A high-throughput approach to protein structure analysis.
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Biophysical Phenomena↗
Biomedical subjects
Mark R Chance
Publications and source records attributed to Mark R Chance.
Biochemical implications of a three-dimensional model of monomeric actin bound to magnesium-chelated ATP.
Actin structure is of intense interest in biology due to its importance in cell function and motility mediated by the spatial and temporal regulation of actin monomer-filament interconversions in a wide range of developmental and disease states. Despite this interest, the structure of many functionally important actin forms has eluded high-resolution analysis. Due to the propensity of actin monomers to assemble into filaments structural analysis of Mg-bound actin monomers has proven difficult, whereas high-resolution structures of actin with a diverse array of ligands that preclude polymerization have been quite successful. In this work, we provide a high-resolution structural model of the Mg-ATP-actin monomer using a combination of computational methods and experimental footprinting data that we have previously published. The key conclusion of this study is that the structure of the nucleotide binding cleft defined by subdomains 2 and 4 is essentially closed, with specific contacts between two subdomains predicted by the data.
Actins↗
Beamline X29: a novel undulator source for X-ray crystallography.
A high-flux insertion device and beamline for macromolecular crystallography has been built at the National Synchrotron Light Source (NSLS) that employs a mini-gap undulator source developed by the NSLS. The mini-gap undulator at beamline X29 is a hybrid-magnet device of period 12.5 mm operating at proven gaps of 3.3-10 mm. The beamline provides hard X-rays for macromolecular crystallography experiments from the second and third harmonics over an energy range of 5-15 keV. The X-ray optics is designed to deliver intense and highly collimated X-rays. Horizontal focusing is achieved by a cryogenically cooled sagittally focusing double-crystal monochromator with approximately 4.1:1 demagnification. A vertical focusing mirror downstream of the monochromator is used for harmonic rejection and vertical focusing. The experimental station hosts an Area Detector Systems Quantum 315 CCD detector with 2.2 s readout time between exposures and Crystal Logic goniostat for crystal rotation and detector positioning. An auto-mounter crystal changer has been installed to facilitate the high-throughput data collection required by the major users, which includes structural genomics projects and the Macromolecular Crystallography Research Resource mail-in program. X29 is 10(3) times brighter than any existing bending-magnet beamline at NSLS with an actual flux of 2.5 x 10(11) photons s(-1) through a 0.12 mm square aperture at 11.271 keV.
Crystallography, X-Ray↗
Crystal structure of Escherichia coli L-arabinose isomerase (ECAI), the putative target of biological tagatose production.
Escherichia coli L-arabinose isomerase (ECAI; EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose in vivo. This enzyme is also of commercial interest as it catalyzes the conversion of D-galactose to D-tagatose in vitro. The crystal structure of ECAI was solved and refined at 2.6 A resolution. The subunit structure of ECAI is organised into three domains: an N-terminal, a central and a C-terminal domain. It forms a crystallographic trimeric architecture in the asymmetric unit. Packing within the crystal suggests the idea that ECAI can form a hexameric assembly. Previous electron microscopic and biochemical studies supports that ECAI is hexameric in solution. A comparison with other known structures reveals that ECAI adopts a protein fold most similar to E. coli fucose isomerase (ECFI) despite very low sequence identity 9.7%. The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly, and active site architecture strongly suggests that the enzymes have functional similarities. Further, the crystal structure of ECAI forms a basis for identifying molecular determinants responsible for isomerization of arabinose to ribulose in vivo and galactose to tagatose in vitro.
Aldose-Ketose Isomerases↗
Inner ear proteomics of mouse models for deafness, a discovery strategy.
Inner ear dysfunction is often associated with defective hair cells. Therefore, hair cells are the focus of study in many of the mouse mutants showing auditory and vestibular deficits. However, harvesting sufficient numbers of hair cells from the tiny bony mouse inner ear for proteomic analysis is challenging. New approaches that would take advantage of mouse mutants and avoid processing steps, such as decalcification or microdissetion, would be more suitable for proteomic analysis. Here, we propose a novel approach called SSUMM-Subtractive Strategy Using Mouse Mutants. SSUMM takes advantage of the differences between control and affected or mutant samples. We predict that SSUMM would be a useful method in proteomics, especially in those cases in which the investigator must work with small numbers of diverse cell types from a tiny organ. Here, we discuss the potential utility of SSUMM to unravel the protein expression profiles of hair cells using the Pou4f3 mouse mutant as an example. Pou4f3 mutant mice exhibit a total loss of inner and outer hair cells, but supporting cells remain relatively intact in the cochlea, thus providing an excellent model for identifying proteins and transcripts that are specific to the hair cell at all life stages. SSUMM would maximize the sensitivity of the analyses while obviating the need for tedious sessions of microdissection and collection of hair cells. By comparing the mutant to control ears at specific time points, it is possible to identify direct targets of a gene product of interest. Further, SSUMM could be used to identify and analyze inner ear development markers and other known genes/proteins that are coexpressed in the ear. In this short technical report, we also discuss protein-profiling approaches suitable for SSUMM and briefly discuss other approaches used in the field of proteomics.
Animals↗
Alcohol dehydrogenase restricts the ability of the pathogen Candida albicans to form a biofilm on catheter surfaces through an ethanol-based mechanism.
Candida biofilms formed on indwelling medical devices are increasingly associated with severe infections. In this study, we used proteomics and Western and Northern blotting analyses to demonstrate that alcohol dehydrogenase (ADH) is downregulated in Candida biofilms. Disruption of ADH1 significantly (P = 0.0046) enhanced the ability of Candida albicans to form biofilm. Confocal scanning laser microscopy showed that the adh1 mutant formed thicker biofilm than the parent strain (210 microm and 140 microm, respectively). These observations were extended to an engineered human oral mucosa and an in vivo rat model of catheter-associated biofilm. Inhibition of Candida ADH enzyme using disulfiram and 4-methylpyrazole resulted in thicker biofilm (P < 0.05). Moreover, biofilms formed by the adh1 mutant strain produced significantly smaller amounts of ethanol, but larger amounts of acetaldehyde, than biofilms formed by the parent and revertant strains (P < 0.0001), demonstrating that the effect of Adh1p on biofilm formation is mediated by its enzymatic activity. Furthermore, we found that 10% ethanol significantly inhibited biofilm formation in vitro, with complete inhibition of biofilm formation at ethanol concentrations of >/=20%. Similarly, using a clinically relevant rabbit model of catheter-associated biofilm, we found that ethanol treatment inhibited biofilm formation by C. albicans in vivo (P < 0.05) but not by Staphylococcus spp. (P > 0.05), indicating that ethanol specifically inhibits Candida biofilm formation. Taken together, our studies revealed that Adh1p contributes to the ability of C. albicans to form biofilms in vitro and in vivo and that the protein restricts biofilm formation through an ethanol-dependent mechanism. These results are clinically relevant and may suggest novel antibiofilm treatment strategies.
Alcohol Dehydrogenase↗
Radiolytic protein footprinting with mass spectrometry to probe the structure of macromolecular complexes.
Structural proteomics approaches using mass spectrometry are increasingly used in biology to examine the composition and structure of macromolecules. Hydroxyl radical-mediated protein footprinting using mass spectrometry has recently been developed to define structure, assembly, and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side chain groups with covalent modification reagents. Accurate measurements of side chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side chain modification sites are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes. In this review, we discuss the basic chemistry of hydroxyl radical reactions with peptides and proteins, highlight various approaches to map protein structure using radical oxidation methods, and describe state-of-the-art approaches to combine computational and footprinting data.
Hydroxyl Radical↗
The ybeY protein from Escherichia coli is a metalloprotein.
The three-dimensional crystallographic structure of the ybeY protein from Escherichia coli (SwissProt entry P77385) is reported at 2.7 A 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.
Amino Acid Sequence↗
Structural allostery and binding of the transferrin*receptor complex.
The structural allostery and binding interface for the human serum transferrin (Tf)*transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions (Cheng, Y., Zak, O., Aisen, P., Harrison, S. C. & Walz, T. (2004) Structure of the human transferrin receptor.transferrin complex. Cell 116, 565-576). The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.
Allosteric Regulation↗
Radiolytic modification and reactivity of amino acid residues serving as structural probes for protein footprinting.
Hydroxyl radical-mediated protein footprinting is a convenient and sensitive technique for mapping solvent-accessible surfaces of proteins and examining the structure and dynamics of biological assemblies. In this study, the reactivities and tendencies to form easily detectable products for all 20 (common) amino acid side chains along with cystine are directly compared using various standards. Although we have previously reported on the oxidation of many of these residues, this study includes a detailed examination of the less reactive residues and better defines their usefulness in hydroxyl radical-mediated footprinting experiments. All 20 amino amides along with cystine and a few tripeptides were irradiated by gamma-rays, the products were analyzed by electrospray mass spectrometry, and rate constants of modification were measured. The reactivities of amino acid side chains were compared based on their loss of mass spectral signal normalized to the rate of loss for Phe or Pro that were radiolyzed simultaneously to serve as internal standards. In this way, accurate quantitation of relative rates could be assured. A reactivity order of amino acid side chains was obtained as Cys > Met > Trp > Tyr > Phe > cystine > His > Leu, Ile > Arg, Lys, Val > Ser, Thr, Pro > Gln, Glu > Asp, Asn > Ala > Gly. Ala and Gly are far too unreactive to be useful probes in typical experiments and Asp and Asn are unlikely to be useful as well. Although Ser and Thr are more reactive than Pro, which is known to be a useful probe, their oxidation products are not easily detectable. Thus, it appears that 14 of the 20 side chains (plus cystine) are most likely to be useful in typical experiments. Since these residues comprise approximately 65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.
Amino Acids↗
Secondary reactions and strategies to improve quantitative protein footprinting.
Hydroxyl radical-mediated footprinting permits detailed examination of structure and dynamic processes of proteins and large biological assemblies, as changes in the rate of reaction of radicals with target peptides are governed by changes in the solvent accessibility of the side-chain probe residues. The precise and accurate determination of peptide reaction rates is essential to successfully probing protein structure using footprinting. In this study, we specifically examine the magnitude and mechanisms of secondary oxidation occurring after radiolytic exposure and prior to mass spectrometric analysis. Secondary oxidation results from hydrogen peroxide and other oxidative species generated during radiolysis, significantly impacting the oxidation of Met and Cys but not aromatic or other reactive residues. Secondary oxidation of Met with formation of sulfoxide degrades data reproducibility and inflates the perceived solvent accessibility of Met-containing peptides. It can be suppressed by adding trace amounts of catalase or millimolar Met-NH2 (or Met-OH) buffer immediately after irradiation; this leads to greatly improved adherence to first-order kinetics and more precise observed oxidation rates. The strategy is shown to suppress secondary oxidation in model peptides and improve data quality in examining the reactivity of peptides within the Arp2/3 protein complex. Cysteine is also subject to secondary oxidation generating disulfide as the principal product. The disulfides can be reduced before mass spectrometric analysis by reducing agents such as TCEP, while methionine sulfoxide is refractory to reduction by this reagent under typical reducing conditions.
Cysteine↗
Radiolytic modification of sulfur-containing amino acid residues in model peptides: fundamental studies for protein footprinting.
Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using gamma-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the gamma-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic digestions; when these are accounted for, a reactivity order of cysteine > methionine approximately tryptophan > cystine is observed.
Amino Acid Sequence↗
Catching RNA polymerase in the act of binding: intermediates in transcription illuminated by synchrotron footprinting.
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DNA↗
Structure and dynamics of the actin filament.
The structures of filamentous Mg-ATP-actin (F actin) in the presence and absence of KCl have been mapped with hydroxyl radicals (*OH) generated by synchrotron X-ray radiolysis. Proteolysis and mass spectrometry (MS) analysis revealed 52 reactive side-chain sites from 27 distinct peptides within actin. The reactivities of these probe sites with *OH in the F-actin states are compared with those of Mg-ATP-G-actin (monomers) analyzed previously [Guan, J.-Q. et al. (2003) Biochemistry 42, 11992-12000]. Filament-dependent protection within subdomains 2, 3, and 4 and at the C terminus is consistent with longitudinal contacts of monomers within the filament helical structure as predicted by the Holmes model. In the absence of KCl, the extent of filament-dependent protection rarely reached 3-fold, consistent with a highly dynamic filament characterized by relatively weak interactions between actin protomers. However, in the presence of KCl, the extents of protection are significantly increased, consistent with a well-ordered, more tightly packed filament structure. Filament-dependent enhancements of reactivity not predicted by the Holmes model are seen for a peptide that overlaps the "hydrophobic plug" (H-plug) region and for a peptide that forms contacts with the polyphosphate moiety of the bound nucleotide. Overall, these data are both consistent with and complementary to a recent deuterium-exchange MS study of filamentous actin [Chik, J. K., and Schriemer, D.C. (2003) J. Mol. Biol. 334, 373-385], which also did not detect any burial of the H plug upon formation of filaments.
Actin Cytoskeleton↗
New York-Structural GenomiX Research Consortium (NYSGXRC): a large scale center for the protein structure initiative.
Structural GenomiX, Inc. (SGX), four New York area institutions, and two University of California schools have formed the New York Structural GenomiX Research Consortium (NYSGXRC), an industrial/academic Research Consortium that exploits individual core competencies to support all aspects of the NIH-NIGMS funded Protein Structure Initiative (PSI), including protein family classification and target selection, generation of protein for biophysical analyses, sample preparation for structural studies, structure determination and analyses, and dissemination of results. At the end of the PSI Pilot Study Phase (PSI-1), the NYSGXRC will be capable of producing 100-200 experimentally determined protein structures annually. All Consortium activities can be scaled to increase production capacity significantly during the Production Phase of the PSI (PSI-2). The Consortium utilizes both centralized and de-centralized production teams with clearly defined deliverables and hand-off procedures that are supported by a web-based target/sample tracking system (SGX Laboratory Information Data Management System, LIMS, and NYSGXRC Internal Consortium Experimental Database, ICE-DB). Consortium management is provided by an Executive Committee, which is composed of the PI and all Co-PIs. Progress to date is tracked on a publicly available Consortium web site (http://www.nysgxrc.org) and all DNA/protein reagents and experimental protocols are distributed freely from the New York City Area institutions. In addition to meeting the requirements of the Pilot Study Phase and preparing for the Production Phase of the PSI, the NYSGXRC aims to develop modular technologies that are transferable to structural biology laboratories in both academe and industry. The NYSGXRC PI and Co-PIs intend the PSI to have a transforming effect on the disciplines of X-ray crystallography and NMR spectroscopy of biological macromolecules. Working with other PSI-funded Centers, the NYSGXRC seeks to create the structural biology laboratory of the future. Herein, we present an overview of the organization of the NYSGXRC and describe progress toward development of a high-throughput Gene-->Structure platform. An analysis of current and projected consortium metrics reflects progress to date and delineates opportunities for further technology development.
Cloning, Molecular↗
Metalloproteomics: high-throughput structural and functional annotation of proteins in structural genomics.
A high-throughput method for measuring transition metal content based on quantitation of X-ray fluorescence signals was used to analyze 654 proteins selected as targets by the New York Structural GenomiX Research Consortium. Over 10% showed the presence of transition metal atoms in stoichiometric amounts; these totals as well as the abundance distribution are similar to those of the Protein Data Bank. Bioinformatics analysis of the identified metalloproteins in most cases supported the metalloprotein annotation; identification of the conserved metal binding motif was also shown to be useful in verifying structural models of the proteins. Metalloproteomics provides a rapid structural and functional annotation for these sequences and is shown to be approximately 95% accurate in predicting the presence or absence of stoichiometric metal content. The project's goal is to assay at least 1 member from each Pfam family; approximately 500 Pfam families have been characterized with respect to transition metal content so far.
Binding Sites↗
Structural proteomics of macromolecular assemblies using oxidative footprinting and mass spectrometry.
Understanding the composition, structure and dynamics of macromolecules and their assemblies is at the forefront of biological science today. Hydroxyl-radical-mediated protein footprinting using mass spectrometry can define macromolecular structure, macromolecular assembly and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side-chain groups with covalent-modification reagents. Subsequent to oxidation by reactive oxygen species, proteins are digested by specific proteases to generate peptides for analysis by mass spectrometry. Accurate measurements of side-chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side-chain sites within the macromolecular probes are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes.
Chromatography, Liquid↗
Principles of RNA compaction: insights from the equilibrium folding pathway of the P4-P6 RNA domain in monovalent cations.
Counterions are required for RNA folding, and divalent metal ions such as Mg(2+) are often critical. To dissect the role of counterions, we have compared global and local folding of wild-type and mutant variants of P4-P6 RNA derived from the Tetrahymena group I ribozyme in monovalent and in divalent metal ions. A remarkably simple picture of the folding thermodynamics emerges. The equilibrium folding pathway in monovalent ions displays two phases. In the first phase, RNA molecules that are initially in an extended conformation enforced by charge-charge repulsion are relaxed by electrostatic screening to a state with increased flexibility but without formation of long-range tertiary contacts. At higher concentrations of monovalent ions, a state that is nearly identical to the native folded state in the presence of Mg(2+) is formed, with tertiary contacts that involve base and backbone interactions but without the subset of interactions that involve specific divalent metal ion-binding sites. The folding model derived from these and previous results provides a robust framework for understanding the equilibrium and kinetic folding of RNA.
Animals↗