separation, and has been combined with autoradiography and various atomic spectrometric techniques for in-gel determination of protein-bound metals. Recently, the combination of GE with state-of-the-art inductively coupled plasma-mass spectrometry (ICP-MS), particularly when linked to laser ablation (LA) for direct gel interrogation, has opened up new opportunities for rapid characterization of metalloproteins. The use of GE and atomic spectrometry for the speciation of protein-bound trace elements is reviewed in this paper. Technical requirements for gel electrophoresis/atomic spectrometric measurement are considered in terms of method compatibilities, detection capability and potential usefulness. The literature is also surveyed to illustrate current status and future trends. Copyright 2004 Wiley-VCH Verlag GmbH and Co.

Development of an X-ray fluorescence holographic measurement system for protein crystals

International Nuclear Information System (INIS)

Sato-Tomita, Ayana; Shibayama, Naoya; Okabe, Takahiro; Happo, Naohisa; Kimura, Koji; Matsushita, Tomohiro; Park, Sam-Yong; Sasaki, Yuji C.; Hayashi, Kouichi

2016-01-01

Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α_2β_2 tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm"3) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

Development of an X-ray fluorescence holographic measurement system for protein crystals

Energy Technology Data Exchange (ETDEWEB)

Sato-Tomita, Ayana, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp; Shibayama, Naoya, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp; Okabe, Takahiro [Division of Biophysics, Department of Physiology, Jichi Medical University, Yakushiji, Shimotsuke 329-0498 (Japan); Happo, Naohisa [Department of Computer and Network Engineering, Graduate School of Information Sciences, Hiroshima City University, Asa-Minami-Ku, Hiroshima 731-3194 (Japan); Kimura, Koji [Department of Physical Science and Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan); Matsushita, Tomohiro [Japan Synchrotron Radiation Research Institute (JASRI), SPring-8, Sayo, Hyogo 679-5198 (Japan); Park, Sam-Yong [Drug Design Laboratory, Department of Medical Life Science, Yokohama City University, Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Sasaki, Yuji C. [Department of Advanced Material Science, Graduate School of Frontier Science, The University of Tokyo, Kashiwanoha, Kashiwa 277-8561 (Japan); Hayashi, Kouichi, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp [Department of Physical Science and Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan); Frontier Research Institute for Materials Science, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan)

2016-06-15

Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α{sub 2}β{sub 2} tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm{sup 3}) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

Artificial Metalloproteins for Binding and Stabilization of a Semiquinone Radical

NARCIS (Netherlands)

Segaud, Nathalie; Drienovska, Ivana; Chen, Juan; Browne, Wesley R.; Roelfes, Gerard

2017-01-01

The interaction of a number of first-row transition-metal ions with a 2,2'-bipyridyl alanine (bpyA) unit incorporated into the lactococcal multidrug resistance regulator (LmrR) scaffold is reported. The composition of the active site is shown to influence binding affinities. In the case of Fe(II),

The ybeY protein from Escherichia coli is a metalloprotein

International Nuclear Information System (INIS)

Zhan, Chenyang; Fedorov, Elena V.; Shi, Wuxian; Ramagopal, U. A.; Thirumuruhan, R.; Manjasetty, Babu A.; Almo, Steve C.; Fiser, Andras; Chance, Mark R.; Fedorov, Alexander A.

2005-01-01

The ybeY protein from E. coli is reported at a 2.7 Å resolution with a metal ion. The three-dimensional crystallographic structure of the ybeY protein from Escherichia coli (SwissProt entry P77385) is reported at 2.7 Å resolution. YbeY is a hypothetical protein that belongs to the UPF0054 family. The structure reveals that the protein binds a metal ion in a tetrahedral geometry. Three coordination sites are provided by histidine residues, while the fourth might be a water molecule that is not seen in the diffraction map because of its relatively low resolution. X-ray fluorescence analysis of the purified protein suggests that the metal is a nickel ion. The structure of ybeY and its sequence similarity to a number of predicted metal-dependent hydrolases provides a functional assignment for this protein family. The figures and tables of this paper were prepared using semi-automated tools, termed the Autopublish server, developed by the New York Structural GenomiX Research Consortium, with the goal of facilitating the rapid publication of crystallographic structures that emanate from worldwide Structural Genomics efforts, including the NIH-funded Protein Structure Initiative

Radiation effects and metalloproteins studied by x-ray photoelectron spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Wurzbach, J.A.

1975-07-01

X-ray photoelectron spectroscopy (XPS) is used to study the bonding structure at the iron site of cytochrome c and the bonding of rare earth ions to the phosphate oxygens of ATP. Radiation effects are studied on several amino acid and simple peptide model systems. The emission spectrum of the x-ray source is calculated from literature references. The distributions of photon energy as a function of photon frequency and as a function of take-off angle are obtained. From these distributions, the radiation dose absorbed by an organic sample is found to be 10/sup 6/ rads/sec. The C 1s and N 1s spectra of amino acids and peptides are studied to characterize an internal reference standard for protein XPS spectra. Samples of native cytochrome c prepared from solutions of pH 1.5, 3, 7, and 11 are studied. Control samples include porphyrin cytochrome c (PCC), the metal free analogue of the native protein, and microperoxidase (MP), a mixture of heme peptides derived from the peptic digestion of cytochrome c. These samples show two S 2p peaks. The first peak has a binding energy (BE) of 163 eV, which corresponds to the S containing amino acids; the second peak is shifted to 167 eV. This large shift may be the result of Fe-S binding, or oxidation, or both. Low spin ferricytochrome c and ferri-MP were found to have Fe 3p BE's that are unusually low (51 eV) compared to other ferric compounds (54 to 58 eV) and even Fe metal (53 eV). X-ray crystal structures of these compounds show that low spin heme Fe lies in the porphyrin plane; while, high spin heme Fe is displaced above the plane. The N 1s and P 2p spectra of ATP show no change except slight broadening when Nd/sup 3 +/ is substituted for Na/sup +/. Thus, there is no inconsistency with proposals that rare earth ions might be useful as substitutes for alkali metal ions and alkaline earth ions in proteins.

Radiation effects and metalloproteins studied by x-ray photoelectron spectroscopy

International Nuclear Information System (INIS)

Wurzbach, J.A.

1975-07-01

X-ray photoelectron spectroscopy (XPS) is used to study the bonding structure at the iron site of cytochrome c and the bonding of rare earth ions to the phosphate oxygens of ATP. Radiation effects are studied on several amino acid and simple peptide model systems. The emission spectrum of the x-ray source is calculated from literature references. The distributions of photon energy as a function of photon frequency and as a function of take-off angle are obtained. From these distributions, the radiation dose absorbed by an organic sample is found to be 10 6 rads/sec. The C 1s and N 1s spectra of amino acids and peptides are studied to characterize an internal reference standard for protein XPS spectra. Samples of native cytochrome c prepared from solutions of pH 1.5, 3, 7, and 11 are studied. Control samples include porphyrin cytochrome c (PCC), the metal free analogue of the native protein, and microperoxidase (MP), a mixture of heme peptides derived from the peptic digestion of cytochrome c. These samples show two S 2p peaks. The first peak has a binding energy (BE) of 163 eV, which corresponds to the S containing amino acids; the second peak is shifted to 167 eV. This large shift may be the result of Fe-S binding, or oxidation, or both. Low spin ferricytochrome c and ferri-MP were found to have Fe 3p BE's that are unusually low (51 eV) compared to other ferric compounds (54 to 58 eV) and even Fe metal (53 eV). X-ray crystal structures of these compounds show that low spin heme Fe lies in the porphyrin plane; while, high spin heme Fe is displaced above the plane. The N 1s and P 2p spectra of ATP show no change except slight broadening when Nd 3+ is substituted for Na + . Thus, there is no inconsistency with proposals that rare earth ions might be useful as substitutes for alkali metal ions and alkaline earth ions in proteins

Lack of radiation protective effect of orgotein in normal and malignant mammalian cells

International Nuclear Information System (INIS)

Overgaard, J.; Nielsen, O.S.; Overgaard, M.; Steenholdt, S.; Jakobsen, A.; Sell, A.

1979-01-01

The potential radiation protective effect of orgotein, a metalloprotein with superoxide dismutase activity, was investigated in L 1 A 2 tumour cells in vitro, jejunal crypt cells and C 3 H mouse mammary carcinoma in vivo. No effect of orgotein, given either 2 hours before irradiation or 30 min after, was observed compared to the effect of irradiation alone. Thus, it was concluded that orgotein did not influence the primary radiation response in air in mammalian cells. (Auth.)

The Effects of Nitroxyl (HNO) on H2O2 Metabolism and Possible Mechanisms of HNO Signaling

OpenAIRE

Jackson, Matthew I.; Fields, Hannah F.; Lujan, Timothy S.; Cantrell, Megan M.; Lin, Joseph; Fukuto, Jon M.

2013-01-01

Nitroxyl (HNO) possesses unique and potentially important biological/physiological activity that is currently mechanistically ill-defined. Previous work has shown that the likely biological targets for HNO are thiol proteins, oxidized metalloproteins (i.e. ferric heme proteins) and, most likely, selenoproteins. Interestingly, these are the same classes of proteins that interact with H2O2. In fact, these classes of proteins not only react with H2O2, and thus potentially responsible for the sig...

LARGE SCALE PRODUCTION, PURIFICATION, AND 65CU SOLID STATE NMR OF AZURIN

Energy Technology Data Exchange (ETDEWEB)

Gao, A.; Heck, R.W.

2008-01-01

This paper details a way to produce azurin with an effi ciency over 10 times greater than previously described and demonstrates the fi rst solid state nuclear magnetic resonance spectrum of 65Cu(I) in a metalloprotein. A synthetic gene for azurin based upon the DNA sequence from Pseudomonas aeruginosa including the periplasmic targeting sequence was subcloned into a T7 overexpression vector to create the plasmid pGS-azurin, which was transformed into BL21 (DE3) competent cells. The leader sequence on the expressed protein causes it to be exported to the periplasmic space of Escherichia coli. Bacteria grown in a fermentation unit were induced to overexpress the azurin, which was subsequently purifi ed through an endosmotic shock procedure followed by high performance liquid chromatography (HPLC). 1,500 mg of azurin were purifi ed per liter of culture. 65Cu(II) was added to apo-azurin and then reduced. The 65Cu metal cofactor in azurin was observed with solid state nuclear magnetic resonance (NMR) to determine any structural variations that accompanied copper reduction. This is the fi rst solid state NMR spectra of a copper(I) metalloprotein. Analysis of the NMR spectra is being used to complement hypotheses set forth by x-ray diffraction and computational calculations of electron transfer mechanisms in azurin.

Sulfur analysis by inductively coupled plasma-mass spectrometry: A review

Energy Technology Data Exchange (ETDEWEB)

Giner Martínez-Sierra, J.; Galilea San Blas, O.; Marchante Gayón, J.M.; García Alonso, J.I., E-mail: jiga@uniovi.es

2015-06-01

In recent years the number of applications of sulfur (S) analysis using inductively coupled plasma mass spectrometry (ICP-MS) as detector has increased significantly. In this article we describe in some depth the application of ICP-MS for S analysis with emphasis placed on the sulfur-specific detection by hyphenated techniques such as LC, GC, CE and LA coupled on-line to ICP-MS. The different approaches available for sulfur isotope ratio measurements by ICP-MS are also detailed. Particular attention has been paid to the quantification of peptides/proteins and the analysis of metallopeptides/metalloproteins via sulfur by LC–ICP-MS. Likewise, the speciation analysis of metal-based pharmaceuticals and metallodrugs and non-metal selective detection of pharmaceuticals via S are highlighted. Labeling procedures for metabolic applications are also included. Finally, the measurement of natural variations in S isotope composition with multicollector ICP-MS instruments is also covered in this review. - Highlights: • Emphasis placed on the sulfur-specific detection by chromatographic techniques coupled on-line to ICP-MS. • Different instrumental approaches available for sulfur measurements by ICP-MS. • Quantification of proteins and the analysis of metalloproteins via sulfur by LC-ICP-MS. • Labelling procedures for metabolic applications are also included. • The measurement of natural variations in S isotope composition with multicollector ICP-MS.

MtZIP6 is a novel metal transporter required for symbiotic nitrogen fixation in nodules of Medicago truncatula plants

OpenAIRE

Saez Somolinos, Ángela; Imperial Ródenas, Juan; Gonzalez Guerrero, Manuel

2015-01-01

Symbiotic nitrogen fixation (SNF) carried out by the interaction rhizobia-legumes takes place in legume root nodules. Many of the enzymes involved in SNF are metalloproteins that obtain their metal cofactor from the host plant. Metals reach the nodule through the vasculature, where they are released in the apoplast on the infection/differentiation zone (zone II) of the nodule (Rodriguez-Haas et al., 2013). From there, these oligonutrients have to cross a number of membranes to be used for met...

Metal-molecular assembly for functional materials

CERN Document Server

Matsuo, Yutaka; Negishi, Yuichi; Yoshizawa, Michito; Uemura, Takashi; Takaya, Hikaru; Takeuchi, Masayuki; Yoshimoto, Soichiro

2013-01-01

This book focuses on modern coordination chemistry, covering porous coordination polymers, metalloproteins, metallopeptides, nanoclusters, nanocapsules, aligned polymers, and fullerenes. As well, it deals with applications to electronic devices and surface characterization. These wide-ranging topics are integrally described from the perspectives of dimensionality (one-, two-, and three-dimension), new materials design, synthesis, molecular assembly, function and application. The nine chapters making up this book have been authored by scientists who are at the cutting edge of research in this p

Lack of radiation protective effect of orgotein in normal and malignant mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Overgaard, J; Nielsen, O S; Overgaard, M; Steenholdt, S; Jakobsen, A; Sell, A [Institute of Cancer Research and The Department of Radiation Therapy and Oncology, The Radium Centre, Aarhus, Denmark

1979-01-01

The potential radiation protective effect of orgotein, a metalloprotein with superoxide dismutase activity, was investigated in L/sub 1/A/sub 2/ tumour cells in vitro, jejunal crypt cells and C/sub 3/H mouse mammary carcinoma in vivo. No effect of orgotein, given either 2 hours before irradiation or 30 min after, was observed compared to the effect of irradiation alone. Thus, it was concluded that orgotein did not influence the primary radiation response in air in mammalian cells.

A synthetic redox biofilm made from metalloprotein-prion domain chimera nanowires

Science.gov (United States)

Altamura, Lucie; Horvath, Christophe; Rengaraj, Saravanan; Rongier, Anaëlle; Elouarzaki, Kamal; Gondran, Chantal; Maçon, Anthony L. B.; Vendrely, Charlotte; Bouchiat, Vincent; Fontecave, Marc; Mariolle, Denis; Rannou, Patrice; Le Goff, Alan; Duraffourg, Nicolas; Holzinger, Michael; Forge, Vincent

2017-02-01

Engineering bioelectronic components and set-ups that mimic natural systems is extremely challenging. Here we report the design of a protein-only redox film inspired by the architecture of bacterial electroactive biofilms. The nanowire scaffold is formed using a chimeric protein that results from the attachment of a prion domain to a rubredoxin (Rd) that acts as an electron carrier. The prion domain self-assembles into stable fibres and provides a suitable arrangement of redox metal centres in Rd to permit electron transport. This results in highly organized films, able to transport electrons over several micrometres through a network of bionanowires. We demonstrate that our bionanowires can be used as electron-transfer mediators to build a bioelectrode for the electrocatalytic oxygen reduction by laccase. This approach opens opportunities for the engineering of protein-only electron mediators (with tunable redox potentials and optimized interactions with enzymes) and applications in the field of protein-only bioelectrodes.

Teoretická bioanorganická chemie a spektroskopie

Czech Academy of Sciences Publication Activity Database

Bím, Daniel; Gutten, Ondrej; Chalupský, Jakub; Srnec, Martin; Rulíšek, Lubomír

2016-01-01

Roč. 110, č. 5 (2016), s. 354-364 ISSN 0009-2770 R&D Projects: GA ČR(CZ) GA14-31419S; GA ČR GA15-19143S; GA ČR(CZ) GJ15-10279Y Institutional support: RVO:61388963 ; RVO:61388955 Keywords : theoretical bioinorganic chemistry * transition metal ion containing systems * quantum chemistry methods * reaction mechanism * metalloproteins * metal ions binding selectivity Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 0.387, year: 2016 http://www.chemicke-listy.cz/docs/full/2016_05_354-364.pdf

Radiation chemistry

International Nuclear Information System (INIS)

Swallow, A.J.

1983-01-01

The subject is covered in chapters, entitled: introduction (defines scope of article as dealing with the chemistry of reactive species, (e.g. excess electrons, excited states, free radicals and inorganic ions in unusual valency states) as studied using radiation with radiation chemistry in its traditional sense and with biological and industrial applications); gases; water and simple inorganic systems; aqueous metallo-organic compounds and metalloproteins; small organic molecules in aqueous solution; microheterogeneous systems; non-aqueous liquids and solutions; solids; biological macromolecules; synthetic polymers. (U.K.)

Electron tunnelling through single azurin molecules can be on/off switched by voltage pulses

Energy Technology Data Exchange (ETDEWEB)

Baldacchini, Chiara [Biophysics and Nanoscience Centre, DEB-CNISM, Università della Tuscia, I-01100 Viterbo (Italy); Institute of Agro-Environmental and Forest Biology, CNR, I-05010 Porano (Italy); Kumar, Vivek; Bizzarri, Anna Rita; Cannistraro, Salvatore, E-mail: cannistr@unitus.it [Biophysics and Nanoscience Centre, DEB-CNISM, Università della Tuscia, I-01100 Viterbo (Italy)

2015-05-04

Redox metalloproteins are emerging as promising candidates for future bio-optoelectronic and nano-biomemory devices, and the control of their electron transfer properties through external signals is still a crucial task. Here, we show that a reversible on/off switching of the electron current tunnelling through a single protein can be achieved in azurin protein molecules adsorbed on gold surfaces, by applying appropriate voltage pulses through a scanning tunnelling microscope tip. The observed changes in the hybrid system tunnelling properties are discussed in terms of long-sustained charging of the protein milieu.

The Rossendorf Beamline at ESRF (ROBL-CRG). Bi-annual report 2003/04

Energy Technology Data Exchange (ETDEWEB)

Scheinost, A.C.; Schell, N. (eds.)

2005-01-01

In this report the work performed at the Rossendorf beam-line at the ESRF is described. It concerns neptunium (IV) uptake by iron metalloproteins, in-situ speciation of actinides using a newly developed spectro-electrochemical cell, quantitative antimony speciation in Swiss shooting-range soils, in-situ studies of ITO film properties and structure during annealing in vacuum, high-temperature investigations of Si/SiGe based quantum cascade structures using X-ray diffraction and reflectivity, and in-situ characterization of stress states in copper dual inlaid interconnects at high temperatures by synchrotron X-ray diffraction. (HSI)

Electron transfer and redox metalloenzyme catalysis at the single-molecule level

DEFF Research Database (Denmark)

Hansen, Allan Glargaard; Zhang, Jingdong; Christensen, Hans Erik Mølager

2004-01-01

transfer (ET). Image interpretation requires, however, theoretical support, as STM represents both electronic and topographic features. Molecules with accessible redox levels offer other insight into electron tunneling mechanisms, addressed in detail for ET metalloproteins. We present here in situ STM...... of the blue redox metalloenzyme copper nitrite reductase (Achromobacter xylosoxidans, AxCuNiR) on Au(111) electrode surfaces modified by a self-assembled cysteamine monolayer. AxCuNiR displays strong nitrite reduction waves in this environment. AxCuNiR/cysteamine/ Au(111) surfaces were imaged at KNO2...

Metals in proteins: correlation between the metal-ion type, coordination number and the amino-acid residues involved in the coordination.

Science.gov (United States)

Dokmanić, Ivan; Sikić, Mile; Tomić, Sanja

2008-03-01

Metal ions are constituents of many metalloproteins, in which they have either catalytic (metalloenzymes) or structural functions. In this work, the characteristics of various metals were studied (Cu, Zn, Mg, Mn, Fe, Co, Ni, Cd and Ca in proteins with known crystal structure) as well as the specificity of their environments. The analysis was performed on two data sets: the set of protein structures in the Protein Data Bank (PDB) determined with resolution metal ion and its electron donors and the latter was used to assess the preferred coordination numbers and common combinations of amino-acid residues in the neighbourhood of each metal. Although the metal ions considered predominantly had a valence of two, their preferred coordination number and the type of amino-acid residues that participate in the coordination differed significantly from one metal ion to the next. This study concentrates on finding the specificities of a metal-ion environment, namely the distribution of coordination numbers and the amino-acid residue types that frequently take part in coordination. Furthermore, the correlation between the coordination number and the occurrence of certain amino-acid residues (quartets and triplets) in a metal-ion coordination sphere was analysed. The results obtained are of particular value for the identification and modelling of metal-binding sites in protein structures derived by homology modelling. Knowledge of the geometry and characteristics of the metal-binding sites in metalloproteins of known function can help to more closely determine the biological activity of proteins of unknown function and to aid in design of proteins with specific affinity for certain metals.

Metallothioneins are multipurpose neuroprotectants during brain pathology

DEFF Research Database (Denmark)

Penkowa, Milena

2006-01-01

Metallothioneins (MTs) constitute a family of cysteine-rich metalloproteins involved in cytoprotection during pathology. In mammals there are four isoforms (MT-I - IV), of which MT-I and -II (MT-I + II) are the best characterized MT proteins in the brain. Accumulating studies have demonstrated MT......-I overexpression demonstrated the importance of MT-I + II for coping with brain pathology. In addition, exogenous MT-I or MT-II injected intraperitoneally is able to promote similar effects as those of endogenous MT-I + II, which indicates that MT-I + II have both extra- and intracellular actions. In injured brain...

Engineered Proteins: Redox Properties and Their Applications

Science.gov (United States)

Prabhulkar, Shradha; Tian, Hui; Wang, Xiaotang; Zhu, Jun-Jie

2012-01-01

Abstract Oxidoreductases and metalloproteins, representing more than one third of all known proteins, serve as significant catalysts for numerous biological processes that involve electron transfers such as photosynthesis, respiration, metabolism, and molecular signaling. The functional properties of the oxidoreductases/metalloproteins are determined by the nature of their redox centers. Protein engineering is a powerful approach that is used to incorporate biological and abiological redox cofactors as well as novel enzymes and redox proteins with predictable structures and desirable functions for important biological and chemical applications. The methods of protein engineering, mainly rational design, directed evolution, protein surface modifications, and domain shuffling, have allowed the creation and study of a number of redox proteins. This review presents a selection of engineered redox proteins achieved through these methods, resulting in a manipulation in redox potentials, an increase in electron-transfer efficiency, and an expansion of native proteins by de novo design. Such engineered/modified redox proteins with desired properties have led to a broad spectrum of practical applications, ranging from biosensors, biofuel cells, to pharmaceuticals and hybrid catalysis. Glucose biosensors are one of the most successful products in enzyme electrochemistry, with reconstituted glucose oxidase achieving effective electrical communication with the sensor electrode; direct electron-transfer-type biofuel cells are developed to avoid thermodynamic loss and mediator leakage; and fusion proteins of P450s and redox partners make the biocatalytic generation of drug metabolites possible. In summary, this review includes the properties and applications of the engineered redox proteins as well as their significance and great potential in the exploration of bioelectrochemical sensing devices. Antioxid. Redox Signal. 17, 1796–1822. PMID:22435347

Determination of trace elements and screening of metalloproteins in human blood and tissues

International Nuclear Information System (INIS)

Prohaska, K.

2003-02-01

Sequential and simultaneous atomic spectrometric detection was applied for the determination of metals in human whole blood, blood fractions and joint tissues. For this purpose ICP-OES (inductively coupled plasma - optical emission spectrometry) and GFAAS (graphite furnace - atomic absorption spectrometry) were optimized. The nebulizing technique of the ICP-OES causes a high consumption of the sample (2.4 mL /min uptake rate). Using the simultaneous modus of measurement it is possible to determine up to 20 elements in the samples of interest. Using GFAAS the elements can be detected sequentially only, the method is more time consuming in comparison. For the direct sample injection into the graphite tube only 20 aeL are needed. Since 1 mL sample has to be filled in the sample vessel of the autosampler, up to six elements can be determined in this volume. The most important advantage of the simultaneous multi-element detection is the low sample consumption, which is essential for analysis of biological samples. Normally only small amounts of human blood or tissues can be collected, and the concentrations of analytes are usually very low. Therefore in many cases a sample preparation is of advantage, which enables a pre-concentration of the analyte. The sample digestion was optimized with respect to the possible pre-concentration of the analytes. Ashing of the freeze-dried blood enabled a six fold higher concentration in the measuring solution compared with wet digestion of blood. For the ICP-OES sample introduction system two different nebulizers were tested in the complex matrix of the digested blood samples. A Meinhard and the microconcentric nebulizer were compared according to their analytical performance. The matrix of the samples caused LODs three to five times higher than in the aqueous standards. The application of the Meinhard nebulizer enabled sufficiently low LODs in the matrix for some elements of interest (Ca, Cu, Fe, Mg, P, S, Zn in freeze-dried blood). For the very low concentrations of trace element in blood fractions (e.g. Co, Cr, or Ni) the GFAAS had to be used. The simultaneous ICP-OES was applied for the determination of Ca, Mg, P, Fe, Cu, and Zn in whole blood. Line selection was performed in the complex sample matrix. In digested freeze-dried blood the simultaneous lines showed no interferences for Ca, Cu, Fe, Mg, P, S, and Zn. Only for Co serious interference occurred in the matrix. The method was not suitable for the determination of Cd, Cr, Mn, Mo, Nb, Ni, Pb, Ti, and V in the blood samples, because the LODs of the ICP-OES method were too high for this purpose. (author)

'DRF-G - Grenoble Department of Fundamental Research. Activity report 1985, Nr 20. Volume II: 'Chemical Physics' 'Biology'

International Nuclear Information System (INIS)

1983-01-01

This volume contains synthetic reports of researches performed in chemistry, in the field of biological and medical applications of nuclear magnetic resonance, and in biology during the 1981-1983 period or only during 1983. As far as chemistry is concerned, the following topics have been addressed: conducting organic polymers, organic and analytic electrochemistry, coordination chemistry, molecular dynamics, vegetal macromolecules, nucleic acids. As far as biology is concerned, the following topics have been addressed: systems associated with membranes, metalloproteins, cell biology and differentiation, immuno-chemistry, haematology, vegetal physiology, structural studies of proteins. Staff lists of researchers are provided for chemistry laboratories and biology laboratories, as well a list of publications

Biological X-ray absorption spectroscopy (BioXAS): a valuable tool for the study of trace elements in the life sciences.

Science.gov (United States)

Strange, Richard W; Feiters, Martin C

2008-10-01

Using X-ray absorption spectroscopy (XAS) the binding modes (type and number of ligands, distances and geometry) and oxidation states of metals and other trace elements in crystalline as well as non-crystalline samples can be revealed. The method may be applied to biological systems as a 'stand-alone' technique, but it is particularly powerful when used alongside other X-ray and spectroscopic techniques and computational approaches. In this review, we highlight how biological XAS is being used in concert with crystallography, spectroscopy and computational chemistry to study metalloproteins in crystals, and report recent applications on relatively rare trace elements utilised by living organisms and metals involved in neurodegenerative diseases.

Nanoscale and single-molecule interfacial electron transfer

DEFF Research Database (Denmark)

Hansen, Allan Glargaard; Wackerbarth, Hainer; Nielsen, Jens Ulrik

2003-01-01

for comprehensive later theoretical work and data interpretation in many areas of chemistry, electrochemistry, and biology. We discuss here some new areas of theoretical electrochemical ET science, with focus on nanoscale electrochemical and bioelectrochemical sciences. Particular attention is given to in situ...... scanning tunneling microscopy (STM) and single-electron tunneling (SET, or Coulomb blockade) in electrochemical. systems directly in aqueous electrolyte solution and at room temperature. We illustrate the new theoretical formalism and its perspectives by recent cases of electrochemical SET, negative...... differential resistance patterns, and by ET dynamics of organized assemblies of biological macromolecules, such as redox metalloproteins and oligonucleotides on single-crystal Au(III)-electrode surfaces....

EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

Energy Technology Data Exchange (ETDEWEB)

Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard [Laboratoire de Chimie Nucleaire Analytique et Bioenvironnementale, CNRS UMR5084, Universite Bordeaux 1, Chemin du Solarium, F-33175 Gradignan cedex (France); Solari, Pier Lorenzo [Synchrotron SOLEIL, L' Orme des Merisiers, BP 48, F-91192 Gif-sur-Yvette cedex, Saint-Aubin (France); Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis, E-mail: ortega@cenbg.in2p3.f [FAME, ESRF, 6 rue Jules Horowitz, BP220, F-38043 Grenoble cedex (France)

2009-11-15

Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

Understanding how cells allocate metals using metal sensors and metallochaperones.

Science.gov (United States)

Tottey, Stephen; Harvie, Duncan R; Robinson, Nigel J

2005-10-01

Each metalloprotein must somehow acquire the correct metal. We review the insights into metal specificity in cells provided by studies of ArsR-SmtB DNA binding, metal-responsive transcriptional repressors, and a bacterial copper chaperone. Cyanobacteria are the one bacterial group that have known enzymatic demand for cytoplasmic copper import. The copper chaperone and ATPases that supply cyanobacterial plastocyanin and cytochrome oxidase are reviewed, along with related ATPases for cobalt and zinc. These studies highlight the contributions of protein-protein interactions to metal speciation. Metal sensors and metallochaperones, along with metal transporters and metal-storage proteins, act in concert not only to supply the correct metals but also to withhold the wrong ones.

EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

Science.gov (United States)

Chevreux, Sylviane; Solari, Pier Lorenzo; Roudeau, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

2009-11-01

Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

International Nuclear Information System (INIS)

Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard; Solari, Pier Lorenzo; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis

2009-01-01

Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

Characterization of Metalloproteins and Biomaterials by X-ray Absorption Spectroscopy and X-ray Diffraction

DEFF Research Database (Denmark)

Frankær, Christian Grundahl

This thesis presents thework on combining complementary X-rays techniques for studying the structures of proteins and other biomaterials, and consists of three different projects: (i) Characterization of protein powders with X-ray powder diffraction (XRPD). (ii) The combination of X-ray...... crystallography and X-ray absorption spectroscopy (XAS) applied to studying different hexameric insulin conformations. (iii) The structures of polymorphs of strontium ranelate and the distribution of strontium in bone tissue. A procedure for fast identification and verification of protein powders using XRPD...... was correction for disordered bulk-solvent, but also correction for background and optimization of unit cell parameters have to be taken into account. A sample holder was designed for collecting powder diffraction data on a standard laboratory X-ray powder diffractometer. The background was reduced by use...

Evaluation of iron and selenium losses in metalloproteins separated by gel electrophoresis by ICPMS

International Nuclear Information System (INIS)

Gherghel, I.; Fernandez, M.L.; Fernandez, B.; Pereiro, R.; Sanz-Medel, A.

2009-01-01

Full text: Metallomics addresses the study of metabolism, transport, and metal-protein interactions aiming to obtain relevant information of physiological and pathological alterations in living organisms. Gel electrophoresis is widely employed in proteomics and its use is actually extending to metallomics. Unfortunately, analysis of proteins by molecular techniques does not offer quantitative information. So, a good alternative could be their determination via the quantification of (semi)metal bound to the protein by ICPMS. In this work, we will show a detailed study of possible losses of protein and/or metal in Fe-bound and selenium proteins (transferrin and glutathione peroxidase, respectively) to evaluate the behaviour of the protein-metal interactions during the electrophoresis process. (author)

Broad-temperature range spectroscopy of the two-centre modular redox metalloprotein Desulfovibrio desulfuricans desulfoferrodoxin

DEFF Research Database (Denmark)

Andersen, Niels Højmark; Harnung, S.E.; Trabjerg, I.

2003-01-01

/VIS, MCD, CD, and EPR spectroscopy. The UV/VIS spectra of grey DFx at room temperature is characterised by broad charge transfer (CT) transitions associated with oxidised centre 1 (495 and 368 nm) and II (335 and 635 nm). The transitions are resolved at 78 K, substantiated by VT-MCD and -CD. The data offer...

Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

Science.gov (United States)

Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

2017-08-04

Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterizing millisecond intermediates in hemoproteins using rapid-freeze-quench resonance Raman spectroscopy.

Science.gov (United States)

Matsumura, Hirotoshi; Moënne-Loccoz, Pierre

2014-01-01

The combination of rapid freeze quenching (RFQ) with resonance Raman (RR) spectroscopy represents a unique tool with which to investigate the nature of short-lived intermediates formed during the enzymatic reactions of metalloproteins. Commercially available equipment allows trapping of intermediates within a millisecond to second time scale for low-temperature RR analysis resulting in the direct detection of metal-ligand vibrations and porphyrin skeletal vibrations in hemoproteins. This chapter briefly discusses RFQ-RR studies carried out previously in our laboratory and presents, as a practical example, protocols for the preparation of RFQ samples of the reaction of metmyoglobin with nitric oxide (NO) under anaerobic conditions. Also described are important controls and practical procedures for the analysis of these samples by low-temperature RR spectroscopy.

Two-point anchoring of a lanthanide-binding peptide to a target protein enhances the paramagnetic anisotropic effect

International Nuclear Information System (INIS)

Saio, Tomohide; Ogura, Kenji; Yokochi, Masashi; Kobashigawa, Yoshihiro; Inagaki, Fuyuhiko

2009-01-01

Paramagnetic lanthanide ions fixed in a protein frame induce several paramagnetic effects such as pseudo-contact shifts and residual dipolar couplings. These effects provide long-range distance and angular information for proteins and, therefore, are valuable in protein structural analysis. However, until recently this approach had been restricted to metal-binding proteins, but now it has become applicable to non-metalloproteins through the use of a lanthanide-binding tag. Here we report a lanthanide-binding peptide tag anchored via two points to the target proteins. Compared to conventional single-point attached tags, the two-point linked tag provides two to threefold stronger anisotropic effects. Though there is slight residual mobility of the lanthanide-binding tag, the present tag provides a higher anisotropic paramagnetic effect

Electron transfer behaviour of biological macromolecules towards the single-molecule level

DEFF Research Database (Denmark)

Zhang, Jingdong; Grubb, Mikala; Hansen, Allan Glargaard

2003-01-01

is combined with state-of-the-art physical electrochemistry with emphasis on single-crystal, atomically planar electrode surfaces, in situ scanning tunnelling microscopy (STM) and other surface techniques. These approaches have brought bioelectrochemistry important steps forward towards the nanoscale...... and single-molecule levels.We discuss here these advances with reference to two specific redox metalloproteins, the blue single-copper protein Pseudomonas aeruginosa azurin and the single-haem protein Saccharomyces cerevisiae yeast cytochrome c, and a short oligonucleotide. Both proteins can be immobilized...... electron transfer (ET) function retained. In situ STM can also address the microscopic mechanisms for electron tunnelling through the biomolecules and offers novel notions such as coherent multi-ET between the substrate and tip via the molecular redox levels. This differs in important respects from...

Development of High Resolution X-Ray spectrometers for the Investigation of Bioinorganic Chemistry in Metalloproteins

Energy Technology Data Exchange (ETDEWEB)

Drury, Owen Byron [Univ. of California, Davis, CA (United States)

2007-01-01

Metals play as varied a role in biology as the proteins they are part of. They are involved in structure formation, they help transfer material and information, and they catalyze chemical reactions. Other proteins transport material or information, such as hemoglobin that distributes O₂ and takes up CO₂, or insulin that signals cells to increase glucose uptake in response to high blood glucose levels. Again other proteins promote chemical reactions, such as photosystem II responsible for photosynthetic oxygen evolution or nitrogenase which catalyzes the reduction of N₂ to NH₃. All of these proteins require the presence of a metal ion for their activity.

Valence-to-core-detected X-ray absorption spectroscopy

DEFF Research Database (Denmark)

Hall, Eleanor R.; Pollock, Christopher J.; Bendix, Jesper

2014-01-01

X-ray absorption spectroscopy (XAS) can provide detailed insight into the electronic and geometric structures of transition-metal active sites in metalloproteins and chemical catalysts. However, standard XAS spectra inherently represent an average contribution from the entire coordination...... environment with limited ligand selectivity. To address this limitation, we have investigated the enhancement of XAS features using valence-to-core (VtC)-detected XAS, whereby XAS spectra are measured by monitoring fluorescence from valence-to-core X-ray emission (VtC XES) events. VtC emission corresponds...... to transitions from filled ligand orbitals to the metal 1s core hole, with distinct energetic shifts for ligands of differing ionization potentials. VtC-detected XAS data were obtained from multiple valence emission features for a series of well-characterized Mn model compounds; taken together, these data...

The role of vitamin D in asthma.

Science.gov (United States)

Luong, Khanh vinh quoc; Nguyen, Lan Thi Hoàng

2012-04-01

Vitamin D metabolites are important immune-modulatory hormones and are able to suppress Th2-mediated allergic airway disease. Some genetic factors that may contribute to asthma are regulated by vitamin D, such as vitamin D receptor (VDR), human leukocyte antigen genes (HLA), human Toll-like receptors (TLR), matrix metalloproteinases (MMPs), a disintegrin and metalloprotein-33 (ADAM-33), and poly(ADP-ribosyl) polymerase- 1 (PARP-1). Vitamin D has also been implicated in asthma through its effects on the obesity, bacillus Calmettee Guérin (BCG) vaccination and high vitamin D level, vitamin D supplement, checkpoint protein kinase 1 (Chk1), plasminogen activator inhibitor-1 (PAI-1) and gamma delta T cells (gdT). Vitamin D plays a role in asthma and exerts its action through either genomic and/or non-genomic ways.

Electrochemically controlled self-assembled monolayers characterized with molecular and sub-molecular resolution

DEFF Research Database (Denmark)

Zhang, Jingdong; Welinder, Anna Christina; Chi, Qijin

2011-01-01

Self-assembled organization of functional molecules on solid surfaces has developed into a powerful and sophisticated tool for surface chemistry and nanotechnology. A number of reviews on the topic have been available since the mid 1990s. This perspective article aims to focus on recent development...... structures and the experimental operating conditions. This is followed by discussion of two major high-resolution experimental methods, scanning tunnelling microscopy (STM) and single-crystal electrochemistry. In Section 3, we briefly address choice of supporting electrolytes and substrate surfaces......, we give examples of what can be offered by theoretical computations for the detailed understanding of the SAM electronic structures revealed by STM images. A brief summary of the current applications of SAMs in wiring metalloproteins, design and fabrication of sensors, and single-molecule electronics...

Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA

International Nuclear Information System (INIS)

Guzder, S.N.; Sung, P.; Prakash, S.; Prakash, L.

1993-01-01

Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA. Of the seven XP complementation groups, A--G, group A represents a severe and frequent form of the disease. The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene. Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA. The authors have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14. As determined by atomic emission spectroscopy, RAD14 contains one zinc atom. They also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions. In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA. Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites. These findings indicate that RAD14 functions in damage recognition during excision repair. 37 refs., 4 figs

role of some transition metals and metalloproteins on oxidative stress formation among ionizing radiation exposed workers

International Nuclear Information System (INIS)

Michael, M.I.

2004-01-01

this study was established to evaluate the role of working in radiation field for different prolonged periods on some oxidant/antioxidant parameters and to estimate the role of other additional factors such as age, smoking and inflammation on the progress of oxidative stress on the chosen volunteers. one hundred and twenty six male volunteers working in the nuclear research center and hot laboratories center were assessed in the present study, they were arranged as 70 radiation exposed workers and 56 control individuals. the radiation exposed workers were rearranged into 50 non-smokers, non-hypertensive and non-diabetics; 10 individuals were smokers, non-hypertensive, non-diabetic and other 10 volunteers with increased erythrocyte sedimentation rate (esr), non-smokers, non-hypertensive and non-diabetics

Electrochemistry of metalloproteins: protein film electrochemistry for the study of E. coli [NiFe]-hydrogenase-1.

Science.gov (United States)

Evans, Rhiannon M; Armstrong, Fraser A

2014-01-01

Protein film electrochemistry is a technique which allows the direct control of redox-active enzymes, providing particularly detailed information on their catalytic properties. The enzyme is deposited onto a working electrode tip, and through control of the applied potential the enzyme activity is monitored as electrical current, allowing for direct study of inherent activity as electrons are transferred to and from the enzyme redox center(s). No mediators are used. Because the only enzyme present in the experiment is bound at the electrode surface, gaseous and liquid phase inhibitors can be introduced and removed whilst the enzyme remains in situ. Potential control means that kinetics and thermodynamics are explored simultaneously; the kinetics of a reaction can be studied as a function of potential. Steady-state catalytic rates are observed directly as current (for a given potential) and non-steady-state rates (such as interconversions between different forms of the enzyme) are observed from the change in current with time. The more active the enzyme, the higher the current and the better the signal-to-noise. In this chapter we outline the practical aspects of PFE for studying electroactive enzymes, using the Escherichia coli [NiFe]-hydrogenase 1 (Hyd-1) as an example.

Biomolecular Structure Information from High-Speed Quantum Mechanical Electronic Spectra Calculation.

Science.gov (United States)

Seibert, Jakob; Bannwarth, Christoph; Grimme, Stefan

2017-08-30

A fully quantum mechanical (QM) treatment to calculate electronic absorption (UV-vis) and circular dichroism (CD) spectra of typical biomolecules with thousands of atoms is presented. With our highly efficient sTDA-xTB method, spectra averaged along structures from molecular dynamics (MD) simulations can be computed in a reasonable time frame on standard desktop computers. This way, nonequilibrium structure and conformational, as well as purely quantum mechanical effects like charge-transfer or exciton-coupling, are included. Different from other contemporary approaches, the entire system is treated quantum mechanically and neither fragmentation nor system-specific adjustment is necessary. Among the systems considered are a large DNA fragment, oligopeptides, and even entire proteins in an implicit solvent. We propose the method in tandem with experimental spectroscopy or X-ray studies for the elucidation of complex (bio)molecular structures including metallo-proteins like myoglobin.

Development of force-detected THz-ESR measurement system and its application to metal porphyrin complexes

Science.gov (United States)

Takahashi, Hideyuki; Okamoto, Tsubasa; Ohmichi, Eiji; Ohta, Hitoshi

Electron spin resonance spectroscopy in the terahertz region (THz-ESR) is a promising technique to study biological materials such as metalloproteins because it directly probes the metal ion sites that play an important role in the emergence of functionality. By combining THz-ESR with force detection, the samples mass is reduced to the order of ng. This feature is of great advantage because the sample preparation process of biological materials is time-consuming. We developed a force-detected THz-ESR system utilizing optical interferometry for precise cantilever displacement measurement. In order to suppress the sensitivity fluctuation and instability of cantilever dynamics under high magnetic field, the tuning of interferometer is feedback-controlled during a measurement. By using this system, we successfully observed the ESR signal of hemin, which is a model substance of hemoglobin and myoglobin, in THz region.

Cobalt-chromium-molybdenum alloy causes metal accumulation and metallothionein up-regulation in rat liver and kidney

DEFF Research Database (Denmark)

Jakobsen, Stig Storgaard; Danscher, Gorm; Stoltenberg, Meredin

2007-01-01

in liver and kidney. We found that metal ions are liberated from CoCrMo alloys and suggest that they are released by dissolucytosis, a process where macrophages causes the metallic surface to release metal ions. Animals with intramuscular implants accumulated metal in liver and kidney and metallohionein I....../II were elevated in liver tissue. The present data do not tell whether kidney and liver are the primary target organs or what possible toxicological effect the different metal ions might have, but they show that metal ions are liberated from CoCrMo alloys that are not subjected to mechanical wear...... and that they accumulate in liver and kidney tissue. That the liberated metal ions affect the tissues is supported by an up-regulation of the detoxifying/pacifying metalloprotein I/II in the liver. Udgivelsesdato: 2007-Dec...

Myoglobin-Catalyzed Olefination of Aldehydes.

Science.gov (United States)

Tyagi, Vikas; Fasan, Rudi

2016-02-12

The olefination of aldehydes constitutes a most valuable and widely adopted strategy for constructing carbon-carbon double bonds in organic chemistry. While various synthetic methods have been made available for this purpose, no biocatalysts are known to mediate this transformation. Reported herein is that engineered myoglobin variants can catalyze the olefination of aldehydes in the presence of α-diazoesters with high catalytic efficiency (up to 4,900 turnovers) and excellent E diastereoselectivity (92-99.9 % de). This transformation could be applied to the olefination of a variety of substituted benzaldehydes and heteroaromatic aldehydes, also in combination with different alkyl α-diazoacetate reagents. This work provides a first example of biocatalytic aldehyde olefination and extends the spectrum of synthetically valuable chemical transformations accessible using metalloprotein-based catalysts. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The interactions between CdTe quantum dots and proteins: understanding nano-bio interface

Directory of Open Access Journals (Sweden)

Shreeram S. Joglekar

2017-01-01

Full Text Available Despite remarkable developments in the nanoscience, relatively little is known about the physical (electrostatic interactions of nanoparticles with bio macromolecules. These interactions can influence the properties of both nanoparticles and the bio-macromolecules. Understanding this bio-interface is a prerequisite to utilize both nanoparticles and biomolecules for bioengineering. In this study, luminescent, water soluble CdTe quantum dots (QDs capped with mercaptopropionic acid (MPA were synthesized by organometallic method and then interaction between nanoparticles (QDs and three different types of proteins (BSA, Lysozyme and Hemoglobin were investigated by fluorescence spectroscopy at pH= 7.4. Based on fluorescence quenching results, Stern-Volmer quenching constant (Ksv, binding constant (Kq and binding sites (n for proteins were calculated. The results show that protein structure (e.g.,globular, metalloprotein, etc. has a significant role in Protein-Quantum dots interactions and each type of protein influence physicochemical properties of Quantum dots differently.

Site-directed mutagenesis and molecular modelling studies show the role of Asp82 and cysteines in rat acylase 1, a member of the M20 family

International Nuclear Information System (INIS)

Herga, Sameh; Brutus, Alexandre; Vitale, Rosa Maria; Miche, Helene; Perrier, Josette; Puigserver, Antoine; Scaloni, Andrea; Giardina, Thierry

2005-01-01

Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis experiments led to the identification of one functionally important amino acid residue located near one of the zinc coordinating residues, which play a critical role in the enzymatic activity. The D82N- and D82E-substituted forms showed no significant activity and very low activity, respectively, along with a loss of zinc coordination. Molecular modelling investigations indicated a putative role of D82 in ensuring a proper protonation of catalytic histidine. In addition, none of the five cysteine residues present in the rat kidney acylase 1 sequence seemed involved in the catalytic process: the loss of activity induced by the C294A substitution was probably due to a conformational change in the 3D structure

Effects of gamma-ray-induced free radicals on the metal content and amino acid composition of human metallothionein-1

International Nuclear Information System (INIS)

Goossens, Lieven

2011-01-01

Metallothioneins (MTs), a low-mass class of metalloproteins, are characterized by a high thiolate sulphur and metal content. MTs are involved in metal homeostasis and heavy metal detoxification, and are efficient scavengers of free radicals. This article describes zinc release from human MT-1 and modification of its amino acid composition when subjected to free radicals generated during gamma ray radiolysis. The effect of gamma ray radiolysis of untreated and metal-depleted human MT-1 was tested under multiple aerobic and anaerobic conditions at increasing irradiation doses. Under all conditions, a rapid increase of serine in the early stages of irradiation was observed. Irradiation for longer times led to cysteic acid formation, except under argon atmosphere. Several other amino acid concentrations gradually decreased. Formation of limited amounts of hydroxyproline, hydroxylysine and ornithine as well as some less common derivatives such as cystathionine occurred as side-effects. (author)

Biomolecular EPR spectroscopy

CERN Document Server

Hagen, Wilfred Raymond

2008-01-01

Comprehensive, Up-to-Date Coverage of Spectroscopy Theory and its Applications to Biological SystemsAlthough a multitude of books have been published about spectroscopy, most of them only occasionally refer to biological systems and the specific problems of biomolecular EPR (bioEPR). Biomolecular EPR Spectroscopy provides a practical introduction to bioEPR and demonstrates how this remarkable tool allows researchers to delve into the structural, functional, and analytical analysis of paramagnetic molecules found in the biochemistry of all species on the planet. A Must-Have Reference in an Intrinsically Multidisciplinary FieldThis authoritative reference seamlessly covers all important bioEPR applications, including low-spin and high-spin metalloproteins, spin traps and spin lables, interaction between active sites, and redox systems. It is loaded with practical tricks as well as do's and don'ts that are based on the author's 30 years of experience in the field. The book also comes with an unprecedented set of...

Immunoelectron microscopic studies on metallothionein induction in Nile cichlid due to heavy metal stress

International Nuclear Information System (INIS)

Chatterjee, S.; Singh, L.; Das, T.K.; Mukhopadhyay, S.K.

2010-01-01

Heavy metals are important environmental pollutants and many of them are toxic even in low concentrations. The uncontrolled input of such elements in milieu is undesirable because once accumulated, are hard to remove. The release of toxic metals in biologically available forms by human activity may damage or alter both natural and man-made ecosystems. The cellular adaptation to toxicity of metals is one of the important factors for organisms living in the stressful conditions. The major type of cellular effect at the cytoplasmic level involves binding of metals through specific metal binding proteins. One of these metalloproteins is metallothionein (MT), MT is a low-molecular-weight (6-7 kDa) cysteine rich protein ubiquitous in the animal kingdom and can bind with essential (Cu + and Zn 2+ ) and nonessential (Cd 2+ , Hg 2+ and Ag + ) metals with a high thermodynamic and low kinetic stability. Again, the induction of MT by other heavy metals such as Cr, Mn and Pb was also reported by several workers

Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin

International Nuclear Information System (INIS)

Jagadeesan, G.; Malathy, P.; Gunasekaran, K.; Harikrishna Etti, S.; Aravindhan, S.

2014-01-01

The great cormorant hemoglobin has been isolated, purified and crystallized and the three dimensional structure is solved using molecular replacement technique. Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3 1 21, with unit-cell parameters a = b = 55.64, c = 153.38 Å, β = 120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit

An investigation of changes in element distribution and chemical states during differentiation of embryonic stem cells

International Nuclear Information System (INIS)

Sugimoto, T.; Ide-Ektessabi, A.; Ishihara, R.; Tanigaki, M.

2004-01-01

Metallic elements and their organic compounds have dynamic regulatory functions in cells. In this study, we implemented a new approach to investigate the mechanism of differentiation of embryonic stem cells, by measuring and analyzing the change in distribution and chemical states of intracellular trace elements. We anticipate that trace metal elements and metalloproteins play important roles in the direction of differentiation, both as active centers, and as factors in the death of neural cells in neurodegenerative disorders. The aim of this study is to analyze the distribution and chemical states of trace elements during the process of differentiation of mouse embryonic stem cells, and to understand how these factors relate to the differentiation process. Using the experimental results, some previously unexplained points are considered, namely (1) how the intracellular elements change during the process of neuronal differentiation, and (2) what the optimal conditions of such elements are for neuronal differentiation. The information obtained during this study is relevant to nervous system development and evolution

Force modulation and electrochemical gating of conductance in a cytochrome

Science.gov (United States)

Davis, Jason J.; Peters, Ben; Xi, Wang

2008-09-01

Scanning probe methods have been used to measure the effect of electrochemical potential and applied force on the tunnelling conductance of the redox metalloprotein yeast iso-1-cytochrome c (YCC) at a molecular level. The interaction of a proximal probe with any sample under test will, at this scale, be inherently perturbative. This is demonstrated with conductive probe atomic force microscopy (CP-AFM) current-voltage spectroscopy in which YCC, chemically adsorbed onto pristine Au(111) via its surface cysteine residue, is observed to become increasingly compressed as applied load is increased, with concomitant decrease in junction resistance. Electrical contact at minimal perturbation, where probe-molecule coupling is comparable to that in scanning tunnelling microscopy, brings with it the observation of negative differential resistance, assigned to redox-assisted probe-substrate tunnelling. The role of the redox centre in conductance is also resolved in electrochemical scanning tunnelling microscopy assays where molecular conductance is electrochemically gateable through more than an order of magnitude.

A new crystal form of Aspergillus oryzae catechol oxidase and evaluation of copper site structures in coupled binuclear copper enzymes.

Science.gov (United States)

Penttinen, Leena; Rutanen, Chiara; Saloheimo, Markku; Kruus, Kristiina; Rouvinen, Juha; Hakulinen, Nina

2018-01-01

Coupled binuclear copper (CBC) enzymes have a conserved type 3 copper site that binds molecular oxygen to oxidize various mono- and diphenolic compounds. In this study, we found a new crystal form of catechol oxidase from Aspergillus oryzae (AoCO4) and solved two new structures from two different crystals at 1.8-Å and at 2.5-Å resolutions. These structures showed different copper site forms (met/deoxy and deoxy) and also differed from the copper site observed in the previously solved structure of AoCO4. We also analysed the electron density maps of all of the 56 CBC enzyme structures available in the protein data bank (PDB) and found that many of the published structures have vague copper sites. Some of the copper sites were then re-refined to find a better fit to the observed electron density. General problems in the refinement of metalloproteins and metal centres are discussed.

A new crystal form of Aspergillus oryzae catechol oxidase and evaluation of copper site structures in coupled binuclear copper enzymes.

Directory of Open Access Journals (Sweden)

Leena Penttinen

Full Text Available Coupled binuclear copper (CBC enzymes have a conserved type 3 copper site that binds molecular oxygen to oxidize various mono- and diphenolic compounds. In this study, we found a new crystal form of catechol oxidase from Aspergillus oryzae (AoCO4 and solved two new structures from two different crystals at 1.8-Å and at 2.5-Å resolutions. These structures showed different copper site forms (met/deoxy and deoxy and also differed from the copper site observed in the previously solved structure of AoCO4. We also analysed the electron density maps of all of the 56 CBC enzyme structures available in the protein data bank (PDB and found that many of the published structures have vague copper sites. Some of the copper sites were then re-refined to find a better fit to the observed electron density. General problems in the refinement of metalloproteins and metal centres are discussed.

Application of SEC-ICP-MS for comparative analyses of metal-containing species in cancerous and healthy human thyroid samples.

Science.gov (United States)

Boulyga, Sergei F; Loreti, Valeria; Bettmer, Jörg; Heumann, Klaus G

2004-09-01

Size exclusion chromatography (SEC) was coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for speciation study of trace metals in cancerous thyroid tissues in comparison to healthy thyroids aimed to estimation of changes in metalloprotein speciation in pathological tissue. The study showed a presence of species binding Cu, Zn, Cd and Pb in healthy thyroid tissue with a good reproducibility of chromatographic results, whereas the same species could not be detected in cancerous tissues. Thus, remarkable differences with respect to metal-binding species were revealed between healthy and pathological thyroid samples, pointing out a completely different distribution of trace metals in cancerous tissues. The metal-binding species could not be identified in the frame of this work because of a lack of appropriate standards. Nevertheless, the results obtained confirm the suitability of SEC-ICP-MS for monitoring of changes in trace metal distribution in cancerous tissue and will help to better understand the role of metal-containing species in thyroid pathology.

Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin

Energy Technology Data Exchange (ETDEWEB)

Jagadeesan, G. [Presidency College, Chennai 600 005 (India); Malathy, P.; Gunasekaran, K. [University of Madras, Chennai 600 025 (India); Harikrishna Etti, S. [GKM College of Engineering and Technology, Kamaraj Salai, Chennai 600 063 (India); Aravindhan, S., E-mail: aravindhanpresidency@gmail.com [Presidency College, Chennai 600 005 (India)

2014-10-25

The great cormorant hemoglobin has been isolated, purified and crystallized and the three dimensional structure is solved using molecular replacement technique. Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3{sub 1}21, with unit-cell parameters a = b = 55.64, c = 153.38 Å, β = 120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

Studies on selenoproteins in bovine kidneys by gel chromatography and neutron activation

Energy Technology Data Exchange (ETDEWEB)

Chatt, A.; Jayawickreme, C.K.

1986-01-01

Selenium at low concentrations has been claimed to be an element which is essential for life and growth. In recent years, selenium attracted increasing interest from researchers around the world because of its possible biological functions in preventing cancer, enhancing the immune system, slowing the aging process, and stimulating sexual activity. In living matter, selenium is mainly incorporated with macromolecules. Much of the metabolic behavior, biological effects, and involvement in homeostatic mechanism of this element may very well depend on the presence of the particular type of selenoproteins in the system. Neutron activation analysis (NAA) in conjunction with several bioanalytical techniques can be used to characterize metalloproteins in general. In a recent study, the distribution of trace elements in subcellular fractions of bovine kidney has been reported. The present work deals with the application of NAA together with other techniques to the isolation and characterization of selenoproteins in bovine kidneys with particular emphasis on the NAA method developed.

Non-invasive imaging using reporter genes altering cellular water permeability

Science.gov (United States)

Mukherjee, Arnab; Wu, Di; Davis, Hunter C.; Shapiro, Mikhail G.

2016-12-01

Non-invasive imaging of gene expression in live, optically opaque animals is important for multiple applications, including monitoring of genetic circuits and tracking of cell-based therapeutics. Magnetic resonance imaging (MRI) could enable such monitoring with high spatiotemporal resolution. However, existing MRI reporter genes based on metalloproteins or chemical exchange probes are limited by their reliance on metals or relatively low sensitivity. Here we introduce a new class of MRI reporters based on the human water channel aquaporin 1. We show that aquaporin overexpression produces contrast in diffusion-weighted MRI by increasing tissue water diffusivity without affecting viability. Low aquaporin levels or mixed populations comprising as few as 10% aquaporin-expressing cells are sufficient to produce MRI contrast. We characterize this new contrast mechanism through experiments and simulations, and demonstrate its utility in vivo by imaging gene expression in tumours. Our results establish an alternative class of sensitive, metal-free reporter genes for non-invasive imaging.

Reversible S-nitrosylation in an engineered azurin

Energy Technology Data Exchange (ETDEWEB)

Tian, Shiliang; Liu, Jing; Cowley, Ryan E.; Hosseinzadeh, Parisa; Marshall, Nicholas M.; Yu, Yang; Robinson, Howard; Nilges, Mark J.; Blackburn, Ninian J.; Solomon, Edward I.; Lu, Yi

2016-04-25

S-Nitrosothiols are known as reagents for NO storage and transportation and as regulators in many physiological processes. Although the S-nitrosylation catalysed by haem proteins is well known, no direct evidence of S-nitrosylation in copper proteins has been reported. Here, we report reversible insertion of NO into a copper–thiolate bond in an engineered copper centre in Pseudomonas aeruginosa azurin by rational design of the primary coordination sphere and tuning its reduction potential by deleting a hydrogen bond in the secondary coordination sphere. The results not only provide the first direct evidence of S-nitrosylation of Cu(II)-bound cysteine in metalloproteins, but also shed light on the reaction mechanism and structural features responsible for stabilizing the elusive Cu(I)–S(Cys)NO species. The fast, efficient and reversible S-nitrosylation reaction is used to demonstrate its ability to prevent NO inhibition of cytochrome bo3 oxidase activity by competing for NO binding with the native enzyme under physiologically relevant conditions.

The Role of Metal Binding in the Amyotrophic Lateral Sclerosis-Related Aggregation of Copper-Zinc Superoxide Dismutase

Directory of Open Access Journals (Sweden)

Ivana Sirangelo

2017-08-01

Full Text Available Protein misfolding and conformational changes are common hallmarks in many neurodegenerative diseases involving formation and deposition of toxic protein aggregates. Although many players are involved in the in vivo protein aggregation, physiological factors such as labile metal ions within the cellular environment are likely to play a key role. In this review, we elucidate the role of metal binding in the aggregation process of copper-zinc superoxide dismutase (SOD1 associated to amyotrophic lateral sclerosis (ALS. SOD1 is an extremely stable Cu-Zn metalloprotein in which metal binding is crucial for folding, enzymatic activity and maintenance of the native conformation. Indeed, demetalation in SOD1 is known to induce misfolding and aggregation in physiological conditions in vitro suggesting that metal binding could play a key role in the pathological aggregation of SOD1. In addition, this study includes recent advances on the role of aberrant metal coordination in promoting SOD1 aggregation, highlighting the influence of metal ion homeostasis in pathologic aggregation processes.

$^{111m}$Cd- and $^{199m}$Hg-derivatives of blue oxidases

CERN Multimedia

2002-01-01

The rack-induced bonding concept (H.B.Gray & B.G.~Malmstroem, Comments Inorg. Chem, 2, 203, 1983) postulates that the bound metal ion in metalloproteins is forced to adopt a coordination geometry determined by the rigid peptide conformation of the protein. Alternatively, the metal ion could create its own favoured coordination geometry in a soft peptide conformation. In order to decide who is slave or master the changes of coordination and rigidity of metal sites in blue copper proteins due to metal and ligand exchange were studied by $^{111m}$Cd and $^{199m}$Hg $\\gamma$-$\\gamma$-perturbed angular correlation (PAC). To get a better understanding of the so called " Type 1 Copper Site " of the blue oxidases laccase (LAC) and ascorbate oxidase (AO) we concentrated our investigations on the small blue copper proteins azurin and plastocyanin. \\\\ \\\\In azurin~(Az), the metal ligand methionine 121~(M121) was replaced by several amino acids, e.g. asparagine~(N), glutamic acid~(E), via site directed mutagenesis. Di...

A Mini HIP HOP Assay Uncovers a Central Role for Copper and Zinc in the Antifungal Mode of Action of Allicin.

Science.gov (United States)

Prescott, Thomas A K; Panaretou, Barry

2017-05-10

Garlic contains the organosulfur compound allicin which exhibits potent antifungal activity. Here we demonstrate the use of a highly simplified yeast chemical genetic screen to characterize its mode of action. By screening 24 validated yeast gene deletion "signature" strains for which hypersensitivity is characteristic for common antifungal modes of action, yeast lacking the high affinity Cu 2+ transporter Ctr1 was found to be hypersensitive to allicin. Focusing on transition metal related genes identified two more hypersensitive strains lacking the Cu 2+ and Zn 2+ transcription factors Mac1 and Zap1. Hypersensitivity in these strains was reversed by the addition of Cu 2+ and Zn 2+ ions, respectively. The results suggest the antifungal activity of allicin is mediated through restricted Cu 2+ and Zn 2+ uptake or inhibition of Cu 2+ and Zn 2+ metalloproteins. As certain antimicrobial modes of action are much more common than others, the approach taken here provides a useful way to identify them early on.

An investigation of changes in element distribution and chemical states during differentiation of embryonic stem cells

Energy Technology Data Exchange (ETDEWEB)

Sugimoto, T.; Ide-Ektessabi, A. E-mail: h51167@sakura.kudpc.kyoto-u.ac.jp; Ishihara, R.; Tanigaki, M

2004-07-01

Metallic elements and their organic compounds have dynamic regulatory functions in cells. In this study, we implemented a new approach to investigate the mechanism of differentiation of embryonic stem cells, by measuring and analyzing the change in distribution and chemical states of intracellular trace elements. We anticipate that trace metal elements and metalloproteins play important roles in the direction of differentiation, both as active centers, and as factors in the death of neural cells in neurodegenerative disorders. The aim of this study is to analyze the distribution and chemical states of trace elements during the process of differentiation of mouse embryonic stem cells, and to understand how these factors relate to the differentiation process. Using the experimental results, some previously unexplained points are considered, namely (1) how the intracellular elements change during the process of neuronal differentiation, and (2) what the optimal conditions of such elements are for neuronal differentiation. The information obtained during this study is relevant to nervous system development and evolution.

Ebselen, a promising antioxidant drug: mechanisms of action and targets of biological pathways.

Science.gov (United States)

Azad, Gajendra Kumar; Tomar, Raghuvir S

2014-08-01

Ebselen, an organoselenium compound, mimics glutathione peroxidase activity. It is a multifunctional compound, which catalyzes several essential reactions for the protection of cellular components from oxidative and free radical damage. Based on a number of in vitro and in vivo studies, various mechanisms are proposed to understand the biomedical actions of ebselen in health and diseases. It modulates metallo-proteins, enzymatic cofactors, gene expression, epigenetics, antioxidant defenses and immune systems. Owing to these properties, ebselen is currently under clinical trials for the prevention and treatment of various disorders such as cardiovascular diseases, arthritis, stroke, atherosclerosis, and cancer. A few ebselen-based pharmaceutical agents are under extensive investigation. As ebselen has been shown to have significant cellular toxicity, appropriate studies are needed to redesign the ebselen-based therapy for clinical trials. This review summarizes current understanding of the biochemical and molecular properties, and pharmacological applications of ebselen and future directions in this area of research.

Zinc in Cellular Regulation: The Nature and Significance of "Zinc Signals".

Science.gov (United States)

Maret, Wolfgang

2017-10-31

In the last decade, we witnessed discoveries that established Zn 2+ as a second major signalling metal ion in the transmission of information within cells and in communication between cells. Together with Ca 2+ and Mg 2+ , Zn 2+ covers biological regulation with redox-inert metal ions over many orders of magnitude in concentrations. The regulatory functions of zinc ions, together with their functions as a cofactor in about three thousand zinc metalloproteins, impact virtually all aspects of cell biology. This article attempts to define the regulatory functions of zinc ions, and focuses on the nature of zinc signals and zinc signalling in pathways where zinc ions are either extracellular stimuli or intracellular messengers. These pathways interact with Ca 2+ , redox, and phosphorylation signalling. The regulatory functions of zinc require a complex system of precise homeostatic control for transients, subcellular distribution and traffic, organellar homeostasis, and vesicular storage and exocytosis of zinc ions.

Studies on selenoproteins in bovine kidneys by gel chromatography and neutron activation

International Nuclear Information System (INIS)

Chatt, A.; Jayawickreme, C.K.

1986-01-01

Selenium at low concentrations has been claimed to be an element which is essential for life and growth. In recent years, selenium attracted increasing interest from researchers around the world because of its possible biological functions in preventing cancer, enhancing the immune system, slowing the aging process, and stimulating sexual activity. In living matter, selenium is mainly incorporated with macromolecules. Much of the metabolic behavior, biological effects, and involvement in homeostatic mechanism of this element may very well depend on the presence of the particular type of selenoproteins in the system. Neutron activation analysis (NAA) in conjunction with several bioanalytical techniques can be used to characterize metalloproteins in general. In a recent study, the distribution of trace elements in subcellular fractions of bovine kidney has been reported. The present work deals with the application of NAA together with other techniques to the isolation and characterization of selenoproteins in bovine kidneys with particular emphasis on the NAA method developed

Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach

Energy Technology Data Exchange (ETDEWEB)

García-Sevillano, M.A.; García-Barrera, T. [Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Huelva 21007 (Spain); Research Center on Health and Environment (CYSMA), University of Huelva (Spain); International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain); Navarro, F. [International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain); Department of Environmental Biology and Public Health, Cell Biology, Faculty of Experimental Sciences, University of Huelva, Campus El Carmen, Huelva 21007 (Spain); Gómez-Ariza, J.L., E-mail: ariza@uhu.es [Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Huelva 21007 (Spain); Research Center on Health and Environment (CYSMA), University of Huelva (Spain); International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain)

2014-09-09

Highlights: • Identification and quantification of Cu,Zn-superoxide dismutase in mice hepatic cells. • IDA-ICP-MSis applied to obtain a high degree of accuracy, precision and sensibility. • This methodology reduces the time of analysis and avoids clean-up procedures. • The application of this method to Hg-exposed mice reveals perturbations in Cu,Zn-SOD. - Abstract: In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min

Shaping mechanisms of metal specificity in a family of metazoan metallothioneins: evolutionary differentiation of mollusc metallothioneins

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Atrian Sílvia

2011-01-01

Full Text Available Abstract Background The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu+ or Cd2+. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion. Results HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd2+, Zn2+ or Cu2+ and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD and ultra violet-visible (UV-Vis spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd6-HpCdMT and Cu12-HpCuMT with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was

Mechanisms of nickel toxicity in microorganisms

Science.gov (United States)

Macomber, Lee

2014-01-01

Summary Nickel has long been known to be an important human toxicant, including having the ability to form carcinomas, but until recently nickel was believed to be an issue only to microorganisms living in nickel-rich serpentine soils or areas contaminated by industrial pollution. This assumption was overturned by the discovery of a nickel defense system (RcnR/RcnA) found in microorganisms that live in a wide range of environmental niches, suggesting that nickel homeostasis is a general biological concern. To date, the mechanisms of nickel toxicity in microorganisms and higher eukaryotes are poorly understood. In this review, we summarize nickel homeostasis processes used by microorganisms and highlight in vivo and in vitro effects of exposure to elevated concentrations of nickel. On the basis of this evidence we propose four mechanisms of nickel toxicity: 1) nickel replaces the essential metal of metalloproteins, 2) nickel binds to catalytic residues of non-metalloenzymes; 3) nickel binds outside the catalytic site of an enzyme to inhibit allosterically, and 4) nickel indirectly causes oxidative stress. PMID:21799955

The potential roles of metallothionein as a therapeutic target for cerebral ischemia and retinal diseases.

Science.gov (United States)

Ito, Yasushi; Tanaka, Hirotaka; Hara, Hideaki

2013-01-01

Methallothionein (MT) is a low molecular weight cysteine rich metalloprotein. In mammals, there are four isoforms (MT-1, -2, -3, and -4) and they have multiple roles, such as the detoxification of heavy metals, regulating essential metal homeostasis, and protecting against oxidative stress. Recently, accumulating studies have suggested that MTs (especially MT-1, -2, and -3) are an important neuroprotective substance for cerebral ischemia and retinal diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), that are characterized by a progressive retinal degeneration. Oxidative stress and/or zinc toxicity has been implicated as part of the common pathway in these diseases. Studying the expression patterns and functions of MTs may broaden our understanding of the endogenous molecular responses that these diseases trigger, and may help us to develop new therapeutic strategies to treat them. However, the precise roles of MTs within the brain and retina are not fully understood in terms of neuropathological conditions. In this review, we discuss the recent findings focusing on MTs' functions following cerebral ischemia, AMD, and RP.

Effects of macro-nutrient, micro-nutrient composition and cooking conditions on in vitro digestibility of meat and aquatic dietary proteins.

Science.gov (United States)

Luo, Jiaqiang; Taylor, Cheryl; Nebl, Thomas; Ng, Ken; Bennett, Louise E

2018-07-15

Animal and aquatic meats represent important sources of dietary protein and micro-nutrients. Although red and processed meats carry some risks for human health, sensory and nutritional advantages drive meat consumption. Therefore, it is important to understand how meat processing and cooking influence healthiness. The research aim was to investigate relationships of meat composition (proximates, amino acids and minerals) and cooking conditions (raw, 90 s microwave, 200 °C oven for 10 or 30 min) on protein digestibility, for a selection of four animal (beef, chicken, pork, kangaroo) and four aquatic meats (salmon, trout, prawn, oyster). Lean meats were minced before cooking followed by in vitro gastro-intestinal digestion and analysed for progress of hydrolysis, and size ranges of peptides using MALDI-TOF-MS. Correlation matrix analysis between compositional and functional parameters indicated that digestibility was significantly linked with protein and metal concentrations, likely reflecting moisture-dependent solubility and inter-mixing of sarcoplasmic metallo-proteins and insoluble myofibrillar proteins. Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.

Manganese L-edge X-ray absorption spectroscopy of manganese catalase from Lactobacillus plantarum and mixed valence manganese complexes

Energy Technology Data Exchange (ETDEWEB)

Grush, M.M.; Chen, J.; George, S.J. [Univ. of California, Davis, CA (United States)] [and others

1996-01-10

The first Mn L-edge absorption spectra of a Mn metalloprotein are presented in this paper. Both reduced and superoxidized Mn catalase have been examined by fluorescence-detected soft X-ray absorption spectroscopy, and their Mn L-edge spectra are dramatically different. The spectrum of reduced Mn(II)Mn(II) catalase has been interpreted by ligand field atomic multiplet calculations and by comparison to model compound spectra. The analysis finds a 10 Dq value of nearly 1.1 eV, consistent with coordination by predominately nitrogen and oxygen donor ligands. For interpretation of mixed valence Mn spectra, an empirical simulation procedure based on the addition of homovalent model compound spectra has been developed and was tested on a variety of Mn complexes and superoxidized Mn catalase. This routine was also used to determine the oxidation state composition of the Mn in [Ba{sub 8}Na{sub 2}ClMn{sub 16}(OH){sub 8}(CO{sub 3}){sub 4}L{sub 8}] .53 H{sub 2}O (L=1,3-diamino-2-hydroxypropane-N,N,N`N`-tetraacetic acid). 27 refs., 6 figs.

Tunneling explains efficient electron transport via protein junctions.

Science.gov (United States)

Fereiro, Jerry A; Yu, Xi; Pecht, Israel; Sheves, Mordechai; Cuevas, Juan Carlos; Cahen, David

2018-05-15

Metalloproteins, proteins containing a transition metal ion cofactor, are electron transfer agents that perform key functions in cells. Inspired by this fact, electron transport across these proteins has been widely studied in solid-state settings, triggering the interest in examining potential use of proteins as building blocks in bioelectronic devices. Here, we report results of low-temperature (10 K) electron transport measurements via monolayer junctions based on the blue copper protein azurin (Az), which strongly suggest quantum tunneling of electrons as the dominant charge transport mechanism. Specifically, we show that, weakening the protein-electrode coupling by introducing a spacer, one can switch the electron transport from off-resonant to resonant tunneling. This is a consequence of reducing the electrode's perturbation of the Cu(II)-localized electronic state, a pattern that has not been observed before in protein-based junctions. Moreover, we identify vibronic features of the Cu(II) coordination sphere in transport characteristics that show directly the active role of the metal ion in resonance tunneling. Our results illustrate how quantum mechanical effects may dominate electron transport via protein-based junctions.

Using model complexes to augment and advance metalloproteinase inhibitor design.

Science.gov (United States)

Jacobsen, Faith E; Cohen, Seth M

2004-05-17

The tetrahedral zinc complex [(Tp(Ph,Me))ZnOH] (Tp(Ph,Me) = hydrotris(3,5-phenylmethylpyrazolyl)borate) was combined with 2-thenylmercaptan, ethyl 4,4,4-trifluoroacetoacetate, salicylic acid, salicylamide, thiosalicylic acid, thiosalicylamide, methyl salicylate, methyl thiosalicyliate, and 2-hydroxyacetophenone to form the corresponding [(Tp(Ph,Me))Zn(ZBG)] complexes (ZBG = zinc-binding group). X-ray crystal structures of these complexes were obtained to determine the mode of binding for each ZBG, several of which had been previously studied with SAR by NMR (structure-activity relationship by nuclear magnetic resonance) as potential ligands for use in matrix metalloproteinase inhibitors. The [(Tp(Ph,Me))Zn(ZBG)] complexes show that hydrogen bonding and donor atom acidity have a pronounced effect on the mode of binding for this series of ligands. The results of these studies give valuable insight into how ligand protonation state and intramolecular hydrogen bonds can influence the coordination mode of metal-binding proteinase inhibitors. The findings here suggest that model-based approaches can be used to augment drug discovery methods applied to metalloproteins and can aid second-generation drug design.

Interactions of human hemoglobin with charged ligand-functionalized iron oxide nanoparticles and effect of counterions

Energy Technology Data Exchange (ETDEWEB)

Ghosh, Goutam, E-mail: ghoshg@yahoo.com [UGC-DAE Consortium for Scientific Research, Mumbai Centre (India); Panicker, Lata [Bhabha Atomic Research Centre, Solid State Physics Division (India)

2014-12-15

Human hemoglobin is an important metalloprotein. It has tetrameric structure with each subunit containing a ‘heme’ group which carries oxygen and carbon dioxide in blood. In this work, we have investigated the interactions of human hemoglobin (Hb) with charged ligand-functionalized iron oxide nanoparticles and the effect of counterions, in aqueous medium. Several techniques like DLS and ζ-potential measurements, UV–vis, fluorescence, and CD spectroscopy have been used to characterize the interaction. The nanoparticle size was measured to be in the range of 20–30 nm. Our results indicated the binding of Hb with both positively as well as negatively charged ligand-functionalized iron oxide nanoparticles in neutral aqueous medium which was driven by the electrostatic and the hydrophobic interactions. The electrostatic binding interaction was not seen in phosphate buffer at pH 7.4. We have also observed that the ‘heme’ groups of Hb remained unaffected on binding with charged nanoparticles, suggesting the utility of the charged ligand-functionalized nanoparticles in biomedical applications.

A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants.

Science.gov (United States)

Luchinat, Enrico; Barbieri, Letizia; Banci, Lucia

2017-12-12

Superoxide dismutase 1 (SOD1) is an important metalloprotein for cellular oxidative stress defence, that is mutated in familiar variants of Amyotrophic Lateral Sclerosis (fALS). Some mutations destabilize the apo protein, leading to the formation of misfolded, toxic species. The Copper Chaperone for SOD1 (CCS) transiently interacts with SOD1 and promotes its correct maturation by transferring copper and catalyzing disulfide bond formation. By in vitro and in-cell NMR, we investigated the role of the SOD-like domain of CCS (CCS-D2). We showed that CCS-D2 forms a stable complex with zinc-bound SOD1 in human cells, that has a twofold stabilizing effect: it both prevents the accumulation of unstructured mutant SOD1 and promotes zinc binding. We further showed that CCS-D2 interacts with apo-SOD1 in vitro, suggesting that in cells CCS stabilizes mutant apo-SOD1 prior to zinc binding. Such molecular chaperone function of CCS-D2 is novel and its implications in SOD-linked fALS deserve further investigation.

Phi-value analysis of apo-azurin folding: comparison between experiment and theory.

Science.gov (United States)

Zong, Chenghang; Wilson, Corey J; Shen, Tongye; Wolynes, Peter G; Wittung-Stafshede, Pernilla

2006-05-23

Pseudomonas aeruginosa azurin is a 128-residue beta-sandwich metalloprotein; in vitro kinetic experiments have shown that it folds in a two-state reaction. Here, we used a variational free energy functional to calculate the characteristics of the transition state ensemble (TSE) for folding of the apo-form of P. aeruginosa azurin and investigate how it responds to thermal and mutational changes. The variational method directly yields predicted chevron plots for wild-type and mutant apo-forms of azurin. In parallel, we performed in vitro kinetic-folding experiments on the same set of azurin variants using chemical perturbation. Like the wild-type protein, all apo-variants fold in apparent two-state reactions both in calculations and in stopped-flow mixing experiments. Comparisons of phi (phi) values determined from the experimental and theoretical chevron parameters reveal an excellent agreement for most positions, indicating a polarized, highly structured TSE for folding of P. aeruginosa apo-azurin. We also demonstrate that careful analysis of side-chain interactions is necessary for appropriate theoretical description of core mutants.

Survey of chemical speciation of trace elements using synchrotron radiation

International Nuclear Information System (INIS)

Gordon, B.M.

1985-01-01

Information concerning the chemical state of trace elements in biological systems generally has not been available. Such information for toxic elements and metals in metalloproteins could prove extremely valuable in the elucidation of their metabolism and other biological processes. The shielding of core electrons by binding electrons affect the energy required for creating inner-shell holes. Furthermore, the molecular binding and the symmetry of the local environment of an atom affect the absorption spectrum in the neighborhood of the absorption edge. X-ray absorption near-edge structure (XANES) using synchrotron radiation excitation can be used to provide chemical speciation information for trace elements at concentrations as low as 10 ppM. The structure and position of the absorption curve in the region of an edge can yield vital data about the local structure and oxidation state of the trace element in question. Data are most easily interpreted by comparing the observed edge structure and position with those of model compounds of the element covering the entire range of possible oxidation states. Examples of such analyses are reviewed. 14 refs., 1 fig

Subcellular Iron Localization Mechanisms in Plants

Directory of Open Access Journals (Sweden)

Emre Aksoy

2017-12-01

Full Text Available The basic micro-nutrient element iron (Fe is present as a cofactor in the active sites of many metalloproteins with important roles in the plant. On the other hand, since it is excessively reactive, excess accumulation in the cell triggers the production of reactive oxygen species, leading to cell death. Therefore, iron homeostasis in the cell is very important for plant growth. Once uptake into the roots, iron is distributed to the subcellular compartments. Subcellular iron transport and hence cellular iron homeostasis is carried out through synchronous control of different membrane protein families. It has been discovered that expression levels of these membrane proteins increase under iron deficiency. Examination of the tasks and regulations of these carriers is very important in terms of understanding the iron intake and distribution mechanisms in plants. Therefore, in this review, the transporters responsible for the uptake of iron into the cell and its subcellular distribution between organelles will be discussed with an emphasis on the current developments about these transporters.

Investigation of metalloproteins distributions in cytosol of hepatocellular carcinoma and its surrounding tissues by using synchrotron radiation X-ray fluorescence

International Nuclear Information System (INIS)

Gao Yuxi; Chen Chunying; Li Bai; Chai Zhifang; Huang Yuying; He Wei; Deng Guilong; Liu Yingbin

2004-01-01

Synchrotron radiation X-ray fluorescence (SRXRF) spectroscopy is an advanced quantitative multielemental analytical technique with space resolution of several μm and sensitivities in the μ g/g range. It can be used for keeping track of trace elements in biological samples after an electrophoretic separation. In this paper, proteins in cytosol of human hepatocellular carcinoma and the surrounding 'normal' tissue were separated with thin layer isoelectric focusing (IEF). The contents of metal ions in protein bands were determined by SRXRF. The results showed that the metal-containing proteins detected in the two samples were very much alike, but their distribution patterns were easily distinguishable. The contents of iron, zinc, and copper in bands from the surrounding 'normal' tissue were generally higher than that from hepatoma tissue, especially in Fe-containing proteins with pIs of 6.5, 7.7, 8.0 and less than 3.5, Cu-containing proteins with PIs of 3.2, 4.9, 5.5, 5.9 and 6.5, as well as Zn-containing proteins with pI of 5.5 and 6.5. However, Fe contents in Fe-containing proteins of 4.0, and 7.0 from the hepatoma tissue were slight higher than that from the surrounding 'normal' tissue. Further studies are necessary to validate the universality and the biological meaning of the pattern. (authors)

Buccal alterations in diabetes mellitus

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Negrato Carlos

2010-01-01

Full Text Available Abstract Long standing hyperglycaemia besides damaging the kidneys, eyes, nerves, blood vessels, heart, can also impair the function of the salivary glands leading to a reduction in the salivary flow. When salivary flow decreases, as a consequence of an acute hyperglycaemia, many buccal or oral alterations can occur such as: a increased concentration of mucin and glucose; b impaired production and/or action of many antimicrobial factors; c absence of a metalloprotein called gustin, that contains zinc and is responsible for the constant maturation of taste papillae; d bad taste; e oral candidiasis f increased cells exfoliation after contact, because of poor lubrication; g increased proliferation of pathogenic microorganisms; h coated tongue; i halitosis; and many others may occur as a consequence of chronic hyperglycaemia: a tongue alterations, generally a burning mouth; b periodontal disease; c white spots due to demineralization in the teeth; d caries; e delayed healing of wounds; f greater tendency to infections; g lichen planus; h mucosa ulcerations. Buccal alterations found in diabetic patients, although not specific of this disease, have its incidence and progression increased when an inadequate glycaemic control is present.

An ArsR/SmtB family member is involved in the regulation by arsenic of the arsenite oxidase operon in Thiomonas arsenitoxydans.

Science.gov (United States)

Moinier, Danielle; Slyemi, Djamila; Byrne, Deborah; Lignon, Sabrina; Lebrun, Régine; Talla, Emmanuel; Bonnefoy, Violaine

2014-10-01

The genetic organization of the aioBA operon, encoding the arsenite oxidase of the moderately acidophilic and facultative chemoautotrophic bacterium Thiomonas arsenitoxydans, is different from that of the aioBA operon in the other arsenite oxidizers, in that it encodes AioF, a metalloprotein belonging to the ArsR/SmtB family. AioF is stabilized by arsenite, arsenate, or antimonite but not molybdate. Arsenic is tightly attached to AioF, likely by cysteine residues. When loaded with arsenite or arsenate, AioF is able to bind specifically to the regulatory region of the aio operon at two distinct positions. In Thiomonas arsenitoxydans, the promoters of aioX and aioB are convergent, suggesting that transcriptional interference occurs. These results indicate that the regulation of the aioBA operon is more complex in Thiomonas arsenitoxydans than in the other aioBA containing arsenite oxidizers and that the arsenic binding protein AioF is involved in this regulation. On the basis of these data, a model to explain the tight control of aioBA expression by arsenic in Thiomonas arsenitoxydans is proposed. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Buccal alterations in diabetes mellitus.

Science.gov (United States)

Negrato, Carlos Antonio; Tarzia, Olinda

2010-01-15

Long standing hyperglycaemia besides damaging the kidneys, eyes, nerves, blood vessels, heart, can also impair the function of the salivary glands leading to a reduction in the salivary flow. When salivary flow decreases, as a consequence of an acute hyperglycaemia, many buccal or oral alterations can occur such as: a) increased concentration of mucin and glucose; b) impaired production and/or action of many antimicrobial factors; c) absence of a metalloprotein called gustin, that contains zinc and is responsible for the constant maturation of taste papillae; d) bad taste; e) oral candidiasis f) increased cells exfoliation after contact, because of poor lubrication; g) increased proliferation of pathogenic microorganisms; h) coated tongue; i) halitosis; and many others may occur as a consequence of chronic hyperglycaemia: a) tongue alterations, generally a burning mouth; b) periodontal disease; c) white spots due to demineralization in the teeth; d) caries; e) delayed healing of wounds; f) greater tendency to infections; g) lichen planus; h) mucosa ulcerations. Buccal alterations found in diabetic patients, although not specific of this disease, have its incidence and progression increased when an inadequate glycaemic control is present.

New developments in high field electron paramagnetic resonance with applications in structural biology

International Nuclear Information System (INIS)

Bennati, Marina; Prisner, Thomas F

2005-01-01

Recent developments in microwave technologies have led to a renaissance of electron paramagnetic resonance (EPR) due to the implementation of new spectrometers operating at frequencies ≥90 GHz. EPR at high fields and high frequencies (HF-EPR) has been established up to THz (very high frequency (VHF) EPR) in continuous wave (cw) operation and up to about 300 GHz in pulsed operation. To date, its most prominent application field is structural biology. This review article first gives an overview of the theoretical basics and the technical aspects of HF-EPR methodologies, such as cw and pulsed HF-EPR, as well as electron nuclear double resonance at high fields (HF-ENDOR). In the second part, the article illustrates different application areas of HF-EPR in studies of protein structure and function. In particular, HF-EPR has delivered essential contributions to disentangling complex spectra of radical cofactors or reaction intermediates in photosynthetic reaction centres, radical enzymes (such as ribonucleotide reductase) and in metalloproteins. Furthermore, HF-EPR combined with site-directed spin labelling in membranes and soluble proteins provides new methods of investigating complex molecular dynamics and intermolecular distances

Global gene response in Saccharomyces cerevisiae exposed to silver nanoparticles.

Science.gov (United States)

Niazi, Javed H; Sang, Byoung-In; Kim, Yeon Seok; Gu, Man Bock

2011-08-01

Silver nanoparticles (AgNPs), exhibiting a broad size range and morphologies with highly reactive facets, which are widely applicable in real-life but not fully verified for biosafety and ecotoxicity, were subjected to report transcriptome profile in yeast Saccharomyces cerevisiae. A large number of genes accounted for ∼3% and ∼5% of the genome affected by AgNPs and Ag-ions, respectively. Principal component and cluster analysis suggest that the different physical forms of Ag were the major cause in differential expression profile. Among 90 genes affected by both AgNPs and Ag-ions, metalloprotein mediating high resistance to copper (CUP1-1 and CUP1-2) were strongly induced by AgNPs (∼45-folds) and Ag-ions (∼22-folds), respectively. A total of 17 genes, responsive to chemical stimuli, stress, and transport processes, were differentially induced by AgNPs. The differential expression was also seen with Ag-ions that affected 73 up- and 161 down-regulating genes, and most of these were involved in ion transport and homeostasis. This study provides new information on the knowledge for impact of nanoparticles on living microorganisms that can be extended to other nanoparticles.

Zinc Binding by Lactic Acid Bacteria

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Jasna Mrvčić

2009-01-01

Full Text Available Zinc is an essential trace element in all organisms. A common method for the prevention of zinc deficiency is pharmacological supplementation, especially in a highly available form of a metalloprotein complex. The potential of different microbes to bind essential and toxic heavy metals has recently been recognized. In this work, biosorption of zinc by lactic acid bacteria (LAB has been investigated. Specific LAB were assessed for their ability to bind zinc from a water solution. Significant amount of zinc ions was bound, and this binding was found to be LAB species-specific. Differences among the species in binding performance at a concentration range between 10–90 mg/L were evaluated with Langmuir model for biosorption. Binding of zinc was a fast process, strongly influenced by ionic strength, pH, biomass concentration, and temperature. The most effective metal-binding LAB species was Leuconostoc mesenteroides (27.10 mg of Zn2+ per gram of dry mass bound at pH=5 and 32 °C, during 24 h. FT-IR spectroscopy analysis and electron microscopy demonstrated that passive adsorption and active uptake of the zinc ions were involved.

Mimic Carbonic Anhydrase Using Metal-Organic Frameworks for CO2 Capture and Conversion.

Science.gov (United States)

Jin, Chaonan; Zhang, Sainan; Zhang, Zhenjie; Chen, Yao

2018-02-19

Carbonic anhydrase (CA) is a zinc-containing metalloprotein, in which the Zn active center plays the key role to transform CO 2 into carbonate. Inspired by nature, herein we used metal-organic frameworks (MOFs) to mimic CA for CO 2 conversion, on the basis of the structural similarity between the Zn coordination in MOFs and CA active center. The biomimetic activity of MOFs was investigated by detecting the hydrolysis of para-nitrophenyl acetate, which is a model reaction used to evaluate CA activity. The biomimetic materials (e.g., CFA-1) showed good catalytic activity, and excellent reusability, and solvent and thermal stability, which is very important for practical applications. In addition, ZIF-100 and CFA-1 were used to mimic CA to convert CO 2 gas, and exhibited good efficiency on CO 2 conversion compared with those of other porous materials (e.g., MCM-41, active carbon). This biomimetic study revealed a novel CO 2 treatment method. Instead of simply using MOFs to absorb CO 2 , ZIF-100 and CFA-1 were used to mimic CA for in situ CO 2 conversion, which provides a new prospect in the biological and industrial applications of MOFs.

Preparation of biological samples for transmission X-ray microanalysis: a review of alternative procedures to the use of sectioned material

International Nuclear Information System (INIS)

Sigee, D.C.

1988-01-01

Although transmission X-ray microanalysis of biological material has traditionally been carried out mainly on sectioned preparations, a number of alternative procedures exist. These are considered under three major headings - whole cell preparations, analysis of cell homogenates and biological fluids, and applications of the technique to microsamples of purified biochemicals. These three aspects provide a continuous range of investigative level - from the cellular to the molecular. The use of X-ray microanalysis with whole cell preparations is considered in reference to eukaryote (animal) cells and prokaryotes - where it has particular potential in environmental studies on bacteria. In the case of cell homogenates and biological fluids, the technique has been used mainly with microdroplets of animal material. The use of X-ray microanalysis with purified biochemicals is considered in relation to both particulate and non-particulate samples. In the latter category, the application of this technique for analysis of thin films of metalloprotein is particularly emphasised. It is concluded that wider use could be made of the range of preparative techniques available - both within a particular investigation, and in diverse fields of study. Transmission X-ray microanalysis has implications for environmental, physiological and molecular biology as well as cell biology

Heavy metals and metalloids as a cause for protein misfolding and aggregation.

Science.gov (United States)

Tamás, Markus J; Sharma, Sandeep K; Ibstedt, Sebastian; Jacobson, Therese; Christen, Philipp

2014-02-25

While the toxicity of metals and metalloids, like arsenic, cadmium, mercury, lead and chromium, is undisputed, the underlying molecular mechanisms are not entirely clear. General consensus holds that proteins are the prime targets; heavy metals interfere with the physiological activity of specific, particularly susceptible proteins, either by forming a complex with functional side chain groups or by displacing essential metal ions in metalloproteins. Recent studies have revealed an additional mode of metal action targeted at proteins in a non-native state; certain heavy metals and metalloids have been found to inhibit the in vitro refolding of chemically denatured proteins, to interfere with protein folding in vivo and to cause aggregation of nascent proteins in living cells. Apparently, unfolded proteins with motile backbone and side chains are considerably more prone to engage in stable, pluridentate metal complexes than native proteins with their well-defined 3D structure. By interfering with the folding process, heavy metal ions and metalloids profoundly affect protein homeostasis and cell viability. This review describes how heavy metals impede protein folding and promote protein aggregation, how cells regulate quality control systems to protect themselves from metal toxicity and how metals might contribute to protein misfolding disorders.

Insight into the Local Solvent Environment of Biologically Relevant Iron-nitroysl Systems through Two-Dimensional Infrared Spectroscopy

Science.gov (United States)

Brookes, Jennifer Faith

Iron-nitrosyl systems, particularly in the form of heme proteins, with their iron metal active sites play an important role in biological systems. Heme proteins act as storage, transporters, and receptors for nitric oxide (NO), a signaling molecule that is important in immune, nervous, and cardiovascular systems of mammals. By better understanding the local environment of the active site of NO binding heme proteins we can gain insight into disease in which the NO pathways have been implicated. This is an important step to being able to develop pharmaceuticals targeting NO pathways in humans. Sodium nitroprusside ((SNP, Na2[Fe(CN)5is NO]·2H 2O) investigated as a model system for the active site of nitric oxide binding heme proteins. Using two-dimensional infrared spectroscopy (2D IR) to obtain dephasing dynamics of the nitrosyl stretch (nuNO) in a series of solvents we are able to better understand the local environment of the more complicated metalloproteins. Rigorous line shape analysis is performed by using nonlinear response theory to simulate 2D IR spectra which are then fit to experimental data in an iterative process to extract frequency-frequency correlation functions (FFCFs). The time scales obtained are then correlated to empirical solvent polarity parameters. The analysis of the 2D IR lineshapes reveal that the spectral diffusion timescale of the nuNO in SNP varies from 0.8 -- 4 ps and is negatively correlated with the empirical solvent polarity scales. We continue to investigate NO binding of metalloproteins through 2D IR experiments on nitrophorin 4 (NP4). NP4 is a pH-sensitive NO transporter protein present in the salivary gland of the blood sucking insect Rhodius prolixus which undergoes a pH sensitive structural change between a closed and open conformation allowing for the storage and delivery of NO. The two structures are observed spectroscopically as two distinct pH-dependent nu NO frequencies at ~1904 and ~1917 cm-1. We obtain FFCFs by globally

Applicability of a field-portable toxic heavy metal detector, using a radioisotope-tagged metalloprotein, to DOE environmental remediation and waste minimization initiatives

International Nuclear Information System (INIS)

Randles, K.E.; Bragg, D.J.; Bodette, D.E.; Lipinski, R.J.; Luera, T.F.

1998-08-01

A system based on the metal-binding kidney protein, metallothionein, bound with a trace quantity of radioactive metal, has been shown to be capable of detecting parts-per-million (ppm) to parts-per-billion (ppb) concentrations of some heavy metals in liquid solution. The main objective of this study was to determine if this type of system has adequate sensitivity and selectivity for application in detecting a number of metallic species of concern to DOE, such as mercury, lead, and chromium. An affinity-displacement study is reported here using the heavy metal radiotracers 65 Zn and 109 Cd bound to metallothionein immobilized on an Affi-Gel 10 filter support. When a heavy metal solution with a greater affinity than the tracer for the protein is poured through the filter the radiotracer is displaced by a mechanism similar to ion exchange. The main objective of this study was to verify previous internal experimental parameters and results, and to determine the specific affinities of metallothionein for the metallic species of most concern to DOE

The Au-S bond and SAM-protein contact in long-range electron transfer of pure and biomimetic metalloproteins via functionalized alkanethiol linkers

DEFF Research Database (Denmark)

Chi, Qijin; Ford, Michael J.; Halder, Arnab

disentangled a wealth of data to identify the nature of the crucial Au-S contact, all suggesting prevalence of a Au(0)-thiyl radical unit. Molecular packing is further determined by the SAM molecular structure and involves binding either to Au-atoms mined out of the surface or directly to a flat surface. We...... functionalized alkanethiols have emerged as core linkers. We have studied molecular linking in the long-range ET (LRET) processes in detail using electrochemistry, in situ STM and AFM, and electronic structure computations. A focus is the electronic structure of the Au-S link and the SAM packing. We have...... is exceedingly sensitive to the structure of the thiol-based SAM molecules, testifying to the crucial importance of SAM packing and Au-S binding, and of the SAM link to the protein. Some of the subtleties are illustrated simpler by similar size (5-6 nm) nanoparticles (NPs). Biomimetic NPs must possess a certain...

Effects of phototherapy on cartilage structure and inflammatory markers in an experimental model of osteoarthritis

Science.gov (United States)

Oliveira, Poliani; Santos, Anderson Amaro; Rodrigues, Tamara; Tim, Carla Roberta; Pinto, Karina Zambone; Magri, Angela Maria Paiva; Fernandes, Kelly Rossetti; Mattiello, Stela M.; Parizotto, Nivaldo Antonio; Anibal, Fernanda Freitas; Rennó, Ana Claudia Muniz

2013-12-01

The aim of this study was to evaluate the effects of laser phototherapy on the degenerative modifications on the articular cartilage after the anterior cruciate ligament transection (ACLT) in the knee of rats. Eighty male rats (Wistar) were distributed into four groups: intact control group (IG), injured control group (CG), injured laser treated group at 10 J/cm2 (L10), and injured laser treated group at 50 J/cm2 (L50). Animals were distributed into two subgroups, sacrificed in 5 and 8 weeks postsurgery. The ACLT was used to induce knee osteoarthritis in rats. After 2 weeks postsurgery, laser phototherapy initiated and it was performed for 15 and 30 sessions. The histological findings revealed that laser irradiation, especially at 10 J/cm2, modulated the progression of the degenerative process, showing a better cartilage structure and lower number of condrocytes compared to the other groups. Laser phototherapy was not able to decrease the degenerative process measured by Mankin score and prevent the increase of cartilage thickness related to the degenerative process. Moreover, it did not have any effect in the biomodulation of the expression of markers IL1β, tumor necrosis factor-α, and metalloprotein-13. Furthermore, laser irradiated animals, at 50 J/cm2 showed a lower amount of collagen type 1.

The Effect of Salts in Promoting Specific and Competitive Interactions between Zinc Finger Proteins and Metals

Science.gov (United States)

Li, Gongyu; Yuan, Siming; Zheng, Shihui; Chen, Yuting; Zheng, Zhen; Liu, Yangzhong; Huang, Guangming

2017-12-01

Specific protein-metal interactions (PMIs) fulfill essential functions in cells and organic bodies, and activation of these functions in vivo are mostly modulated by the complex environmental factors, including pH value, small biomolecules, and salts. Specifically, the role of salts in promoting specific PMIs and their competition among various metals has remained untapped mainly due to the difficulty to distinguish nonspecific PMIs from specific PMIs by classic spectroscopic techniques. Herein, we report Hofmeister salts differentially promote the specific PMIs by combining nanoelectrospray ionization mass spectrometry and spectroscopic techniques (fluorescence measurement and circular dichroism). Furthermore, to explore the influence of salts in competitive binding between metalloproteins and various metals, we designed a series of competitive experiments and applied to a well-defined model system, the competitive binding of zinc (II) and arsenic (III) to holo-promyelocytic leukemia protein (PML). These experiments not only provided new insights at the molecular scale as complementary to previous NMR and spectroscopic results, but also deduced the relative binding ability between zinc finger proteins and metals at the molecular scale, which avoids the mass spectrometric titration-based determination of binding constants that is frequently affected and often degraded by variable solution conditions including salt contents. [Figure not available: see fulltext.

Structure And Specificity of a Quorum-Quenching Lactonase (AiiB) From Agrobacterium Tumefaciens

Energy Technology Data Exchange (ETDEWEB)

Liu, D.; Thomas, P.W.; Momb, J.; Hoang, Q.Q.; Petsko, G.A.; Ringe, D.; Fast, W.

2009-06-03

N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called 'quorum-quenching' enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be a metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.

Metallothionein as an Anti-Inflammatory Mediator

Directory of Open Access Journals (Sweden)

Ken-ichiro Inoue

2009-01-01

Full Text Available The integration of knowledge concerning the regulation of MT, a highly conserved, low molecular weight, cystein-rich metalloprotein, on its proposed functions is necessary to clarify how MT affects cellular processes. MT expression is induced/enhanced in various tissues by a number of physiological mediators. The cellular accumulation of MT depends on the availability of cellular zinc derived from the diet. MT modulates the binding and exchange/transport of heavy metals such as zinc, cadmium, or copper under physiological conditions and cytoprotection from their toxicities, and the release of gaseous mediators such as hydroxyl radicals or nitric oxide. In addition, MT reportedly affects a number of cellular processes, such as gene expression, apoptosis, proliferation, and differentiation. Given the genetic approach, the apparently healthy status of MT-deficient mice argues against an essential biological role for MT; however, this molecule may be critical in cells/tissues/organs in times of stress, since MT expression is also evoked/enhanced by various stresses. In particular, because metallothionein (MT is induced by inflammatory stress, its roles in inflammation are implied. Also, MT expression in various organs/tissues can be enhanced by inflammatory stimuli, implicating in inflammatory diseases. In this paper, we review the role of MT of various inflammatory conditions.

Cytochrome c interaction with hematite ({alpha}-Fe{sub 2}O{sub 3}) surfaces

Energy Technology Data Exchange (ETDEWEB)

Eggleston, Carrick M. [Department of Geology and Geophysics, University of Wyoming, Laramie, WY 82071 (United States)]. E-mail: carrick@uwyo.edu; Khare, Nidhi [Department of Geology and Geophysics, University of Wyoming, Laramie, WY 82071 (United States); Lovelace, David M. [Department of Geology and Geophysics, University of Wyoming, Laramie, WY 82071 (United States)

2006-02-15

The interaction of metalloproteins such as cytochromes with oxides is of interest for a number of reasons, including molecular catalysis of environmentally important mineral-solution electron transfer reactions (e.g., dehalogenations) and photovoltaic applications. Iron reduction by bacteria, thought to be cytochrome mediated, is of interest for geochemical and environmental remediation reasons. As a baseline for understanding cytochrome interaction with ferric oxide surfaces, we report on the interaction of mitochondrial cytochrome c (Mcc), a well-studied protein, with hematite ({alpha}-Fe{sub 2}O{sub 3}) surfaces. Mcc sorbs strongly to hematite from aqueous solution in a narrow pH range corresponding to opposite charge on Mcc and hematite (between pH 8.5 and 10, Mcc is positively charged and hematite surfaces are negatively charged). Cyclic voltammetry of Mcc using hematite electrodes gives redox potentials characteristic of Mcc in a native conformational state, with no evidence for unfolding on the hematite surface. Atomic force microscopy imaging is consistent with a loosely attached adsorbate that is easily deformed by the AFM tip. In phosphate-containing solution, Mcc adhers to the surface more strongly. These results establish hematite as a viable material for electrochemical and spectroscopic characterization of cytochrome-mineral interaction.

Fast Atomic Charge Calculation for Implementation into a Polarizable Force Field and Application to an Ion Channel Protein

Directory of Open Access Journals (Sweden)

Raiker Witter

2015-01-01

Full Text Available Polarization of atoms plays a substantial role in molecular interactions. Class I and II force fields mostly calculate with fixed atomic charges which can cause inadequate descriptions for highly charged molecules, for example, ion channels or metalloproteins. Changes in charge distributions can be included into molecular mechanics calculations by various methods. Here, we present a very fast computational quantum mechanical method, the Bond Polarization Theory (BPT. Atomic charges are obtained via a charge calculation method that depend on the 3D structure of the system in a similar way as atomic charges of ab initio calculations. Different methods of population analysis and charge calculation methods and their dependence on the basis set were investigated. A refined parameterization yielded excellent correlation of R=0.9967. The method was implemented in the force field COSMOS-NMR and applied to the histidine-tryptophan-complex of the transmembrane domain of the M2 protein channel of influenza A virus. Our calculations show that moderate changes of side chain torsion angle χ1 and small variations of χ2 of Trp-41 are necessary to switch from the inactivated into the activated state; and a rough two-side jump model of His-37 is supported for proton gating in accordance with a flipping mechanism.

Transcriptional Response of Desulfovibrio vulgaris Hildenborough to Oxidative Stress Mimicking Environmental Conditions

Energy Technology Data Exchange (ETDEWEB)

Pereira, Patricia M.; He, Qiang; Xavier, Antonio V.; Zhou, Jizhong; Pereira, Ines A.C.; Louro, Ricardo O.

2008-03-12

Sulphate-reducing bacteria are anaerobes readily found in oxic-anoxic interfaces. Multiple defence pathways against oxidative conditions were identified in these organisms and proposed to be differentially expressed under different concentrations of oxygen, contributing to their ability to survive oxic conditions. In this study, Desulfovibrio vulgaris Hildenborough cells were exposed to the highest concentration of oxygen that sulphate-reducing bacteria are likely to encounter in natural habitats, and the global transcriptomic response was determined. 307 genes were responsive, with cellular roles in energy metabolism, protein fate, cell envelope and regulatory functions, including multiple genes encoding heat shock proteins, peptidases and proteins with heat shock promoters. Of the oxygen reducing mechanisms of D. vulgaris only the periplasmic hydrogen-dependent mechanism is up-regulated, involving the [NiFeSe]hydrogenase, formate dehydrogenase(s) and the Hmc membrane complex. The oxidative defence response concentrates on damage repair by metal-free enzymes. These data, together with the down regulation of the Fur operon, which restricts the availability of iron, and the lack of response of the PerR operon, suggest that a major effect of this oxygen stress is the inactivation and/or degradation of multiple metalloproteins present in D. vulgaris as a consequence of oxidative damage to their metal clusters.

Studies on the effect of divalent metal ions on exfoliative toxins from Staphylococcus hyicus: indications of ExhA and ExhB being metalloproteins

DEFF Research Database (Denmark)

Andresen, Lars Ole

1999-01-01

determined in ELISA. Other metal chelators had varied effect in the detection of the toxins in ELISA. It was, however, not possible to restore the recognition of toxins by the monoclonal antibodies by incubation of EDDHA-dialyzed toxin preparations with Co2+, Cu2+ or Zn2+. The results of this study suggest...

Preconcentration and Speciation of Trace Elements and Trace-Element Analogues of Radionuclides by Neutron Activation Analysis

International Nuclear Information System (INIS)

Chatt, A.

1999-01-01

We have developed a number of preconcentration neutron activation analysis (PNAA) methods in our laboratory for the determination of trace elements in a variety of complex sample matrices. We developed a number of cocrystallization and coprecipitation methods for the determination of trace elements in water samples. We developed several methods for the determination of I in foods and diets. We have developed a number of PNAA methods in our laboratory We determined As and Sb in geological materials and natural waters by coprecipitation with Se and Au in silicate rocks and ores by coprecipitation with Te followed by NAA. We developed an indirect NAA method for the determination of B in leachates of borosilicate glass. We have been interested in studying the speciation of Am, Tc, and Np in simulated vitrified groundwater leachates of high-level wastes under oxid and anoxic conditions using a number of techniques. We then used PNAA methods to study speciation of trace-element analogues of radionuclides. We have been able to apply biochemical techniques and NAA for the separation, preconcentration, and characterization of metalloprotein and protein-bound trace-element species in subcellular fractions of bovine kidneys. Lately, we have concentrated our efforts to develop chemical and biochemical methods in conjunction with NAA, NMR, and MS for the separation and identification of extractable organohalogens (EOX) in tissues of beluga whales, cod, and northern pink shrimp

Quantitative investigation of two metallohydrolases by X-ray absorption spectroscopy near-edge spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Zhao, W. [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Chu, W.S.; Yang, F.F.; Yu, M.J.; Chen, D.L.; Guo, X.Y. [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); Zhou, D.W.; Shi, N. [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Marcelli, A. [Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali di Frascati, P.O. Box 13, Frascati 00044 (Italy); Niu, L.W.; Teng, M.K. [Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Gong, W.M. [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Benfatto, M. [Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali di Frascati, P.O. Box 13, Frascati 00044 (Italy); Wu, Z.Y. [Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 (China); Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali di Frascati, P.O. Box 13, Frascati 00044 (Italy)], E-mail: wuzy@ihep.ac.cn

2007-09-21

The last several years have witnessed a tremendous increase in biological applications using X-ray absorption spectroscopy (BioXAS), thanks to continuous advancements in synchrotron radiation (SR) sources and detector technology. However, XAS applications in many biological systems have been limited by the intrinsic limitations of the Extended X-ray Absorption Fine Structure (EXAFS) technique e.g., the lack of sensitivity to bond angles. As a consequence, the application of the X-ray absorption near-edge structure (XANES) spectroscopy changed this scenario that is now continuously changing with the introduction of the first quantitative XANES packages such as Minut XANES (MXAN). Here we present and discuss the XANES code MXAN, a novel XANES-fitting package that allows a quantitative analysis of experimental data applied to Zn K-edge spectra of two metalloproteins: Leptospira interrogans Peptide deformylase (LiPDF) and acutolysin-C, a representative of snake venom metalloproteinases (SVMPs) from Agkistrodon acutus venom. The analysis on these two metallohydrolases reveals that proteolytic activities are correlated to subtle conformation changes around the zinc ion. In particular, this quantitative study clarifies the occurrence of the LiPDF catalytic mechanism via a two-water-molecules model, whereas in the acutolysin-C we have observed a different proteolytic activity correlated to structural changes around the zinc ion induced by pH variations.

Quantitative investigation of two metallohydrolases by X-ray absorption spectroscopy near-edge spectroscopy

International Nuclear Information System (INIS)

Zhao, W.; Chu, W.S.; Yang, F.F.; Yu, M.J.; Chen, D.L.; Guo, X.Y.; Zhou, D.W.; Shi, N.; Marcelli, A.; Niu, L.W.; Teng, M.K.; Gong, W.M.; Benfatto, M.; Wu, Z.Y.

2007-01-01

The last several years have witnessed a tremendous increase in biological applications using X-ray absorption spectroscopy (BioXAS), thanks to continuous advancements in synchrotron radiation (SR) sources and detector technology. However, XAS applications in many biological systems have been limited by the intrinsic limitations of the Extended X-ray Absorption Fine Structure (EXAFS) technique e.g., the lack of sensitivity to bond angles. As a consequence, the application of the X-ray absorption near-edge structure (XANES) spectroscopy changed this scenario that is now continuously changing with the introduction of the first quantitative XANES packages such as Minut XANES (MXAN). Here we present and discuss the XANES code MXAN, a novel XANES-fitting package that allows a quantitative analysis of experimental data applied to Zn K-edge spectra of two metalloproteins: Leptospira interrogans Peptide deformylase (LiPDF) and acutolysin-C, a representative of snake venom metalloproteinases (SVMPs) from Agkistrodon acutus venom. The analysis on these two metallohydrolases reveals that proteolytic activities are correlated to subtle conformation changes around the zinc ion. In particular, this quantitative study clarifies the occurrence of the LiPDF catalytic mechanism via a two-water-molecules model, whereas in the acutolysin-C we have observed a different proteolytic activity correlated to structural changes around the zinc ion induced by pH variations

Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones.

Science.gov (United States)

Maianti, Juan Pablo; McFedries, Amanda; Foda, Zachariah H; Kleiner, Ralph E; Du, Xiu Quan; Leissring, Malcolm A; Tang, Wei-Jen; Charron, Maureen J; Seeliger, Markus A; Saghatelian, Alan; Liu, David R

2014-07-03

Despite decades of speculation that inhibiting endogenous insulin degradation might treat type-2 diabetes, and the identification of IDE (insulin-degrading enzyme) as a diabetes susceptibility gene, the relationship between the activity of the zinc metalloprotein IDE and glucose homeostasis remains unclear. Although Ide(-/-) mice have elevated insulin levels, they exhibit impaired, rather than improved, glucose tolerance that may arise from compensatory insulin signalling dysfunction. IDE inhibitors that are active in vivo are therefore needed to elucidate IDE's physiological roles and to determine its potential to serve as a target for the treatment of diabetes. Here we report the discovery of a physiologically active IDE inhibitor identified from a DNA-templated macrocycle library. An X-ray structure of the macrocycle bound to IDE reveals that it engages a binding pocket away from the catalytic site, which explains its remarkable selectivity. Treatment of lean and obese mice with this inhibitor shows that IDE regulates the abundance and signalling of glucagon and amylin, in addition to that of insulin. Under physiological conditions that augment insulin and amylin levels, such as oral glucose administration, acute IDE inhibition leads to substantially improved glucose tolerance and slower gastric emptying. These findings demonstrate the feasibility of modulating IDE activity as a new therapeutic strategy to treat type-2 diabetes and expand our understanding of the roles of IDE in glucose and hormone regulation.

Purification, crystallization and preliminary X-ray diffraction studies on avian haemoglobin from pigeon (Columba livia)

Science.gov (United States)

Sathya Moorthy, Pon.; Neelagandan, K.; Balasubramanian, M.; Ponnuswamy, M. N.

2009-01-01

Haemoglobin is a physiologically significant metalloprotein that is involved in the exchange of gases for sustaining life. The respiratory system of birds is unique and complex compared with that of mammals. Many investigations of avian haemoglobins have revealed the presence of inositol pentaphosphate (IP5), a principal allosteric effector that is involved in regulation of their function. Structural investigations of avian haemoglobins are presently not adequate to explain their function. Efforts have been made in this direction in order to understand the oxygen-binding affinity involved in adapting to hypoxia in avian haemoglobins. Fresh whole blood was collected from pigeon (Columba livia) and purified using a DEAE cellulose anion-exchange chromatographic column. Crystallization of pigeon haemoglobin was accomplished using the hanging-drop vapour-diffusion method using PEG 3350 as a precipitant in 50 mM sodium acetate buffer pH 5.5 with 1 M NaCl. Data collection was carried out using a MAR345 image-plate detector system. The crystals diffracted to 2 Å resolution. Pigeon haemoglobin crystallizes in a triclinic space group, with two whole biological molecules in the asymmetric unit and with unit-cell parameters a = 55.005, b = 65.528, c = 104.370 Å, α = 78.742, β = 89.819, γ = 65.320°. PMID:19194000

Physics-based scoring of protein-ligand interactions: explicit polarizability, quantum mechanics and free energies.

Science.gov (United States)

Bryce, Richard A

2011-04-01

The ability to accurately predict the interaction of a ligand with its receptor is a key limitation in computer-aided drug design approaches such as virtual screening and de novo design. In this article, we examine current strategies for a physics-based approach to scoring of protein-ligand affinity, as well as outlining recent developments in force fields and quantum chemical techniques. We also consider advances in the development and application of simulation-based free energy methods to study protein-ligand interactions. Fuelled by recent advances in computational algorithms and hardware, there is the opportunity for increased integration of physics-based scoring approaches at earlier stages in computationally guided drug discovery. Specifically, we envisage increased use of implicit solvent models and simulation-based scoring methods as tools for computing the affinities of large virtual ligand libraries. Approaches based on end point simulations and reference potentials allow the application of more advanced potential energy functions to prediction of protein-ligand binding affinities. Comprehensive evaluation of polarizable force fields and quantum mechanical (QM)/molecular mechanical and QM methods in scoring of protein-ligand interactions is required, particularly in their ability to address challenging targets such as metalloproteins and other proteins that make highly polar interactions. Finally, we anticipate increasingly quantitative free energy perturbation and thermodynamic integration methods that are practical for optimization of hits obtained from screened ligand libraries.

Development of METAL-ACTIVE SITE and ZINCCLUSTER tool to predict active site pockets.

Science.gov (United States)

Ajitha, M; Sundar, K; Arul Mugilan, S; Arumugam, S

2018-03-01

The advent of whole genome sequencing leads to increasing number of proteins with known amino acid sequences. Despite many efforts, the number of proteins with resolved three dimensional structures is still low. One of the challenging tasks the structural biologists face is the prediction of the interaction of metal ion with any protein for which the structure is unknown. Based on the information available in Protein Data Bank, a site (METALACTIVE INTERACTION) has been generated which displays information for significant high preferential and low-preferential combination of endogenous ligands for 49 metal ions. User can also gain information about the residues present in the first and second coordination sphere as it plays a major role in maintaining the structure and function of metalloproteins in biological system. In this paper, a novel computational tool (ZINCCLUSTER) is developed, which can predict the zinc metal binding sites of proteins even if only the primary sequence is known. The purpose of this tool is to predict the active site cluster of an uncharacterized protein based on its primary sequence or a 3D structure. The tool can predict amino acids interacting with a metal or vice versa. This tool is based on the occurrence of significant triplets and it is tested to have higher prediction accuracy when compared to that of other available techniques. © 2017 Wiley Periodicals, Inc.

Decarboxylation of oxalacetate to pyruvate by purified avian liver phosphoenolpyruvate carboxykinase

Energy Technology Data Exchange (ETDEWEB)

Noce, P S; Utter, M F

1975-01-01

Phosphoenolpyruvate carboxykinase, which has been isolated from chicken liver mitochondria in essentially homogenous form, carries out the irreversible decarboxylation of oxalacetate to pyruvate in the presence of catalytic amounts of GDP or IDP, as well as the reversible decarboxylation of oxalacetate to phosphoenolpyruvate in the presence of substrate amounts of GTP or ITP. The pyruvate- and phosphoenolpyruvate-forming reactions are similar in their nucleoside specificity and appear to be carried out by the same protein. However, the two activities vary markedly in their response to added metal ions and sulfhydryl reagents. Phosphoenolpyruvate formation is completely dependent on the presence of a divalent metal ion, with Mn/sup 2 +/ the most effective species. This reaction is also stimulated by sulfhydryl reagents such as 2-mercaptoethanol. In contrast, the pyruvate-forming reaction is strongly inhibited by divalent metal ions, including Mn/sup 2 +/, and also by moderate concentrations of sulfhydryl reagents. These observations and the demonstration that pyruvate kinase-like activity is very low or absent make it unlikely that pyruvate formation proceeds via phosphoenolpyruvate as an intermediate. Although the pyruvate-forming reaction is inhibited by added metal ions, the reaction is also inhibited by metal-chelating agents such as 8-hydroxyquinoline and o-phenanthroline, suggesting that the reaction is dependent on the presence of a metal ion. It has not been possible, however, to demonstrate that the enzyme is a metalloprotein.

Biosynthesis, isolation and characterization of {sup 57}Fe-enriched Phaseolus vulgaris ferritin after heterologous expression in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Hoppler, Matthias [ETH Zurich, Laboratory of Human Nutrition, Zurich (Switzerland); Meile, Leo [ETH Zurich, Laboratory of Food Biotechnology, Zurich (Switzerland); Walczyk, Thomas [National University of Singapore, Department of Chemistry and Department of Biochemistry, Singapore (Singapore)

2008-01-15

Ferritin is the major iron storage protein in the biosphere. Iron stores of an organism are commonly assessed by measuring the concentration of the protein shell of the molecule in fluids and tissues. The amount of ferritin-bound iron, the more desirable information, still remains inaccessible owing to the lack of suitable techniques. Iron saturation of ferritin is highly variable, with a maximum capacity of 4,500 iron atoms per molecule. This study describes the direct isotopic labeling of a complex metalloprotein in vivo by biosynthesis, in order to measure ferritin-bound iron by isotope dilution mass spectrometry. [{sup 57}Fe]ferritin was produced by cloning and overexpressing the Phaseolus vulgaris ferritin gene pfe in Escherichia coli in the presence of {sup 57}FeCl{sub 2}. Recombinant ferritin was purified in a fully assembled form and contained approximately 1,000 iron atoms per molecule at an isotopic enrichment of more than 95% {sup 57}Fe. We did not find any evidence of species conversion of the isotopic label for at least 5 months of storage at -20 C. Transfer efficiency of enriched iron into [{sup 57}Fe]ferritin of 20% was sufficient to be economically feasible. Negligible amounts of non-ferritin-bound iron in the purified [{sup 57}Fe]ferritin solution allows for use of this spike for quantification of ferritin-bound iron by isotope dilution mass spectrometry. (orig.)

XFEL diffraction: developing processing methods to optimize data quality

Energy Technology Data Exchange (ETDEWEB)

Sauter, Nicholas K., E-mail: nksauter@lbl.gov [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States)

2015-01-29

Bragg spots recorded from a still crystal necessarily give partial measurements of the structure factor intensity. Correction to the full-spot equivalent, relying on both a physical model for crystal disorder and postrefinement of the crystal orientation, improves the electron density map in serial crystallography. Serial crystallography, using either femtosecond X-ray pulses from free-electron laser sources or short synchrotron-radiation exposures, has the potential to reveal metalloprotein structural details while minimizing damage processes. However, deriving a self-consistent set of Bragg intensities from numerous still-crystal exposures remains a difficult problem, with optimal protocols likely to be quite different from those well established for rotation photography. Here several data processing issues unique to serial crystallography are examined. It is found that the limiting resolution differs for each shot, an effect that is likely to be due to both the sample heterogeneity and pulse-to-pulse variation in experimental conditions. Shots with lower resolution limits produce lower-quality models for predicting Bragg spot positions during the integration step. Also, still shots by their nature record only partial measurements of the Bragg intensity. An approximate model that corrects to the full-spot equivalent (with the simplifying assumption that the X-rays are monochromatic) brings the distribution of intensities closer to that expected from an ideal crystal, and improves the sharpness of anomalous difference Fourier peaks indicating metal positions.

Metallomics of two microorganisms relevant to heavy metal bioremediation reveal fundamental differences in metal assimilation and utilization

Energy Technology Data Exchange (ETDEWEB)

Lancaster, Andrew [Univ. of Georgia, Athens, GA (United States); Menon, Angeli [Univ. of Georgia, Athens, GA (United States); Scott, Israel [Univ. of Georgia, Athens, GA (United States); Poole, Farris [Univ. of Georgia, Athens, GA (United States); Vaccaro, Brian [Univ. of Georgia, Athens, GA (United States); Thorgersen, Michael P. [Univ. of Georgia, Athens, GA (United States); Geller, Jil [Lawrence Berkeley National Laboratory (LBNL); Hazen, Terry C. [Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Hurt Jr., Richard Ashley [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Brown, Steven D. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Elias, Dwayne A. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Adams, Michael W. W. [Univ. of Georgia, Athens, GA (United States)

2014-03-26

Although as many as half of all proteins are thought to require a metal cofactor, the metalloproteomes of microorganisms remain relatively unexplored. Microorganisms from different environments are likely to vary greatly in the metals that they assimilate, not just among the metals with well-characterized roles but also those lacking any known function. Herein we investigated the metal utilization of two microorganisms that were isolated from very similar environments and are of interest because of potential roles in the immobilization of heavy metals, such as uranium and chromium. The metals assimilated and their concentrations in the cytoplasm of Desulfovibrio vulgaris strain Hildenborough (DvH) and Enterobacter cloacae strain Hanford (EcH) varied dramatically, with a larger number of metals present in Enterobacter. For example, a total of 9 and 19 metals were assimilated into their cytoplasmic fractions, respectively, and DvH did not assimilate significant amounts of zinc or copper whereas EcH assimilated both. However, bioinformatic analysis of their genome sequences revealed a comparable number of predicted metalloproteins, 813 in DvH and 953 in EcH. These allowed some rationalization of the types of metal assimilated in some cases (Fe, Cu, Mo, W, V) but not in others (Zn, Nd, Ce, Pr, Dy, Hf and Th). It was also shown that U binds an unknown soluble protein in EcH but this incorporation was the result of extracellular U binding to cytoplasmic components after cell lysis.

Purification, crystallization and preliminary X-ray diffraction studies on avian haemoglobin from pigeon (Columba livia)

International Nuclear Information System (INIS)

Sathya Moorthy, Pon.; Neelagandan, K.; Balasubramanian, M.; Ponnuswamy, M. N.

2009-01-01

Crystallization of pigeon haemoglobin at low pH (5.5) and high ionic concentration (1 M) using the hanging-drop vapour-diffusion method is reported. Haemoglobin is a physiologically significant metalloprotein that is involved in the exchange of gases for sustaining life. The respiratory system of birds is unique and complex compared with that of mammals. Many investigations of avian haemoglobins have revealed the presence of inositol pentaphosphate (IP5), a principal allosteric effector that is involved in regulation of their function. Structural investigations of avian haemoglobins are presently not adequate to explain their function. Efforts have been made in this direction in order to understand the oxygen-binding affinity involved in adapting to hypoxia in avian haemoglobins. Fresh whole blood was collected from pigeon (Columba livia) and purified using a DEAE cellulose anion-exchange chromatographic column. Crystallization of pigeon haemoglobin was accomplished using the hanging-drop vapour-diffusion method using PEG 3350 as a precipitant in 50 mM sodium acetate buffer pH 5.5 with 1 M NaCl. Data collection was carried out using a MAR345 image-plate detector system. The crystals diffracted to 2 Å resolution. Pigeon haemoglobin crystallizes in a triclinic space group, with two whole biological molecules in the asymmetric unit and with unit-cell parameters a = 55.005, b = 65.528, c = 104.370 Å, α = 78.742, β = 89.819, γ = 65.320°

Development of an x-ray beam line at the NSLS for studies in materials science using x-ray absorption spectroscopy: Annual progress report

International Nuclear Information System (INIS)

Sayers, D.E.

1986-01-01

Although only in operation since May, 1985, the X-11 participation research team (PRT) at the NSLS has already demonstrated that it is one of the leading centers of x-ray absorption spectroscopy (XAS). During this time, results have been obtained and programs initiated in a number of areas, for example: interfaces, including deposited metal-metal and metal-semiconductor systems, multilayers and ion implanted layers; electrochemical systems, including Pt electrode fuel cells, Ni oxide battery electrodes, conducting polymers, passivation and corrosion; catalysts, including highly-dispersed supported metal catalysts and zeolite systems; quasi-crystals, heavy fermion systems, uranium and neptunium compounds, rare gas clusters, disordered metals and semiconductors, ferroelectric transition; and, biological systems and related models, including synthetic porphyrins and a number of metalloproteins. In concert with these scientific results have been a number of developments involving the technique itself. These include implementation of unique optical systems on both the A and B lines for optical performance over their designed energy ranges, advances in experimental capability, particular in glancing angle studies, optimization of ion chambers for surface studies, the improvement of electron yield detectors, and improved software for data acquisition and analysis. This report emphasizes some of the research highlights and significant developments of our PRT which occurred during the past year. A detailed bibliography of papers and talks resulting from work done at our beamline and the progress reports for our PRT which were in the 1985 NSLS Annual Report are appended

Assessment of Quantum Mechanical Methods for Copper and Iron Complexes by Photoelectron Spectroscopy.

Science.gov (United States)

Niu, Shuqiang; Huang, Dao-Ling; Dau, Phuong D; Liu, Hong-Tao; Wang, Lai-Sheng; Ichiye, Toshiko

2014-03-11

Broken-symmetry density functional theory (BS-DFT) calculations are assessed for redox energetics [Cu(SCH 3 ) 2 ] 1-/0 , [Cu(NCS) 2 ] 1-/0 , [FeCl 4 ] 1-/0 , and [Fe(SCH 3 ) 4 ] 1-/0 against vertical detachment energies (VDE) from valence photoelectron spectroscopy (PES), as a prelude to studies of metalloprotein analogs. The M06 and B3LYP hybrid functionals give VDE that agree with the PES VDE for the Fe complexes, but both underestimate it by ∼400 meV for the Cu complexes; other hybrid functionals give VDEs that are an increasing function of the amount of Hartree-Fock (HF) exchange and so cannot show good agreement for both Cu and Fe complexes. Range-separated (RS) functionals appear to give a better distribution of HF exchange since the negative HOMO energy is approximately equal to the VDEs but also give VDEs dependent on the amount of HF exchange, sometimes leading to ground states with incorrect electron configurations; the LRC- ω PBEh functional reduced to 10% HF exchange at short-range give somewhat better values for both, although still ∼150 meV too low for the Cu complexes and ∼50 meV too high for the Fe complexes. Overall, the results indicate that while HF exchange compensates for self-interaction error in DFT calculations of both Cu and Fe complexes, too much may lead to more sensitivity to nondynamical correlation in the spin-polarized Fe complexes.

Theoretical calculations of physico-chemical and spectroscopic properties of bioinorganic systems: current limits and perspectives.

Science.gov (United States)

Rokob, Tibor András; Srnec, Martin; Rulíšek, Lubomír

2012-05-21

In the last decade, we have witnessed substantial progress in the development of quantum chemical methodologies. Simultaneously, robust solvation models and various combined quantum and molecular mechanical (QM/MM) approaches have become an integral part of quantum chemical programs. Along with the steady growth of computer power and, more importantly, the dramatic increase of the computer performance to price ratio, this has led to a situation where computational chemistry, when exercised with the proper amount of diligence and expertise, reproduces, predicts, and complements the experimental data. In this perspective, we review some of the latest achievements in the field of theoretical (quantum) bioinorganic chemistry, concentrating mostly on accurate calculations of the spectroscopic and physico-chemical properties of open-shell bioinorganic systems by wave-function (ab initio) and DFT methods. In our opinion, the one-to-one mapping between the calculated properties and individual molecular structures represents a major advantage of quantum chemical modelling since this type of information is very difficult to obtain experimentally. Once (and only once) the physico-chemical, thermodynamic and spectroscopic properties of complex bioinorganic systems are quantitatively reproduced by theoretical calculations may we consider the outcome of theoretical modelling, such as reaction profiles and the various decompositions of the calculated parameters into individual spatial or physical contributions, to be reliable. In an ideal situation, agreement between theory and experiment may imply that the practical problem at hand, such as the reaction mechanism of the studied metalloprotein, can be considered as essentially solved.

Metalloproteinase 11, potential marker and molecular target in advanced and castration-resistant prostate cancer. Culture study of peritumoral fibroblasts.

Science.gov (United States)

Fernandez-Gomez, J M; Eiro, N; García-Rodríguez, J J; Quintás-Blanco, A; Gonzalez-Ruiz de León, C; Perez de Haro, M L; Vizoso-Piñero, F

To analyze the expression of metalloprotein 11 (MMP11) in cultured fibroblasts obtained from human prostate tumors with different clinical and pathological characteristics. For this study we analyzed samples of transrectal prostate biopsies from tumors with different characteristics, treated with or whithout androgen deprivation (AD). After optimization of the culture method, fibroblasts were isolated and cultured to perform the study (PCR) of MMP11 mRNA. Finally, 37 cases were studied: 5 samples of benign prostatic hyperplasia, 14 cases with localized neoplasms (7 high-risk according to the D'Amico classification), 5 with metastasic tumors (bone metastases), and 13 treated with AD therapy, of which 6 fulfilled the requirements to be defined as resistant to castration. In tumors without AD therapy, MMP11 expression was significantly higher (P=.001) in fibroblasts of higher grade tumors. A significant (P=.001) correlation was found between PSA and expression of MMP11 in fibroblast s and a significant increase of MMP11 expression in metastatic tumors. In tumors with AD therapy, a significantly greater expression of MMP11 was observed in resistant to castration patients than in those sensitive to castration (P=.003). In advanced prostate tumors or in stages of increased tumor aggressiveness, the production of MMP11 by fibroblasts is significantly greater than in non-metastatic tumors or in AD sensitive tumors. Copyright © 2016 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

2008 GRC Iron Sulfur Enzymes-Conference to be held June 8-13, 2008

Energy Technology Data Exchange (ETDEWEB)

Cramer, Stephen [Univ. of California, Davis, CA (United States); Gray, Nancy Ryan [Gordon Research Conferences, West Kingston, RI (United States)

2009-01-01

Iron-sulfur proteins are among the most common and ancient enzymes and electron-transfer agents in nature. They play key roles in photosynthesis, respiration, and the metabolism of small molecules such as H2, CO, and N2. The Iron Sulfur Enzyme Gordon Research Conference evolved from an earlier GRC on Nitrogen Fixation that began in 1994. The scope of the current meeting has broadened to include all enzymes or metalloproteins in which Fe-S bonds play a key role. This year's meeting will focus on the biosynthesis of Fe-S clusters, as well as the structure and mechanism of key Fe-S enzymes such as hydrogenase, nitrogenase and its homologues, radical SAM enzymes, and aconitase-related enzymes. Recent progress on the role of Fe-S enzymes in health, disease, DNA/RNA-processing, and alternative bio-energy systems will also be highlighted. This conference will assemble a broad, diverse, and international group of biologists and chemists who are investigating fundamental issues related to Fe-S enzymes, on atomic, molecular, organism, and environmental scales. The topics to be addressed will include: Biosynthesis & Genomics of Fe-S Enzymes; Fundamental Fe-S Chemistry; Hydrogen and Fe-S Enzymes; Nitrogenase & Homologous Fe-S Enzymes; Fe-S Enzymes in Health & Disease; Radical SAM and Aconitase-Related Fe-S Enzymes; Fe-S Enzymes and Synthetic Analogues in BioEnergy; and Fe-S Enzymes in Geochemistry and the Origin of Life.

Cupryphans, metal-binding, redox-active, redesigned conopeptides.

Science.gov (United States)

Barba, Marco; Sobolev, Anatoli P; Romeo, Cristina; Schininà, M Eugenia; Pietraforte, Donatella; Mannina, Luisa; Musci, Giovanni; Polticelli, Fabio

2009-03-01

Contryphans are bioactive peptides, isolated from the venom of marine snails of the genus Conus, which are characterized by the short length of the polypeptide chain and the high degree of unusual post-translational modifications. The cyclization of the polypeptide chain through a single disulphide bond, the presence of two conserved Pro residues, and the epimerization of a Trp/Leu residue confer to Contryphans a stable and well-defined structure in solution, conserved in all members of the family, and tolerant to multiple substitutions. The potential of Contryphans as scaffolds for the design of redox-active (macro)molecules was tested by engineering a copper-binding site on two different variants of the natural peptide Contryphan-Vn. The binding site was designed by computational modeling, and the redesigned peptides were synthesized and characterized by optical, fluorescence, electron spin resonance, and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupryphan and Arg-Cupryphan, bind Cu(2+) ions with a 1:1 stoichiometry and a K(d) in the 100 nM range. Other divalent metals (e.g., Zn(2+) and Mg(2+)) are bound with much lower affinity. In addition, Cupryphans catalyze the dismutation of superoxide anions with an activity comparable to other nonpeptidic superoxide dismutase mimics. We conclude that the Contryphan motif represents a natural robust scaffold which can be engineered to perform different functions, providing additional means for the design of catalytically active mini metalloproteins.

Feasibility of asymmetric flow field-flow fractionation coupled to ICP-MS for the characterization of wear metal particles and metalloproteins in biofluids from hip replacement patients

DEFF Research Database (Denmark)

Löschner, Katrin; Harrington, Chris F.; Kearney, Jacque-Lucca

2015-01-01

or other elements, but the current analytical methods used to investigate the processes involved do not provide sufficient information to understand the size or composition of the wear particles generated in vivo. In this qualitative feasibility study, asymmetric flow field-flow fractionation (AF4) coupled...... to ICP-MS was used to confirm the metal–protein associations in the serum samples. Off-line single particle ICP-MS (spICP-MS) analysis was used to confirm the approximate size distribution indicated by AF4 of the wear particles in hip aspirates. In the serum samples, AF4–ICP-MS suggested that Cr...... unidentified compounds; AEC analysis confirmed the Cr results and the association of Co with Alb and a second compound. Enzymatic digestion of the hip aspirate sample, followed by separation using AF4 with detection by UV absorption (280 nm), multi-angle light scattering and ICP-MS, suggested that the sizes...

Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex

Science.gov (United States)

Zhou, Mowei; Yan, Jing; Romano, Christine A.; Tebo, Bradley M.; Wysocki, Vicki H.; Paša-Tolić, Ljiljana

2018-01-01

Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase, Mnx, from Bacillus sp. PL-12 has been successfully overexpressed in active form as a protein complex with a molecular mass of 211 kDa. We have recently used surface induced dissociation (SID) and ion mobility-mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data from the native mass spectrometry experiments led to a plausible structural model of this multicopper oxidase, which has been difficult to study by conventional structural biology methods. It was also revealed that each Mnx subunit binds a variable number of copper ions. Becasue of the heterogeneity of the protein and limited mass resolution, ambiguities in assigning some of the observed peaks remained as a barrier to fully understanding the role of metals and potential unknown ligands in Mnx. In this study, we performed SID in a modified Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer. The high mass accuracy and resolution offered by FTICR unveiled unexpected artificial modifications on the protein that had been previously thought to be iron bound species based on lower resolution spectra. Additionally, isotopically resolved spectra of the released subcomplexes revealed the metal binding stoichiometry at different structural levels. This method holds great potential for in-depth characterization of metalloproteins and protein-ligand complexes. [Figure not available: see fulltext.

Project in determination of crystal structure of nitrogen fixation proteins from azospirilum brasiliense and herbaspirilum seropedicae by synchrotron x-ray diffraction

International Nuclear Information System (INIS)

Barbosa, Valma M.; Leggs, Luciana A.; Delboni, Luis F.; Chubatsu, LedaS.; Souza, Emanuel M.; Machado, Hidevaldo B.; Yates, Geoffrey M.; Pedrosa, Fabio O.

1996-01-01

Full text. Biological nitrogen fixation is essential for maintaining the nitrogen cycle on earth and of high importance for Brazilian agriculture. The nitrogenase enzyme system, which provides the biochemical machinery for nitrogen fixation, consists of two component metalloproteins, the molybdenumiron (Mo Fe) protein and the iron (Fe) protein. Nitrogen fixation is a very energy-intensive process, requiring around 16 moles of ATP for each mol of N 2 fixed (reduced). As a consequence, synthesis and activity of nitrogenase is tighty regulated at two levels: general and specific. The general level regulation is mediated by the ntr (nitrogen regulation) system. Two gene products are involved: the ntrB gene product (NtrB) is responsible for the activation of the ntrC gene product (NtrC) by phosphorylating a conserved Asp54, which activates the expression of the nifA gene. The nif specific control system is mediated by the NifA protein, which binds to a DNA specific sequence (UAS, Upstream Activator Sequence) and activates nif promoter transcriptions by RNA polymerase- α54 , following ATP hydrolysis. The aim of this project is to solve the crystal structure of dinitrogenase reductase (iron protein) and dinitrogenase (molybdenum-iron protein) from Azospirilim brasiliense and the regulatory proteins NifA from Herbaspirillum seropedicae and NtrC Azospirillum brasiliense. The three dimensional structure of the proteins involved in this project will allow a better understanding of the mechanism of biological nitrogen fixation. To this end, the data collection will probably be done at the LNLS facilities which will be available in the near future. (author)

Stepwise O-Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening.

Science.gov (United States)

Shin, Inchul; Ambler, Brett R; Wherritt, Daniel; Griffith, Wendell P; Maldonado, Amanda C; Altman, Ryan A; Liu, Aimin

2018-03-28

Heme-based tryptophan dioxygenases are established immunosuppressive metalloproteins with significant biomedical interest. Here, we synthesized two mechanistic probes to specifically test if the α-amino group of the substrate directly participates in a critical step of the O atom transfer during catalysis in human tryptophan 2,3-dioxygenase (TDO). Substitution of the nitrogen atom of the substrate to a carbon (probe 1) or oxygen (probe 2) slowed the catalytic step following the first O atom transfer such that transferring the second O atom becomes less likely to occur, although the dioxygenated products were observed with both probes. A monooxygenated product was also produced from probe 2 in a significant quantity. Analysis of this new product by HPLC coupled UV-vis spectroscopy, high-resolution mass spectrometry, 1 H NMR, 13 C NMR, HSQC, HMBC, and infrared (IR) spectroscopies concluded that this monooxygenated product is a furoindoline compound derived from an unstable epoxyindole intermediate. These results prove that small molecules can manipulate the stepwise O atom transfer reaction of TDO and provide a showcase for a tunable mechanism by synthetic compounds. The product analysis results corroborate the presence of a substrate-based epoxyindole intermediate during catalysis and provide the first substantial experimental evidence for the involvement of the substrate α-amino group in the epoxide ring-opening step during catalysis. This combined synthetic, biochemical, and biophysical study establishes the catalytic role of the α-amino group of the substrate during the O atom transfer reactions and thus represents a substantial advance to the mechanistic comprehension of the heme-based tryptophan dioxygenases.

Substrate analog interaction with MCR-1 offers insight into the rising threat of the plasmid-mediated transferable colistin resistance.

Science.gov (United States)

Wei, Pengcheng; Song, Guangji; Shi, Mengyang; Zhou, Yafei; Liu, Yang; Lei, Jun; Chen, Peng; Yin, Lei

2018-02-01

Colistin is considered a last-resort antibiotic against most gram-negative bacteria. Recent discoveries of a plasmid-mediated, transferable mobilized colistin-resistance gene ( mcr-1) on all continents have heralded the imminent emergence of pan-drug-resistant superbacteria. The inner-membrane protein MCR-1 can catalyze the transfer of phosphoethanolamine (PEA) to lipid A, resulting in colistin resistance. However, little is known about the mechanism, and few drugs exist to address this issue. We present crystal structures revealing the MCR-1 catalytic domain (cMCR-1) as a monozinc metalloprotein with ethanolamine (ETA) and d-glucose, respectively, thus highlighting 2 possible substrate-binding pockets in the MCR-1-catalyzed PEA transfer reaction. Mutation of the residues involved in ETA and d-glucose binding impairs colistin resistance in recombinant Escherichia coli containing full-length MCR-1. Partial analogs of the substrate are used for cocrystallization with cMCR-1, providing valuable information about the family of PEA transferases. One of the analogs, ETA, causes clear inhibition of polymyxin B resistance, highlighting its potential for drug development. These data demonstrate the crucial role of the PEA- and lipid A-binding pockets and provide novel insights into the structure-based mechanisms, important drug-target hot spots, and a drug template for further drug development to combat the urgent, rising threat of MCR-1-mediated antibiotic resistance.-Wei, P., Song, G., Shi, M., Zhou, Y., Liu, Y., Lei, J., Chen, P., Yin, L. Substrate analog interaction with MCR-1 offers insight into the rising threat of the plasmid-mediated transferable colistin resistance.

Inhibition of the myostatin/Smad signaling pathway by short decorin-derived peptides.

Science.gov (United States)

El Shafey, Nelly; Guesnon, Mickaël; Simon, Françoise; Deprez, Eric; Cosette, Jérémie; Stockholm, Daniel; Scherman, Daniel; Bigey, Pascal; Kichler, Antoine

2016-02-15

Myostatin, also known as growth differentiation factor 8, is a member of the transforming growth factor-beta superfamily that has been shown to play a key role in the regulation of the skeletal muscle mass. Indeed, while myostatin deletion or loss of function induces muscle hypertrophy, its overexpression or systemic administration causes muscle atrophy. Since myostatin blockade is effective in increasing skeletal muscle mass, myostatin inhibitors have been actively sought after. Decorin, a member of the small leucine-rich proteoglycan family is a metalloprotein that was previously shown to bind and inactivate myostatin in a zinc-dependent manner. Furthermore, the myostatin-binding site has been shown to be located in the decorin N-terminal domain. In the present study, we investigated the anti-myostatin activity of short and soluble fragments of decorin. Our results indicate that the murine decorin peptides DCN48-71 and 42-65 are sufficient for inactivating myostatin in vitro. Moreover, we show that the interaction of mDCN48-71 to myostatin is strictly zinc-dependent. Binding of myostatin to activin type II receptor results in the phosphorylation of Smad2/3. Addition of the decorin peptide 48-71 decreased in a dose-dependent manner the myostatin-induced phosphorylation of Smad2 demonstrating thereby that the peptide inhibits the activation of the Smad signaling pathway. Finally, we found that mDCN48-71 displays a specificity towards myostatin, since it does not inhibit other members of the transforming growth factor-beta family. Copyright © 2016 Elsevier Inc. All rights reserved.

High resolution x-ray fluorescence spectroscopy - a new technique for site- and spin-selectivity

International Nuclear Information System (INIS)

Wang, Xin

1996-12-01

X-ray spectroscopy has long been used to elucidate electronic and structural information of molecules. One of the weaknesses of x-ray absorption is its sensitivity to all of the atoms of a particular element in a sample. Through out this thesis, a new technique for enhancing the site- and spin-selectivity of the x-ray absorption has been developed. By high resolution fluorescence detection, the chemical sensitivity of K emission spectra can be used to identify oxidation and spin states; it can also be used to facilitate site-selective X-ray Absorption Near Edge Structure (XANES) and site-selective Extended X-ray Absorption Fine Structure (EXAFS). The spin polarization in K fluorescence could be used to generate spin selective XANES or spin-polarized EXAFS, which provides a new measure of the spin density, or the nature of magnetic neighboring atoms. Finally, dramatic line-sharpening effects by the combination of absorption and emission processes allow observation of structure that is normally unobservable. All these unique characters can enormously simplify a complex x-ray spectrum. Applications of this novel technique have generated information from various transition-metal model compounds to metalloproteins. The absorption and emission spectra by high resolution fluorescence detection are interdependent. The ligand field multiplet model has been used for the analysis of Kα and Kβ emission spectra. First demonstration on different chemical states of Fe compounds has shown the applicability of site selectivity and spin polarization. Different interatomic distances of the same element in different chemical forms have been detected using site-selective EXAFS

Project in determination of crystal structure of nitrogen fixation proteins from azospirilum brasiliense and herbaspirilum seropedicae by synchrotron x-ray diffraction

Energy Technology Data Exchange (ETDEWEB)

Barbosa, Valma M.; Leggs, Luciana A.; Delboni, Luis F.; Chubatsu, LedaS.; Souza, Emanuel M.; Machado, Hidevaldo B.; Yates, Geoffrey M.; Pedrosa, Fabio O. [Parana Univ., Curitiba, PR (Brazil). Dept. de Bioquimica

1996-12-31

Full text. Biological nitrogen fixation is essential for maintaining the nitrogen cycle on earth and of high importance for Brazilian agriculture. The nitrogenase enzyme system, which provides the biochemical machinery for nitrogen fixation, consists of two component metalloproteins, the molybdenumiron (Mo Fe) protein and the iron (Fe) protein. Nitrogen fixation is a very energy-intensive process, requiring around 16 moles of ATP for each mol of N{sub 2} fixed (reduced). As a consequence, synthesis and activity of nitrogenase is tighty regulated at two levels: general and specific. The general level regulation is mediated by the ntr (nitrogen regulation) system. Two gene products are involved: the ntrB gene product (NtrB) is responsible for the activation of the ntrC gene product (NtrC) by phosphorylating a conserved Asp54, which activates the expression of the nifA gene. The nif specific control system is mediated by the NifA protein, which binds to a DNA specific sequence (UAS, Upstream Activator Sequence) and activates nif promoter transcriptions by RNA polymerase-{sup {alpha}54}, following ATP hydrolysis. The aim of this project is to solve the crystal structure of dinitrogenase reductase (iron protein) and dinitrogenase (molybdenum-iron protein) from Azospirilim brasiliense and the regulatory proteins NifA from Herbaspirillum seropedicae and NtrC Azospirillum brasiliense. The three dimensional structure of the proteins involved in this project will allow a better understanding of the mechanism of biological nitrogen fixation. To this end, the data collection will probably be done at the LNLS facilities which will be available in the near future. (author)

Effects of treatment with Maraviroc a CCR5 inhibitor on a human hepatic stellate cell line.

Science.gov (United States)

Coppola, Nicola; Perna, Angelica; Lucariello, Angela; Martini, Salvatore; Macera, Margherita; Carleo, Maria A; Guerra, Germano; Esposito, Vincenzo; De Luca, Antonio

2018-08-01

After an acute liver damage, tissue regeneration repairs lesions with degradation of deposed fibrotic material, while mechanisms of tissue restoration are persistently activated following several repeated injuries, inducing deposition of extracellular matrix. (ECM). Factors responsible for ECM remodeling have been identified in a pathway involving a family of zinc-dependent enzyme matrix metalloproteinases (MMPs), together with tissue inhibitor of metalloproteinases (TIMPs). Recent experimental models suggested a role of CCR5 receptor in the genesis of liver fibrosis. Drawing from these background we decided to evaluate the effects of the treatment with the CCR5 inhibitor Maraviroc on LX-2, a human hepatic stellate cell line (HSC). Treatment with Maraviroc resulted in a block in S phase of LX-2 cells with increased expression levels of cyclin D1 and p21 while the expression of p53 was reduced. Treatment with Maraviroc was also able to block the accumulation of fibrillar collagens and extracellular matrix proteins (ECM), as demonstrated by the decrease of specific markers as Collagen type I, α-SMA, and TGF-β1. In addition we observed a down regulation of both metalloproteins (MMP-2, MMP-9), used for the degradation of the extracellular matrix and their inhibitors (TIMP-1, TIMP-2). The identification of a compound that may modulate the dynamic of liver fibrosis could be crucial in all chronic liver diseases. Maraviroc could play an important role because, in addition to its own anti-HIV activity, it could reduce the release of pro-inflammatory citokynes implicated in liver fibrogenesis. © 2018 Wiley Periodicals, Inc.

Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

KAUST Repository

Vogler, Malvina M.

2018-01-01

Heme proteins, also termed cytochromes, are a widespread class of metalloproteins containing an Fe-protoporphyrin IX cofactor. They perform numerous functions in nature such as oxygen-transport by hemoglobin, monooxygenation reactions catalyzed by Cytochrome P-450, and electron transfer reactions during photosynthesis. The differences between proteincofactor binding characteristics and the cofactor environment greatly influence the extensive range of functions. In this dissertation, proteins from the Mtr pathway of Shewanella oneidensis are characterized. These c-type cytochromes contain multiple heme cofactors per protein molecule that covalently attach to the protein amino acid sequence and are involved in electron transfer to extracellular metal oxides during anaerobic conditions. Successful recombinant expression of pathway components MtrC and MtrA is achieved in Escherichia coli. Heme-dependent gel staining and UV/Vis spectroscopy show characteristic c-type cytochrome characteristics. Mass spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy measurements of MtrA provide intriguing structural information and highlight the strong influence of the heme cofactors within the protein structure. Next, an Arabidopsis thaliana protein is analyzed. It was previously identified via a motif search of the plant genome, based on conserved residues in the H4 NOX pocket. Here, the incorporation of a heme b cofactor is confirmed. UV/Vis spectroscopy under anaerobic conditions demonstrates reversible binding of nitric oxide to the heme iron and depicts the previously published characteristic absorption maxima for other H-NOX proteins.

Molecular biology of microbial hydrogenases.

Science.gov (United States)

Vignais, P M; Colbeau, A

2004-07-01

Hydrogenases (H2ases) are metalloproteins. The great majority of them contain iron-sulfur clusters and two metal atoms at their active center, either a Ni and an Fe atom, the [NiFe]-H2ases, or two Fe atoms, the [FeFe]-H2ases. Enzymes of these two classes catalyze the reversible oxidation of hydrogen gas (H2 2 H+ + 2 e-) and play a central role in microbial energy metabolism; in addition to their role in fermentation and H2 respiration, H2ases may interact with membrane-bound electron transport systems in order to maintain redox poise, particularly in some photosynthetic microorganisms such as cyanobacteria. Recent work has revealed that some H2ases, by acting as H2-sensors, participate in the regulation of gene expression and that H2-evolving H2ases, thought to be involved in purely fermentative processes, play a role in membrane-linked energy conservation through the generation of a protonmotive force. The Hmd hydrogenases of some methanogenic archaea constitute a third class of H2ases, characterized by the absence of Fe-S cluster and the presence of an iron-containing cofactor with catalytic properties different from those of [NiFe]- and [FeFe]-H2ases. In this review, we emphasise recent advances that have greatly increased our knowledge of microbial H2ases, their diversity, the structure of their active site, how the metallocenters are synthesized and assembled, how they function, how the synthesis of these enzymes is controlled by external signals, and their potential use in biological H2 production.

Simultaneous speciation neutron activation analysis for trace elements

International Nuclear Information System (INIS)

Chatt, A.; Kiceniuk, J.W.; Menendez Sanchez, W.; Bottaro, C.

2006-01-01

Among the various forms of neutron activation technique being developed in our laboratory, much emphasis has been placed over the last ten years or so on the development of simultaneous speciation neutron activation analysis (SSNAA). This technique can now be used for the simultaneous determination of various species of a number of elements. Almost all speciation techniques consist of two steps. The first step involves the separation of species from the sample followed by the second step of element-specific detection. A number of characteristic features of NAA, which other techniques normally do not possess, can be advantageously exploited in SSNAA. For example, SSNAA can be used for: (i) multielement speciation with high specificity, (ii) speciation of chemically dissimilar elements such as Cd, Mn and Se, (iii) speciation of elements such as Cl, Br and I which are rather difficult to determine by most other techniques, etc. We have developed SSNAA methods for assaying various arsenic species, namely As(III), As(V), dimethyl arsonic acid (DMA), monomethylarsinic acid (MMA), arsenobetaine (AsB), organically bound arsenic (OBAs), and lipid-soluble arsenic (LSAs) in marine fish samples. We have extended these methods to include simultaneous determination of various species of As, Sb and Se in water. We have also developed SSNAA methods employing biochemical techniques for the simultaneous separation, preconcentration and characterization of metalloproteins and protein-bound trace element species of As, Br, Cd, Cu, Mn, Se, and Zn. We have developed methods for the simultaneous separation and characterization of organohalogen compounds in fish. An overview of the SSNAA methods being developed in our laboratory will be presented. (author)

Theoretical Analysis of the Relative Significance of Thermodynamic and Kinetic Dispersion in the dc and ac Voltammetry of Surface-Confined Molecules

KAUST Repository

Morris, Graham P.; Baker, Ruth E.; Gillow, Kathryn; Davis, Jason J.; Gavaghan, David J.; Bond, Alan M.

2015-01-01

© 2015 American Chemical Society. Commonly, significant discrepancies are reported in theoretical and experimental comparisons of dc voltammograms derived from a monolayer or close to monolayer coverage of redox-active surface-confined molecules. For example, broader-than-predicted voltammetric wave shapes are attributed to the thermodynamic or kinetic dispersion derived from distributions in reversible potentials (E⁰) and electrode kinetics (k⁰), respectively. The recent availability of experimentally estimated distributions of E⁰ and k⁰ values derived from the analysis of data for small numbers of surface-confined modified azurin metalloprotein molecules now allows more realistic modeling to be undertaken, assuming the same distributions apply under conditions of high surface coverage relevant to voltammetric experiments. In this work, modeling based on conventional and stochastic kinetic theory is considered, and the computationally far more efficient conventional model is shown to be equivalent to the stochastic one when large numbers of molecules are present. Perhaps unexpectedly, when experimentally determined distributions of E⁰ and k⁰ are input into the model, thermodynamic dispersion is found to be unimportant and only kinetic dispersion contributes significantly to the broadening of dc voltammograms. Simulations of ac voltammetric experiments lead to the conclusion that the ac method, particularly when the analysis of kinetically very sensitive higher-order harmonics is undertaken, are far more sensitive to kinetic dispersion than the dc method. ac methods are therefore concluded to provide a potentially superior strategy for addressing the inverse problem of determining the k⁰ distribution that could give rise to the apparent anomalies in surface-confined voltammetry.

Theoretical Analysis of the Relative Significance of Thermodynamic and Kinetic Dispersion in the dc and ac Voltammetry of Surface-Confined Molecules

KAUST Repository

Morris, Graham P.

2015-05-05

© 2015 American Chemical Society. Commonly, significant discrepancies are reported in theoretical and experimental comparisons of dc voltammograms derived from a monolayer or close to monolayer coverage of redox-active surface-confined molecules. For example, broader-than-predicted voltammetric wave shapes are attributed to the thermodynamic or kinetic dispersion derived from distributions in reversible potentials (E⁰) and electrode kinetics (k⁰), respectively. The recent availability of experimentally estimated distributions of E⁰ and k⁰ values derived from the analysis of data for small numbers of surface-confined modified azurin metalloprotein molecules now allows more realistic modeling to be undertaken, assuming the same distributions apply under conditions of high surface coverage relevant to voltammetric experiments. In this work, modeling based on conventional and stochastic kinetic theory is considered, and the computationally far more efficient conventional model is shown to be equivalent to the stochastic one when large numbers of molecules are present. Perhaps unexpectedly, when experimentally determined distributions of E⁰ and k⁰ are input into the model, thermodynamic dispersion is found to be unimportant and only kinetic dispersion contributes significantly to the broadening of dc voltammograms. Simulations of ac voltammetric experiments lead to the conclusion that the ac method, particularly when the analysis of kinetically very sensitive higher-order harmonics is undertaken, are far more sensitive to kinetic dispersion than the dc method. ac methods are therefore concluded to provide a potentially superior strategy for addressing the inverse problem of determining the k⁰ distribution that could give rise to the apparent anomalies in surface-confined voltammetry.

Analysis of NFU-1 metallocofactor binding-site substitutions-impacts on iron-sulfur cluster coordination and protein structure and function.

Science.gov (United States)

Wesley, Nathaniel A; Wachnowsky, Christine; Fidai, Insiya; Cowan, J A

2017-11-01

Iron-sulfur (Fe/S) clusters are ancient prosthetic groups found in numerous metalloproteins and are conserved across all kingdoms of life due to their diverse, yet essential functional roles. Genetic mutations to a specific subset of mitochondrial Fe/S cluster delivery proteins are broadly categorized as disease-related under multiple mitochondrial dysfunction syndrome (MMDS), with symptoms indicative of a general failure of the metabolic system. Multiple mitochondrial dysfunction syndrome 1 (MMDS1) arises as a result of the missense mutation in NFU1, an Fe/S cluster scaffold protein, which substitutes a glycine near the Fe/S cluster-binding pocket to a cysteine (p.Gly208Cys). This substitution has been shown to promote protein dimerization such that cluster delivery to NFU1 is blocked, preventing downstream cluster trafficking. However, the possibility of this additional cysteine, located adjacent to the cluster-binding site, serving as an Fe/S cluster ligand has not yet been explored. To fully understand the consequences of this Gly208Cys replacement, complementary substitutions at the Fe/S cluster-binding pocket for native and Gly208Cys NFU1 were made, along with six other variants. Herein, we report the results of an investigation on the effect of these substitutions on both cluster coordination and NFU1 structure and function. The data suggest that the G208C substitution does not contribute to cluster binding. Rather, replacement of the glycine at position 208 changes the oligomerization state as a result of global structural alterations that result in the downstream effects manifest as MMDS1, but does not perturb the coordination chemistry of the Fe-S cluster. © 2017 Federation of European Biochemical Societies.

Structural changes caused by radiation-induced reduction and radiolysis: the effect of X-ray absorbed dose in a fungal multicopper oxidase

International Nuclear Information System (INIS)

De la Mora, Eugenio; Lovett, Janet E.; Blanford, Christopher F.; Garman, Elspeth F.; Valderrama, Brenda; Rudino-Pinera, Enrique

2012-01-01

Radiation-induced reduction, radiolysis of copper sites and the effect of pH value together with the concomitant geometrical distortions of the active centres were analysed in several fungal (C. gallica) laccase structures collected at cryotemperature. This study emphasizes the importance of careful interpretation when the crystallographic structure of a metalloprotein is described. X-ray radiation induces two main effects at metal centres contained in protein crystals: radiation-induced reduction and radiolysis and a resulting decrease in metal occupancy. In blue multicopper oxidases (BMCOs), the geometry of the active centres and the metal-to-ligand distances change depending on the oxidation states of the Cu atoms, suggesting that these alterations are catalytically relevant to the binding, activation and reduction of O 2 . In this work, the X-ray-determined three-dimensional structure of laccase from the basidiomycete Coriolopsis gallica (Cg L), a high catalytic potential BMCO, is described. By combining spectroscopic techniques (UV–Vis, EPR and XAS) and X-ray crystallography, structural changes at and around the active copper centres were related to pH and absorbed X-ray dose (energy deposited per unit mass). Depletion of two of the four active Cu atoms as well as low occupancies of the remaining Cu atoms, together with different conformations of the metal centres, were observed at both acidic pH and high absorbed dose, correlating with more reduced states of the active coppers. These observations provide additional evidence to support the role of flexibility of copper sites during O 2 reduction. This study supports previous observations indicating that interpretations regarding redox state and metal coordination need to take radiation effects explicitly into account

Study of solid/liquid and solid/gas interfaces in Cu–isoleucine complex by surface X-ray diffraction

International Nuclear Information System (INIS)

Ferrer, Pilar; Rubio-Zuazo, Juan; Castro, German R.

2013-01-01

The enzymes could be understood like structures formed by amino acids bonded with metals, which act as active sites. The research on the coordination of metal–amino acid complexes will bring light on the behavior of metal enzymes, due to the close relation existing between the atomic structure and the functionality. The Cu–isoleucine bond is considered as a good model system to attain a better insight into the characteristics of naturally occurring copper metalloproteins. The surface structure of metal–amino acid complex could be considered as a more realistic model for real systems under biologic working conditions, since the molecular packing is decreased. In the surface, the structural constrains are reduced, keeping the structural capability of surface complex to change as a function of the surrounding environment. In this work, we present a surface X-ray diffraction study on Cu–isoleucine complex under different ambient conditions. Cu(Ile) 2 crystals of about 5 mm × 5 mm × 1 mm have been growth, by seeding method in a supersaturated solution, presenting a surface of high quality. The sample for the surface diffraction study was mounted on a cell specially designed for solid/liquid or solid/gas interface analysis. The Cu–isoleucine crystal was measured under a protective dry N 2 gas flow and in contact with a saturated metal amino acid solution. The bulk and the surface signals were compared, showing different atomic structures. In both cases, from surface diffraction data, it is observed that the atomic structure of the top layer undergoes a clear structural deformation. A non-uniform surface relaxation is observed producing an inhomogeneous displacement of the surface atoms towards the surface normal.

In-vitro free radical scavenging activity of biosynthesized gold and silver nanoparticles using Prunus armeniaca (apricot) fruit extract

Energy Technology Data Exchange (ETDEWEB)

Dauthal, Preeti; Mukhopadhyay, Mausumi, E-mail: mausumi_mukhopadhyay@yahoo.com [S.V. National Institute of Technology, Department of Chemical Engineering (India)

2013-01-15

In-vitro free radical scavenging activity of biosynthesized gold (Au-NPs) and silver (Ag-NPs) nanoparticles was investigated in the present study. Natural precursor Prunus armeniaca (apricot) fruit extract was used as a reducing agent for the nanoparticle synthesis. The free radical scavenging activity of the nanoparticles were observed by modified 1,1 Prime -diphynyl-2-picrylhydrazyl, DPPH and 2,2 Prime -azinobis (3-ethylbenzothiazoline-6-sulfonic acid), ABTS assay. The synthesized nanoparticles were characterized by UV-Visible spectroscopy, dynamic light scattering, transmission electron microscopy (TEM), X-ray diffraction (XRD), energy dispersive spectroscopy, and fourier transform infrared spectroscopy (FTIR). Appearance of optical absorption peak at 537 nm (2.20 keV) and 435 nm (3 keV) within 0.08 and 0.5 h of reaction time was confirmed the presence of metallic Au and Ag nanoclusters, respectively. Nearly spherical nanoparticles with majority of particle below 20 nm (TEM) for both Au-NPs and Ag-NPs were synthesized. XRD pattern confirmed the existence of pure nanocrystalline Au-NPs while few additional peaks in the vicinity of fcc silver-speculated crystallization of metalloproteins of fruit extract on the surface of the Ag-NPs and vice versa. FTIR spectra was supported the role of amino acids of protein/enzymes of fruit extract for synthesis and stabilization of nanoparticles. Dose-dependent scavenging activity was observed for Au-NPs and Ag-NPs in both DPPH and ABTS in-vitro assay. 50 % scavenging activity for DPPH were 11.27 and 16.18 mg and for ABTS 3.40 and 7.12 mg with Au-NPs and Ag-NPs, respectively.

Identification and Analysis of a Novel Gene Cluster Involves in Fe2+ Oxidation in Acidithiobacillus ferrooxidans ATCC 23270, a Typical Biomining Acidophile.

Science.gov (United States)

Ai, Chenbing; Liang, Yuting; Miao, Bo; Chen, Miao; Zeng, Weimin; Qiu, Guanzhou

2018-07-01

Iron-oxidizing Acidithiobacillus spp. are applied worldwide in biomining industry to extract metals from sulfide minerals. They derive energy for survival through Fe 2+ oxidation and generate Fe 3+ for the dissolution of sulfide minerals. However, molecular mechanisms of their iron oxidation still remain elusive. A novel two-cytochrome-encoding gene cluster (named tce gene cluster) encoding a high-molecular-weight cytochrome c (AFE_1428) and a c 4 -type cytochrome c 552 (AFE_1429) in A. ferrooxidans ATCC 23270 was first identified in this study. Bioinformatic analysis together with transcriptional study showed that AFE_1428 and AFE_1429 were the corresponding paralog of Cyc2 (AFE_3153) and Cyc1 (AFE_3152) which were encoded by the extensively studied rus operon and had been proven involving in ferrous iron oxidation. Both AFE_1428 and AFE_1429 contained signal peptide and the classic heme-binding motif(s) as their corresponding paralog. The modeled structure of AFE_1429 showed high resemblance to Cyc1. AFE_1428 and AFE_1429 were preferentially transcribed as their corresponding paralogs in the presence of ferrous iron as sole energy source as compared with sulfur. The tce gene cluster is highly conserved in the genomes of four phylogenetic-related A. ferrooxidans strains that were originally isolated from different sites separated with huge geographical distance, which further implies the importance of this gene cluster. Collectively, AFE_1428 and AFE_1429 involve in Fe 2+ oxidation like their corresponding paralog by integrating with the metalloproteins encoded by rus operon. This study provides novel insights into the Fe 2+ oxidation mechanism in Fe 2+ -oxidizing A. ferrooxidans ssp.

Relation between some environmental pollutants and recurrent spontaneous abortion

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Aziza A. Saad

2016-09-01

Full Text Available Reproductive health is exquisitely sensitive to characteristics of an individual’s environment including physical, biological, behavioral, cultural and socioeconomic factors. This study was launched to elucidate the effect of the exposure to chemical pollutants as aromatic amines viz. (benzidine, mono-acetyl benzidine, diacetyl benzidine, α,β-naphthylamine as well as the biological pollutants e.g., human cytomegalovirus (HCMV as risk factors for recurrent spontaneous abortion (RSA through determination of MDA as a marker of oxidative stress and determination of some antioxidant markers. The results of the current study revealed that the aborter mothers were being exposed to environmental pollutants as aromatic amines which were manifested by the presence of benzidine, mono-acetyl-benzidine, di-acetyl-benzidine, α,β-naphthylamine in most of their urine samples, where the level of aromatic amines were more 13.6, 10, 15, and 4-folds than the control group, respectively. Also, the data suggest that in early pregnancy failure there is an increase in markers of oxidative stress and a probable decrease in maternal antioxidant defenses (22 nmol/ml and 17 mg/l, 550 U/l, respectively. Generation of ROS in large quantities, in the first trimester placenta which has limited antioxidant defenses may cause DNA damage, oxidation of protein and lipid resulting in extensive cell death. Also, it was demonstrated that high elevation of HCMV inhibits cytotrophoblasts proliferation, migration invasion and matrix metalloproteins (MMP expression. Obviously, placental toxicological responses are partly due to pharmaco/toxico dynamic responses to the chemicals. Conclusively, the aforementioned findings emphasis that, the exposures to environmental chemical and/or biological risk factors are implicated in the pathogenesis of recurrent spontaneous abortion.

A microscopic insight from conformational thermodynamics to functional ligand binding in proteins.

Science.gov (United States)

Sikdar, Samapan; Chakrabarti, J; Ghosh, Mahua

2014-12-01

We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for β-1,4-galactosyltransferase (β4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock β4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.

The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism.

Science.gov (United States)

Lill, Roland; Hoffmann, Bastian; Molik, Sabine; Pierik, Antonio J; Rietzschel, Nicole; Stehling, Oliver; Uzarska, Marta A; Webert, Holger; Wilbrecht, Claudia; Mühlenhoff, Ulrich

2012-09-01

Mitochondria play a key role in iron metabolism in that they synthesize heme, assemble iron-sulfur (Fe/S) proteins, and participate in cellular iron regulation. Here, we review the latter two topics and their intimate connection. The mitochondrial Fe/S cluster (ISC) assembly machinery consists of 17 proteins that operate in three major steps of the maturation process. First, the cysteine desulfurase complex Nfs1-Isd11 as the sulfur donor cooperates with ferredoxin-ferredoxin reductase acting as an electron transfer chain, and frataxin to synthesize an [2Fe-2S] cluster on the scaffold protein Isu1. Second, the cluster is released from Isu1 and transferred toward apoproteins with the help of a dedicated Hsp70 chaperone system and the glutaredoxin Grx5. Finally, various specialized ISC components assist in the generation of [4Fe-4S] clusters and cluster insertion into specific target apoproteins. Functional defects of the core ISC assembly machinery are signaled to cytosolic or nuclear iron regulatory systems resulting in increased cellular iron acquisition and mitochondrial iron accumulation. In fungi, regulation is achieved by iron-responsive transcription factors controlling the expression of genes involved in iron uptake and intracellular distribution. They are assisted by cytosolic multidomain glutaredoxins which use a bound Fe/S cluster as iron sensor and additionally perform an essential role in intracellular iron delivery to target metalloproteins. In mammalian cells, the iron regulatory proteins IRP1, an Fe/S protein, and IRP2 act in a post-transcriptional fashion to adjust the cellular needs for iron. Thus, Fe/S protein biogenesis and cellular iron metabolism are tightly linked to coordinate iron supply and utilization. This article is part of a Special Issue entitled: Cell Biology of Metals. Copyright © 2012 Elsevier B.V. All rights reserved.

Do we see what we should see? Describing non-covalent interactions in protein structures including precision

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Manickam Gurusaran

2014-01-01

Full Text Available The power of X-ray crystal structure analysis as a technique is to `see where the atoms are'. The results are extensively used by a wide variety of research communities. However, this `seeing where the atoms are' can give a false sense of security unless the precision of the placement of the atoms has been taken into account. Indeed, the presentation of bond distances and angles to a false precision (i.e. to too many decimal places is commonplace. This article has three themes. Firstly, a basis for a proper representation of protein crystal structure results is detailed and demonstrated with respect to analyses of Protein Data Bank entries. The basis for establishing the precision of placement of each atom in a protein crystal structure is non-trivial. Secondly, a knowledge base harnessing such a descriptor of precision is presented. It is applied here to the case of salt bridges, i.e. ion pairs, in protein structures; this is the most fundamental place to start with such structure-precision representations since salt bridges are one of the tenets of protein structure stability. Ion pairs also play a central role in protein oligomerization, molecular recognition of ligands and substrates, allosteric regulation, domain motion and α-helix capping. A new knowledge base, SBPS (Salt Bridges in Protein Structures, takes these structural precisions into account and is the first of its kind. The third theme of the article is to indicate natural extensions of the need for such a description of precision, such as those involving metalloproteins and the determination of the protonation states of ionizable amino acids. Overall, it is also noted that this work and these examples are also relevant to protein three-dimensional structure molecular graphics software.

Formation of a homocitrate-free iron-molybdenum cluster on NifEN: implications for the role of homocitrate in nitrogenase assembly.

Science.gov (United States)

Fay, Aaron Wolfe; Blank, Michael Aaron; Yoshizawa, Janice Mariko; Lee, Chi Chung; Wiig, Jared Andrew; Hu, Yilin; Hodgson, Keith Owen; Hedman, Britt; Ribbe, Markus Walter

2010-03-28

Molybdenum (Mo)-dependent nitrogenase is a complex metalloprotein that catalyzes the biological reduction of dinitrogen (N(2)) to ammonia (NH(3)) at the molybdenum-iron cofactor (FeMoco) site of its molybdenum-iron (MoFe) protein component. Here we report the formation of a homocitrate-free, iron-molybdenum ("FeMo") cluster on the biosynthetic scaffold of FeMoco, NifEN. Such a NifEN-associated "FeMo" cluster exhibits EPR features similar to those of the NifEN-associated, fully-complemented "FeMoco", which originate from the presence of Mo in both cluster species; however, "FeMo" cluster and "FeMoco" display different temperature-dependent changes in the line shape and the signal intensity of their respective EPR features, which reflect the impact of homocitrate on the redox properties of these clusters. XAS/EXAFS analysis reveals that the Mo centers in both "FeMo" cluster and "FeMoco" are present in a similar coordination environment, although Mo in "FeMo" cluster is more loosely coordinated as compared to that in "FeMoco" with respect to the Mo-O distances in the cluster, likely due to the absence of homocitrate that normally serves as an additional ligand for the Mo in the cluster. Subsequent biochemical investigation of the "FeMo" cluster not only facilitates the determination of the sequence of events in the mobilization of Mo and homocitrate during FeMoco maturation, but also permits the examination of the role of homocitrate in the transfer of FeMoco between NifEN and MoFe protein. Combined outcome of these studies establishes a platform for future structural analysis of the interactions between NifEN and MoFe protein, which will provide useful insights into the mechanism of cluster transfer between the two proteins.

Lead-zinc interactions in the production of osteocalcin by ROS 17/2.8 osteoblastic bone cells

International Nuclear Information System (INIS)

Pounds, J.G.

1991-01-01

The serum level of osteocalcin, a bone specific protein produced by osteoblasts and used clinically as a marker of osteoblast acceptive, is decreased in lead intoxicated children. Previous studies suggest that the reduced osteocalcin production appears to be the result of impaired transcriptional regulation of this 1,25-dihydroxyvitamin D 3 gene product, and not translation. As part of a study to investigate the potential interaction of Pb 2+ with Zn 2+ , and with the zinc fingers of the vitamin D receptor, ROS cells were treated with 0, 5, 10, or 25 μM lead acetate for 24 hr, in the presence of 10, 30, or 50 μM Zn followed by an additional 24 hr treatment with lead with 1,25-dihydroxyvitamin D 3 (100 pg/ml media). At the end of this period a radioimmunoassay was conducted to determine the amount of osteocalcin in the cells and secreted in the media. 1,25-dihydroxyvitamin D 3 caused an increase in osteocalcin secreted into the media in cultures containing 0 μM lead, but this increase was inhibited by lead in a concentration dependent manner, so that osteocalcin secretion in 10 or 25 μM lead treated groups was less than cultures without 1,25-dihydroxyvitamin D 3 treatment. This inhibitory effect of lead was blocked by increasing the medium zinc concentration of 50 μM. Increasing medium Pb 2+ concentrations decreased the amount of 65 Zn taken up by cells by ∼30%, which was nullified by increasing medium Zn. These results suggest that lead produces a localized and specific Zn deficiency in the vitamin D receptor zinc finger, and perhaps other zinc metalloproteins, and that these effects of lead are not mediated through general effects on RNA or protein synthesis

Effect of interventional treatment with p53 on the invasion and metastasis of VX2 liver tumor in experimental rabbits

International Nuclear Information System (INIS)

Li Caixia; Feng Yan; Gu Tao; Li Chunmei

2010-01-01

Objective: To investigate the effect of interventional treatment with p53 on the invasion and metastasis of VX2 liver tumor in experimental rabbits. Methods: VX2 carcinoma cells were surgically implanted into the left hepatic lobe in 48 New Zealand white rabbits, and the rabbit hepatic carcinoma models were thus established. The rabbits were randomly divided into 4 groups with 12 rabbits in each group. After hepatic arterial catheterization was completed physiological saline (control group), Lipiodol (Group A), Ad-p53 (Group B) and Lipiodol+Ad-p53 (Group C) were respectively infused into the rabbits of four groups via common hepatic artery. One week after the procedure the rabbits were sacrificed and the livers were removed for the determination of matrix metalloprotein-2 (MMP-2), proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) of the tumor with immunohistochemistry technique. Results: The tumor growth in study groups (group A, B and C) was markedly suppressed, which was significantly different in comparison with that in control group (P 0.05). The positive rates of MMP-2, PCNA and VEGF in group B and C were significantly lower than those in control group (P < 0.05). The positive rates of MMP-2, PCNA and VEGF of the rabbits with metastasis were markedly higher than those without metastasis(P < 0.05). MMP-2 bore a certain relationship with VEGF and PCNA (P < 0.05). Conclusion: The increase of the positive rates of MMP-2, PCNA and VEGF indicates that the tumor possesses higher possibility for developing metastasis, proliferation and vascular formation. The interventional treatment with Adp53 or Lipiodol+Ad-p53 can inhibit the growth, metastasis and vascular formation of VX2 liver tumor in experimental rabbits. (J Intervent Radiol, 2010, 19 : 800-804) (authors)

Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3

International Nuclear Information System (INIS)

Ding Zhichun; Teng Xinchen; Cai Bin; Wang Hui; Zheng Qi; Wang Yang; Zhou Guoming; Zhang Mingjie; Wu Houming; Sun Hongzhe; Huang Zhongxian

2006-01-01

Human metallothionein-3 (hMT3), first isolated and identified as a neuronal growth inhibitory factor (GIF), is a metalloprotein expressed predominantly in brain. However, untill now, the exact mechanism of the bioactivity of hMT3 is still unknown. In order to study the influence of acid-base catalysis on S-nitrosylation of hMT3, we constructed the E23K mutant of hMT3. During the course of bioassay, we found out unexpectedly that mutation at E23 of hMT3 eliminates the neuronal growth inhibitory activity completely. To the best of our knowledge, it is First report that other residues, besides the TCPCP motif, in the β-domain can alter the bioactivity of hMT3. In order to figure out the causes for the loss of bioactivity of the E23K mutant, the biochemical properties were characterized by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, and SNOC reaction. All data demonstrated that stability of the metal-thiolate cluster and overall structure of the E23K mutant were not altered too much. However, the reaction of the E23K mutant with SNOC exhibited biphasic kinetics and the mutant protein released zinc ions much faster than hMT3 in the initial step, while hMT3 exhibited single kinetic process. The 2D [ 1 H- 15 N] HSQC was also employed to characterize structural changes during the reaction of hMT3 with varying mounts of nitric oxide. It was shown that the resonance of Glu23 disappeared at a molar ratio of NO to protein of 4. Based on these results, we suggest that mutation at Glu23 may alter the NO metabolism and/or affect zinc homeostasis in brain, thus altering the neuronal growth inhibitory activity

Comparative genomic analyses of nickel, cobalt and vitamin B12 utilization

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Gelfand Mikhail S

2009-02-01

Full Text Available Abstract Background Nickel (Ni and cobalt (Co are trace elements required for a variety of biological processes. Ni is directly coordinated by proteins, whereas Co is mainly used as a component of vitamin B12. Although a number of Ni and Co-dependent enzymes have been characterized, systematic evolutionary analyses of utilization of these metals are limited. Results We carried out comparative genomic analyses to examine occurrence and evolutionary dynamics of the use of Ni and Co at the level of (i transport systems, and (ii metalloproteomes. Our data show that both metals are widely used in bacteria and archaea. Cbi/NikMNQO is the most common prokaryotic Ni/Co transporter, while Ni-dependent urease and Ni-Fe hydrogenase, and B12-dependent methionine synthase (MetH, ribonucleotide reductase and methylmalonyl-CoA mutase are the most widespread metalloproteins for Ni and Co, respectively. Occurrence of other metalloenzymes showed a mosaic distribution and a new B12-dependent protein family was predicted. Deltaproteobacteria and Methanosarcina generally have larger Ni- and Co-dependent proteomes. On the other hand, utilization of these two metals is limited in eukaryotes, and very few of these organisms utilize both of them. The Ni-utilizing eukaryotes are mostly fungi (except saccharomycotina and plants, whereas most B12-utilizing organisms are animals. The NiCoT transporter family is the most widespread eukaryotic Ni transporter, and eukaryotic urease and MetH are the most common Ni- and B12-dependent enzymes, respectively. Finally, investigation of environmental and other conditions and identity of organisms that show dependence on Ni or Co revealed that host-associated organisms (particularly obligate intracellular parasites and endosymbionts have a tendency for loss of Ni/Co utilization. Conclusion Our data provide information on the evolutionary dynamics of Ni and Co utilization and highlight widespread use of these metals in the three

COMe: the ontology of bioinorganic proteins

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Contrino Sergio

2004-02-01

Full Text Available Abstract Background Many characterised proteins contain metal ions, small organic molecules or modified residues. In contrast, the huge amount of data generated by genome projects consists exclusively of sequences with almost no annotation. One of the goals of the structural genomics initiative is to provide representative three-dimensional (3-D structures for as many protein/domain folds as possible to allow successful homology modelling. However, important functional features such as metal co-ordination or a type of prosthetic group are not always conserved in homologous proteins. So far, the problem of correct annotation of bioinorganic proteins has been largely ignored by the bioinformatics community and information on bioinorganic centres obtained by methods other than crystallography or NMR is only available in literature databases. Results COMe (Co-Ordination of Metals represents the ontology for bioinorganic and other small molecule centres in complex proteins. COMe consists of three types of entities: 'bioinorganic motif' (BIM, 'molecule' (MOL, and 'complex proteins' (PRX, with each entity being assigned a unique identifier. A BIM consists of at least one centre (metal atom, inorganic cluster, organic molecule and two or more endogenous and/or exogenous ligands. BIMs are represented as one-dimensional (1-D strings and 2-D diagrams. A MOL entity represents a 'small molecule' which, when in complex with one or more polypeptides, forms a functional protein. The PRX entities refer to the functional proteins as well as to separate protein domains and subunits. The complex proteins in COMe are subdivided into three categories: (i metalloproteins, (ii organic prosthetic group proteins and (iii modified amino acid proteins. The data are currently stored in both XML format and a relational database and are available at http://www.ebi.ac.uk/come/. Conclusion COMe provides the classification of proteins according to their 'bioinorganic' features

Apoprotein Structure and Metal Binding Characterization of a de Novo Designed Peptide, α3DIV, that Sequesters Toxic Heavy Metals.

Science.gov (United States)

Plegaria, Jefferson S; Dzul, Stephen P; Zuiderweg, Erik R P; Stemmler, Timothy L; Pecoraro, Vincent L

2015-05-12

De novo protein design is a biologically relevant approach that provides a novel process in elucidating protein folding and modeling the metal centers of metalloproteins in a completely unrelated or simplified fold. An integral step in de novo protein design is the establishment of a well-folded scaffold with one conformation, which is a fundamental characteristic of many native proteins. Here, we report the NMR solution structure of apo α3DIV at pH 7.0, a de novo designed three-helix bundle peptide containing a triscysteine motif (Cys18, Cys28, and Cys67) that binds toxic heavy metals. The structure comprises 1067 NOE restraints derived from multinuclear multidimensional NOESY, as well as 138 dihedral angles (ψ, φ, and χ1). The backbone and heavy atoms of the 20 lowest energy structures have a root mean square deviation from the mean structure of 0.79 (0.16) Å and 1.31 (0.15) Å, respectively. When compared to the parent structure α3D, the substitution of Leu residues to Cys enhanced the α-helical content of α3DIV while maintaining the same overall topology and fold. In addition, solution studies on the metalated species illustrated metal-induced stability. An increase in the melting temperatures was observed for Hg(II), Pb(II), or Cd(II) bound α3DIV by 18-24 °C compared to its apo counterpart. Further, the extended X-ray absorption fine structure analysis on Hg(II)-α3DIV produced an average Hg(II)-S bond length at 2.36 Å, indicating a trigonal T-shaped coordination environment. Overall, the structure of apo α3DIV reveals an asymmetric distorted triscysteine metal binding site, which offers a model for native metalloregulatory proteins with thiol-rich ligands that function in regulating toxic heavy metals, such as ArsR, CadC, MerR, and PbrR.

Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis.

Science.gov (United States)

Roberts, Blaine R; Lim, Nastasia K H; McAllum, Erin J; Donnelly, Paul S; Hare, Dominic J; Doble, Philip A; Turner, Bradley J; Price, Katherine A; Lim, Sin Chun; Paterson, Brett M; Hickey, James L; Rhoads, Timothy W; Williams, Jared R; Kanninen, Katja M; Hung, Lin W; Liddell, Jeffrey R; Grubman, Alexandra; Monty, Jean-Francois; Llanos, Roxana M; Kramer, David R; Mercer, Julian F B; Bush, Ashley I; Masters, Colin L; Duce, James A; Li, Qiao-Xin; Beckman, Joseph S; Barnham, Kevin J; White, Anthony R; Crouch, Peter J

2014-06-04

Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1. Copyright © 2014 the authors 0270-6474/14/348021-11$15.00/0.

Computational analysis of TRAPPC9: candidate gene for autosomal recessive non-syndromic mental retardation.

Science.gov (United States)

Khattak, Naureen Aslam; Mir, Asif

2014-01-01

Mental retardation (MR)/ intellectual disability (ID) is a neuro-developmental disorder characterized by a low intellectual quotient (IQ) and deficits in adaptive behavior related to everyday life tasks such as delayed language acquisition, social skills or self-help skills with onset before age 18. To date, a few genes (PRSS12, CRBN, CC2D1A, GRIK2, TUSC3, TRAPPC9, TECR, ST3GAL3, MED23, MAN1B1, NSUN1) for autosomal-recessive forms of non syndromic MR (NS-ARMR) have been identified and established in various families with ID. The recently reported candidate gene TRAPPC9 was selected for computational analysis to explore its potentially important role in pathology as it is the only gene for ID reported in more than five different familial cases worldwide. YASARA (12.4.1) was utilized to generate three dimensional structures of the candidate gene TRAPPC9. Hybrid structure prediction was employed. Crystal Structure of a Conserved Metalloprotein From Bacillus Cereus (3D19-C) was selected as best suitable template using position-specific iteration-BLAST. Template (3D19-C) parameters were based on E-value, Z-score and resolution and quality score of 0.32, -1.152, 2.30°A and 0.684 respectively. Model reliability showed 93.1% residues placed in the most favored region with 96.684 quality factor, and overall 0.20 G-factor (dihedrals 0.06 and covalent 0.39 respectively). Protein-Protein docking analysis demonstrated that TRAPPC9 showed strong interactions of the amino acid residues S(253), S(251), Y(256), G(243), D(131) with R(105), Q(425), W(226), N(255), S(233), its functional partner 1KBKB. Protein-protein interacting residues could facilitate the exploration of structural and functional outcomes of wild type and mutated TRAPCC9 protein. Actively involved residues can be used to elucidate the binding properties of the protein, and to develop drug therapy for NS-ARMR patients.

Superoxide reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-rays

Energy Technology Data Exchange (ETDEWEB)

Santos-Silva, Teresa; Trincão, José; Carvalho, Ana L.; Bonifácio, Cecília; Auchère, Françoise; Moura, Isabel; Moura, José J. G.; Romão, Maria J., E-mail: mromao@dq.fct.unl.pt [REQUIMTE Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal)

2005-11-01

Superoxide reductase is a non-haem iron-containing protein involved in resistance to oxidative stress. The oxidized form of the protein has been crystallized and its three-dimensional structure solved. A highly redundant X-ray diffraction data set was collected on a rotating-anode generator using Cu Kα X-ray radiation. Four Fe atoms were located in the asymmetric unit corresponding to four protein molecules arranged as a dimer of homodimers. Superoxide reductase is a 14 kDa metalloprotein containing a catalytic non-haem iron centre [Fe(His){sub 4}Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K{sub 3}Fe(CN){sub 6} belonged to space group P2{sub 1} (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 Å, β = 106.9°) and diffracted beyond 1.60 Å resolution, while crystals grown in the presence of Na{sub 2}IrCl{sub 6} belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 Å, β = 104.9°) and diffracted beyond 1.55 Å. A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator (λ = 1.542 Å) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P2{sub 1} data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.

Superoxide reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-rays

International Nuclear Information System (INIS)

Santos-Silva, Teresa; Trincão, José; Carvalho, Ana L.; Bonifácio, Cecília; Auchère, Françoise; Moura, Isabel; Moura, José J. G.; Romão, Maria J.

2005-01-01

Superoxide reductase is a non-haem iron-containing protein involved in resistance to oxidative stress. The oxidized form of the protein has been crystallized and its three-dimensional structure solved. A highly redundant X-ray diffraction data set was collected on a rotating-anode generator using Cu Kα X-ray radiation. Four Fe atoms were located in the asymmetric unit corresponding to four protein molecules arranged as a dimer of homodimers. Superoxide reductase is a 14 kDa metalloprotein containing a catalytic non-haem iron centre [Fe(His) 4 Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K 3 Fe(CN) 6 belonged to space group P2 1 (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 Å, β = 106.9°) and diffracted beyond 1.60 Å resolution, while crystals grown in the presence of Na 2 IrCl 6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 Å, β = 104.9°) and diffracted beyond 1.55 Å. A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator (λ = 1.542 Å) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P2 1 data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed

An integrative computational framework based on a two-step random forest algorithm improves prediction of zinc-binding sites in proteins.

Directory of Open Access Journals (Sweden)

Cheng Zheng

Full Text Available Zinc-binding proteins are the most abundant metalloproteins in the Protein Data Bank where the zinc ions usually have catalytic, regulatory or structural roles critical for the function of the protein. Accurate prediction of zinc-binding sites is not only useful for the inference of protein function but also important for the prediction of 3D structure. Here, we present a new integrative framework that combines multiple sequence and structural properties and graph-theoretic network features, followed by an efficient feature selection to improve prediction of zinc-binding sites. We investigate what information can be retrieved from the sequence, structure and network levels that is relevant to zinc-binding site prediction. We perform a two-step feature selection using random forest to remove redundant features and quantify the relative importance of the retrieved features. Benchmarking on a high-quality structural dataset containing 1,103 protein chains and 484 zinc-binding residues, our method achieved >80% recall at a precision of 75% for the zinc-binding residues Cys, His, Glu and Asp on 5-fold cross-validation tests, which is a 10%-28% higher recall at the 75% equal precision compared to SitePredict and zincfinder at residue level using the same dataset. The independent test also indicates that our method has achieved recall of 0.790 and 0.759 at residue and protein levels, respectively, which is a performance better than the other two methods. Moreover, AUC (the Area Under the Curve and AURPC (the Area Under the Recall-Precision Curve by our method are also respectively better than those of the other two methods. Our method can not only be applied to large-scale identification of zinc-binding sites when structural information of the target is available, but also give valuable insights into important features arising from different levels that collectively characterize the zinc-binding sites. The scripts and datasets are available at http://protein.cau.edu.cn/zincidentifier/.

Quantification of trace elements in protein bands by synchrotron radiation x-ray fluorescence after isoelectric focusing separation of human hemoglobin

International Nuclear Information System (INIS)

Gao Yuxi; Chen Chunying; Li Bai; He Wei; Huang Yuying; Chai Zhifang

2005-01-01

The role and effects of a trace element in a particular organism strongly depend on its particular chemical forms in which the element is present. Therefore, the bulk content or concentration of an element in the organism of interest is often meaningless in judging its biological significance. To understand bioavailability, transportation, cell uptake, metabolism, toxicity, and other biological behaviors of trace elements in the body, information is needed about speciation of trace element, especially about distribution of metal-containing proteins. Development of appropriate methods for speciation analysis is therefore required. Synchrotron radiation x-ray fluorescence (SRXRF) is a sensitive method for multielemental analysis with detection limit of 10 ng/g. It has been successfully used for imaging and quantifying trace elements in various pathological and healthy tissues, even in a single cell, to help understand the mechanism of diseases and the biochemistry of elements. In our previous work, the technique was combined with electrophoresis to study distribution of metalloproteins in biological samples, but the quantitative analysis of trace elements in protein bands after electrophoresis was still unrealized. In this study, a procedure has been proposed for quantification of Fe, Cu, and Zn in protein bands with SRXRF analysis after isoelectric focusing (IEF) separation. Calibration standards were prepared by adding certain amounts of metal ions and free-metal proteins to electrophoresis gel. Human hemoglobin was separated with IEF, and Fe, Cu, and Zn in protein bands were analyzed by SRXRF. The calibration curves can be obtained in a range of 0-8 mg/kg metals and a linear relationship between dosage of metals and fluorescent intensity can be observed (r 2 > 0.99). The method provides the detection limits of 2.43, 1.12, and 0.96 mg/kg for Fe, Cu and Zn, and the recoveries of 90.4 and 115.7 % for Fe and Zn, respectively. The hyphenated technique of SRXRF and IEF

A cleanroom sleeping environment's impact on markers of oxidative stress, immune dysregulation, and behavior in children with autism spectrum disorders.

Science.gov (United States)

Faber, Scott; Zinn, Gregory M; Boggess, Andrew; Fahrenholz, Timothy; Kern, John C; Kingston, H M Skip

2015-03-19

An emerging paradigm suggests children with autism display a unique pattern of environmental, genetic, and epigenetic triggers that make them susceptible to developing dysfunctional heavy metal and chemical detoxification systems. These abnormalities could be caused by alterations in the methylation, sulfation, and metalloprotein pathways. This study sought to evaluate the physiological and behavioral effects of children with autism sleeping in an International Organization for Standardization Class 5 cleanroom. Ten children with autism, ages 3-12, slept in a cleanroom for two weeks to evaluate changes in toxin levels, oxidative stress, immune dysregulation, and behavior. Before and after the children slept in the cleanroom, samples of blood and hair and rating scale scores were obtained to assess these changes. Five children significantly lowered their concentration of oxidized glutathione, a biomarker of oxidative stress. The younger cohort, age 5 and under, showed significantly greater mean decreases in two markers of immune dysregulation, CD3% and CD4%, than the older cohort. Changes in serum magnesium, influencing neuronal regulation, correlated negatively while changes in serum iron, affecting oxygenation of tissues, correlated positively with age. Changes in serum benzene and PCB 28 concentrations showed significant negative correlations with age. The younger children demonstrated significant improvements on behavioral rating scales compared to the older children. In a younger pair of identical twins, one twin showed significantly greater improvements in 4 out of 5 markers of oxidative stress, which corresponded with better overall behavioral rating scale scores than the other twin. Younger children who slept in the cleanroom altered elemental levels, decreased immune dysregulation, and improved behavioral rating scales, suggesting that their detoxification metabolism was briefly enhanced. The older children displayed a worsening in behavioral rating scale

The teratogenicity of cadmium-metallothionein in the rat

International Nuclear Information System (INIS)

Webb, M.; Holt, D.; Brown, N.; Hard, G.C.

1988-01-01

A single dose in the range 0.25-1.9 mg metallothionein-bound cadmium (MT-Cd)/kg body weight, when administered parenterally to the rat between day 8 and day 14 of gestation, is teratogenic. In vitro, the development of the isolated rat conceptus is unaffected by the addition of 1.5 μM MT-Cd to the culture medium whereas the same concentration of ionic Cd (as CdCl 2 ) is lethal. At short times after injection of 0.25 mg MT-Cd/kg body weight on gd 12, the maximal foetal and placental contents of Cd are low in comparison with those after a teratogenic dose of CdCl 2 and are of the same order as those in the embryo and placenta + yolk sac of the rat conceptus, cultured in the presence of the highest no-effect concentration of CdCl 2 . From this evidence, it is concluded that the uptake by the conceptus in vivo of either CdMT, or of Cd liberated therefrom, is unlikely to contribute to the teratogenic response. In the pregnant, as in the non-pregnant rat, the kidney appears to be the only organ that is affected directly by the metalloprotein. All doses in the range 0.25-1.0 mg MT-Cd/kg body weight are nephrotoxic and result in prolonged anorexia in the pregnant animal. While some of the foetal deformities that occur in the CdMT-dosed animal seem to be direct consequences of the renal dysfunction, others apparently are secondary to the maternal anorexia. In rats that are injected i.p on gd 12 with 0.25 mg MT-Cd/kg renal uptake of Cd is slower, but the final concentration is higher than in animals that are given the same dose i.v. At this and the higher dose levels structural and/or functional damage to the kidneys also is greater in i.p.-, than in i.v.-dosed animals. The incidence of foetal malformations, however, is similar in the i.p. and i.v. groups and varies little over the dose range. (orig./MG)

MAGGIE Component 1: Identification and Purification of Native and Recombinant Multiprotein Complexes and Modified Proteins from Pyrococcus furiosus

Energy Technology Data Exchange (ETDEWEB)

Adams, Michael W. [University of Georgia; W. W. Adams, Michael

2014-01-07

Virtualy all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes (PCs), the composition of which is largely unknown. Structural genomics efforts have demonstrated that less than 25% of the genes in a given prokaryotic genome will yield stable, soluble proteins when expressed using a one-ORF-at-a-time approach. We proposed that much of the remaining 75% of the genes encode proteins that are part of multiprotein complexes or are modified post-translationally, for example, with metals. The problem is that PCs and metalloproteins (MPs) cannot be accurately predicted on a genome-wide scale. The only solution to this dilemma is to experimentally determine PCs and MPs in biomass of a model organism and to develop analytical tools that can then be applied to the biomass of any other organism. In other words, organisms themselves must be analyzed to identify their PCs and MPs: “native proteomes” must be determined. This information can then be utilized to design multiple ORF expression systems to produce recombinant forms of PCs and MPs. Moreover, the information and utility of this approach can be enhanced by using a hyperthermophile, one that grows optimally at 100°C, as a model organism. By analyzing the native proteome at close to 100 °C below the optimum growth temperature, we will trap reversible and dynamic complexes, thereby enabling their identification, purification, and subsequent characterization. The model organism for the current study is Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100°C. It is grown up to 600-liter scale and kg quantities of biomass are available. In this project we identified native PCs and MPs using P. furiosus biomass (with MS/MS analyses to identify proteins by component 4). In addition, we provided samples of abundant native PCs and MPs for structural characterization (using SAXS by component 5). We also designed and evaluated generic bioinformatics and

The potential of organic (electrospray- and atmospheric pressure chemical ionisation) mass spectrometric techniques coupled to liquid-phase separation for speciation analysis.

Science.gov (United States)

Rosenberg, Erwin

2003-06-06

environment, health and food. Particular attention is given to the characterisation of biomolecules and metalloproteins (metallothioneins and phytochelatins) and to the investigation of the interaction of metals and biomolecules. Particularly in the latter field, ESI-MS is the ideal technique due to the softness of the ionisation process which allows to assume that the detected gas-phase ions are a true representation of the ions or ion-biomolecule complexes prevalent in solution. It is particularly this field, important to biochemistry, physiology and medical chemistry, where we can expect significant developments also in the future.

Highly selective BSA imprinted polyacrylamide hydrogels facilitated by a metal-coding MIP approach.

Science.gov (United States)

El-Sharif, H F; Yapati, H; Kalluru, S; Reddy, S M

2015-12-01

We report the fabrication of metal-coded molecularly imprinted polymers (MIPs) using hydrogel-based protein imprinting techniques. A Co(II) complex was prepared using (E)-2-((2 hydrazide-(4-vinylbenzyl)hydrazono)methyl)phenol; along with iron(III) chloroprotoporphyrin (Hemin), vinylferrocene (VFc), zinc(II) protoporphyrin (ZnPP) and protoporphyrin (PP), these complexes were introduced into the MIPs as co-monomers for metal-coding of non-metalloprotein imprints. Results indicate a 66% enhancement for bovine serum albumin (BSA) protein binding capacities (Q, mg/g) via metal-ion/ligand exchange properties within the metal-coded MIPs. Specifically, Co(II)-complex-based MIPs exhibited 92 ± 1% specific binding with Q values of 5.7 ± 0.45 mg BSA/g polymer and imprinting factors (IF) of 14.8 ± 1.9 (MIP/non-imprinted (NIP) control). The selectivity of our Co(II)-coded BSA MIPs were also tested using bovine haemoglobin (BHb), lysozyme (Lyz), and trypsin (Tryp). By evaluating imprinting factors (K), each of the latter proteins was found to have lower affinities in comparison to cognate BSA template. The hydrogels were further characterised by thermal analysis and differential scanning calorimetry (DSC) to assess optimum polymer composition. The development of hydrogel-based molecularly imprinted polymer (HydroMIPs) technology for the memory imprinting of proteins and for protein biosensor development presents many possibilities, including uses in bio-sample clean-up or selective extraction, replacement of biological antibodies in immunoassays and biosensors for medicine and the environment. Biosensors for proteins and viruses are currently expensive to develop because they require the use of expensive antibodies. Because of their biomimicry capabilities (and their potential to act as synthetic antibodies), HydroMIPs potentially offer a route to the development of new low-cost biosensors. Herein, a metal ion-mediated imprinting approach was employed to metal-code our

Surface Redox Chemistry of Immobilized Nanodiamond: Effects of Particle Size and Electrochemical Environment

Science.gov (United States)

Gupta, S.; McDonald, B.; Carrizosa, S. B.

2017-07-01

processes in the presence of specific redox-active molecules via feedback mechanism. Apparently, FcMeOH+ tended to have electrostatic affinity for negatively charged ND surface functionalities, corroborated by present experiments. We also attempted to study biocatalytic process using model metalloprotein (cytochrome c; Cyt c) immobilized on ND particles for investigating interfacial electron transfer kinetics and compared with those of functionalized graphene (graphene oxide; GO and reduced GO). The findings are discussed in terms of interplay of sp 3-bonded C (ND core) and sp 2-bonded C (ND shell and graphene-based systems).

Spin-Polarization-Induced Preedge Transitions in the Sulfur K-Edge XAS Spectra of Open-Shell Transition-Metal Sulfates: Spectroscopic Validation of σ-Bond Electron Transfer.

Science.gov (United States)

Frank, Patrick; Szilagyi, Robert K; Gramlich, Volker; Hsu, Hua-Fen; Hedman, Britt; Hodgson, Keith O

2017-02-06

extends across a hydrogen-bond bridge between sulfate and the itao ligand and involves orbitals at energies below the frontier set. This electronic structure feature provides a direct spectroscopic confirmation of the through-bond electron-transfer mechanism of redox-active metalloproteins.

Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential.

Science.gov (United States)

Blakeley, Matthew P; Hasnain, Samar S; Antonyuk, Svetlana V

2015-07-01

crystallography therefore remains the only approach where diffraction data can be collected at room temperature without radiation damage issues and the only approach to locate mobile or highly polarized H atoms and protons. Here a review of the current status of sub-atomic X-ray and neutron macromolecular crystallography is given and future prospects for combined approaches are outlined. New results from two metalloproteins, copper nitrite reductase and cytochrome c', are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å) X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography.

Sub-atomic resolution X-ray crystallography and neutron crystallography: promise, challenges and potential

Directory of Open Access Journals (Sweden)

Matthew P. Blakeley

2015-07-01

. Neutron crystallography therefore remains the only approach where diffraction data can be collected at room temperature without radiation damage issues and the only approach to locate mobile or highly polarized H atoms and protons. Here a review of the current status of sub-atomic X-ray and neutron macromolecular crystallography is given and future prospects for combined approaches are outlined. New results from two metalloproteins, copper nitrite reductase and cytochrome c′, are also included, which illustrate the type of information that can be obtained from sub-atomic-resolution (∼0.8 Å X-ray structures, while also highlighting the need for complementary neutron studies that can provide details of H atoms not provided by X-ray crystallography.

Catalytic zinc site and mechanism of the metalloenzyme PR-AMP cyclohydrolase.

Science.gov (United States)

D'Ordine, Robert L; Linger, Rebecca S; Thai, Carolyn J; Davisson, V Jo

2012-07-24

The enzyme N(1)-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn(2+) metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis, an uncommon ring-opening of a purine heterocycle for use in primary metabolism. A three-dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn(2+) binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate N(1)-(5'-phosphoribosyl)adenosine 5'-monophosphate; (PR-AMP), while Mg(2+), a metal required for catalytic activity, enhanced the rate of inactivation. Site-directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn(2+) content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity, and only the C116A was still capable of binding Zn(2+). Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn(2+) and provides further evidence that the Zn(2+) coordination site involves the

Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9 Purificação e caracterização de uma lipase extracelular produzida por uma nova cepa: Pseudomonas aeruginosa SRT9

Directory of Open Access Journals (Sweden)

Prita S. Borkar

2009-06-01

Full Text Available An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL- 4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of temperature and pH values with optimum temperature of 55ºC and pH 6.9. The lipase preferably acted on triacylglycerols of long chain (C14-C16 fatty acids. The lipase was inhibited strongly by EDTA suggesting the enzyme might be metalloprotein. SDS and metal ions such as Hg2+, Zn2+, Cu2+, Ag2+ and Fe2+ decreased the lipase activity remarkedly. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool with a variety of applications including organo synthetic reactions and preparation of enantiomerically pure pharmaceuticals. The Km and Vmax value of the purified enzyme for triolein hydrolysis were calculated to be 1.11 mmol/L and 0.05 mmol/L/minrespectively.Uma lipase extracelular foi isolada e purificada a partir de um caldo de cultura de Pseudomonas aeruginosa SRT9 até homogeneidade visível empregando-se precipitação com sulfato de amônia, seguida de técnicas cromatográficas em colunas de fenil sefarose CL-4B e Mono Q HR 5/5, obtendo-se um fator de purificação de 98 vezes, e atividade especifica de 12307,8 U/mg. Por SDS_PAGE, estimou-se que o peso molecular da lipase purificada é 29kDa, com um ponto isoelétrico de 4,5. A lipase apresentou atividade máxima em uma ampla faixa de temperatura e pH, com ótimos a 55ºC e pH 6,9. A lípase foi mais ativa sobre triacilglicerois de cadeia longa (C14-C16. A lipase foi fortemente inibida por EDTA, o que sugere que a

Ultracentrifugation and inductively coupled plasma mass spectrometry for metal-protein equilibrium studies

Energy Technology Data Exchange (ETDEWEB)

Arnquist, Isaac J.; Holcombe, James A., E-mail: holcombe@mail.utexas.edu

2012-10-15

The coupling of separation by preparative ultracentrifugation and metal detection by inductively coupled plasma mass spectrometry (ICP-MS) has been explored for metal-protein equilibrium determinations. This study characterizes the stoichiometry as well as apparent (K{sub app}) and intrinsic (K{sub int}) binding affinities of the metal-protein association for a model protein. In particular, the affinity of Cu{sup 2+} for the high affinity binding site in bovine serum albumin (BSA) is determined. Once equilibrium is established between Cu{sup 2+} and BSA, preparative ultracentrifugation moves the metalloprotein away from the meniscus, leaving unbound equilibrium copper in the protein free solution. Since the initial (total) concentrations of purified BSA and Cu{sup 2+} can be determined, the free copper concentration at equilibrium can also be determined by taking a small aliquot above the sedimenting boundary for analysis using ICP-MS. This analysis allows for the determination of free Cu{sup 2+} ion, which is identical to the equilibrium concentration prior to ultracentrifugation. From these data K{sub app} and K{sub int} were determined at two different conditions, 100 mM Tris(hydroxymethyl)aminomethane (Tris) at pH 9.53 and pH 7.93. log K{sub app} values of 17.6 and 14.6 were determined at pH 9.53 and pH 7.93, respectively. Furthermore, pH-independent log K{sub int} values of - 1.43 and - 1.04 were determined at pH 9.53 and 7.93, respectively. While the log K{sub int} at pH 9.53 was in good agreement with literature values obtained from alternative methods, K{sub int} at pH 7.93 was about 2.5 Multiplication-Sign larger than previously reported. BSA undergoes a structural rearrangement between pH 7-9, and the generally accepted pH-dependency of protein tertiary structure may be responsible for the variations in the 'intrinsic' binding constant. The Cu-BSA binding affinity was also monitored in 100 mM Tris 0.1% sodium dodecyl sulfate (SDS) solution at p

Structure and function of proteins investigated by crystallographic and spectroscopic time-resolved methods

Science.gov (United States)

Purwar, Namrta

Biomolecules play an essential role in performing the necessary functions for life. The goal of this thesis is to contribute to an understanding of how biological systems work on the molecular level. We used two biological systems, beef liver catalase (BLC) and photoactive yellow protein (PYP). BLC is a metalloprotein that protects living cells from the harmful effects of reactive oxygen species by converting H2O2 into water and oxygen. By binding nitric oxide (NO) to the catalase, a complex was generated that mimics the Cat-H2O2 adduct, a crucial intermediate in the reaction promoted by the catalase. The Cat-NO complex is obtained by using a convenient NO generator (1-(N,N-diethylamino)diazen-1-ium-1,2-diolate). Concentrations up to 100˜200 mM are reached by using a specially designed glass cavity. With this glass apparatus and DEANO, sufficient NO occupation is achieved and structure determination of the catalase with NO bound to the heme iron becomes possible. Structural changes upon NO binding are minute. NO has a slightly bent geometry with respect to the heme normal, which results in a substantial overlap of the NO orbitals with the iron-porphyrin molecular orbitals. From the structure of the iron-NO complex, conclusions on the electronic properties of the heme iron can be drawn that ultimately lead to an insight into the catalytic properties of this enzyme. Enzyme kinetics is affected by additional parameters such as temperature and pH. Additionally, in crystallography, the absorbed X-ray dose may impair protein function. To address the effect of these parameters, we performed time-resolved crystallographic experiments on a model system, PYP. By collecting multiple time-series on PYP at increasing X-ray dose levels, we determined a kinetic dose limit up to which kinetically meaningful X-ray data sets can be collected. From this, we conclude that comprehensive time-series spanning up to 12 orders of magnitude in time can be collected from a single PYP

Sustainable resource recovery and energy conversion processes using microbial electrochemical technologies

Science.gov (United States)

Yates, Matthew D.

electrodes were found to be derived primarily from metalloproteins produced by G. sulfurreducens and M. barkeri cells. These findings show that hydrogen can be produced in a biocathodic system by abiotic cell material attached to a graphite electrode surface and that it does not require electron uptake by living cells.