共查询到20条相似文献,搜索用时 31 毫秒
1.
Parminder Kaur Sara Tomechko Janna Kiselar Wuxian Shi Galahad Deperalta Aaron T Wecksler Giridharan Gokulrangan Victor Ling Mark R Chance 《MABS-AUSTIN》2014,6(6):1486-1499
Amino acid-specific covalent labeling is well suited to probe protein structure and macromolecular interactions, especially for macromolecules and their complexes that are difficult to examine by alternative means, due to size, complexity, or instability. Here we present a detailed account of carbodiimide-based covalent labeling (with GEE tagging) applied to a glycosylated monoclonal antibody therapeutic, which represents an important class of biologic drugs. Characterization of such proteins and their antigen complexes is essential to development of new biologic-based medicines. In this study, the experiments were optimized to preserve the structural integrity of the protein, and experimental conditions were varied and replicated to establish the reproducibility and precision of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include D, E, and the C-terminus, against the experimental surface accessibility data in order to understand the accuracy of the approach in providing an unbiased assessment of structure. Data from the protein were also compared to reactivity measures of several model peptides to explain sequence or structure-based variations in reactivity. The results highlight several advantages of this approach. These include: the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling (indicating that the label does not significantly perturb the structure of the protein), the high reproducibility of replicate experiments (<2 % variation in modification extent), the similar reactivity of the 3 target probe residues (as suggested by analysis of model peptides), and the overall positive and significant correlation of reactivity and solvent accessible surface area (the latter values predicted by the homology modeling). Attenuation of reactivity, in otherwise solvent accessible probes, is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. The results are also compared with data from hydroxyl radical-mediated oxidative footprinting on the same protein, showing that complementary information is gained from the 2 approaches, although the number of target residues in carbodiimide/GEE labeling is fewer. Overall, this approach is an accurate and precise method for assessing protein structure of biologic drugs. 相似文献
2.
《MABS-AUSTIN》2013,5(3):540-552
Structural characterization of proteins and their antigen complexes is essential to the development of new biologic-based medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta et al using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (<2% variation in modification extent), the similar reactivity of the three target probes, and significant correlation of reactivity and solvent accessible surface area. 相似文献
3.
Parminder Kaur Sara E Tomechko Janna Kiselar Wuxian Shi Galahad Deperalta Aaron T Wecksler Giridharan Gokulrangan Victor Ling Mark R Chance 《MABS-AUSTIN》2015,7(3):540-552
Structural characterization of proteins and their antigen complexes is essential to the development of new biologic-based medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta et al using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (<2% variation in modification extent), the similar reactivity of the three target probes, and significant correlation of reactivity and solvent accessible surface area. 相似文献
4.
Ladner CL Turner RJ Edwards RA 《Protein science : a publication of the Protein Society》2007,16(6):1204-1213
Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction. In this study, tryptophan residues of carbonic anhydrase are reacted with chloroform, 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA), or 3-bromo-1-propanol (BP) under UV irradiation at 280 nm. The light-driven reactions with chloroform, TCE, TCA, and BP attach a formyl, hydroxyethanone, carboxylic acid, and propanol group, respectively, onto the indole ring of tryptophan. Trypsin and chymotrypsin digests of the modified carbonic anhydrase are used to map accessible tryptophan residues using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tryptophan reactivity is determined by identifying peptides with tryptophan residues modified with the appropriate label. The reactivity is calculated from the frequency that the modification is identified and a semiquantitative measure of the amount of products formed. Both of these measures of tryptophan reactivity correlate significantly with the accessible surface area of tryptophan residues in carbonic anhydrase determined from the X-ray crystal structure. Therefore the photochemical reaction of halocompounds with tryptophan residues in carbonic anhydrase indicates the degree of solvent accessibility of these residues. 相似文献
5.
The design and implementation of a new algorithm, known as PROXIMO for protein oxidation interface modeller, is described to predict the structure of protein complexes using data generated in radical probe mass spectrometry (RP-MS) experiments. Photochemical radiolysis and discharge sources can be used to effect RP-MS in which hydroxyl radicals are formed directly from the bulk solvent on millisecond timescales and react with surface accessible residues in footprinting-like experiments. The algorithm utilizes a geometric surface fitting routine to predict likely structures for protein complexes. These structures are scored based on a correlation between the measured solvent accessibility of oxidizable residue side chains and oxidation shielding data obtained by RP-MS. The algorithm has been implemented to predict structures for the ribonuclease S-protein-peptide and calmodulin-melittin complexes using RP-MS data generated in this laboratory. The former is in close agreement with the high-resolution experimental structure available. 相似文献
6.
Hauser M Kauffman S Lee BK Naider F Becker JM 《The Journal of biological chemistry》2007,282(14):10387-10397
In this study of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p, we present data indicating that the first extracellular loop (EL1) of the alpha-factor receptor has tertiary structure that limits solvent accessibility and that its conformation changes in a ligand-dependent manner. The substituted cysteine accessibility method was used to probe the solvent exposure of single cysteine residues engineered to replace residues Tyr(101) through Gln(135) of EL1 in the presence and absence of the tridecapeptide alpha-factor and a receptor antagonist. Surprisingly, many residues, especially those at the N-terminal region, were not solvent-accessible, including residues of the binding-competent yet signal transduction-deficient mutants L102C, N105C, S108C, Y111C, and T114C. In striking contrast, two N-terminal residues, Y101C and Y106C, were readily solvent-accessible, but upon incubation with alpha-factor labeling was reduced, suggesting a pheromone-dependent conformational change limiting solvent accessibility had occurred. Labeling in the presence of the antagonist, which binds Ste2p but does not initiate signal transduction, did not significantly alter reactivity with the Y101C and Y106C receptors, suggesting that the alpha-factor-dependent decrease in solvent accessibility was not because of steric hindrance that prevented the labeling reagent access to these residues. Based on these and previous observations, we propose a model in which the N terminus of EL1 is structured such that parts of the loop are buried in a solvent-inaccessible environment interacting with the extracellular part of the transmembrane domain bundle. This study highlights the essential role of an extracellular loop in activation of a G protein-coupled receptor upon ligand binding. 相似文献
7.
We describe here the use of cysteine substitution mutants in the Alzheimer disease amyloid plaque peptide Abeta-(1-40) to probe amyloid fibril structure and stabilization. In one approach, amyloid fibrils were grown from Cys mutant peptides under reducing conditions and then challenged with an alkylating agent to probe solvent accessibility of different residues in the fibril. In another approach, monomeric Cys mutants, either in the thiol form or modified with iodoacetic acid or methyl iodide, were grown into amyloid fibrils, and the equilibrium position at the end of the amyloid formation reaction was quantified by determining the concentration of monomeric Abeta. The DeltaG values of fibril elongation obtained were then compared in order to provide information on the environment of each residue side chain in the fibril. In general, Cys residues in the N and C termini of Abeta-(1-40) were not only accessible to alkylation in the fibril state but also, when modified in the monomeric state, did not greatly impact fibril stability; these observations were consistent with previous indications that these portions of the peptide are not part of the amyloid core. In contrast, residues 16-19 and 31-34 were not only uniformly inaccessible to alkylation in the fibril state, but their modification with the negatively charged carboxymethyl group in monomeric Abeta also destabilized fibril elongation, confirming other data showing that these segments are likely packed into a hydrophobic amyloid core. Residues 20, 30, and 35, flanking these implicated beta-sandwich regions, are accessible to alkylation in the fibril indicating a location in solvent exposed structure. 相似文献
8.
Vasicek L O'Brien JP Browning KS Tao Z Liu HW Brodbelt JS 《Molecular & cellular proteomics : MCP》2012,11(7):O111.015826-O111.015826-10
A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides. Here a novel hydrazone reagent (NN) is presented that allows facile identification of all modified surface residues through a preferential cleavage upon activation by electron transfer dissociation coupled with a collision activation scan to pinpoint the modified residue in the peptide sequence. Using this approach, the correlation between percent reactivity and surface accessibility is demonstrated for two biologically active proteins, wheat eIF4E and PARP-1 Domain C. 相似文献
9.
Although atomic-resolution crystal structures of the conserved C-terminal domain of several species of TBP and their complexes with DNA have been determined, little information is available concerning the structure in solution of full-length TBP containing both the conserved C-terminal and nonconserved N-terminal domains. Quantitation of the amino acid side chain oxidation products generated by synchrotron X-ray radiolysis by mass spectrometry has been used to determine the solvent accessibility of individual residues in monomeric Saccharomyces cerevisiae TATA binding protein (TBP) free in solution and in the TBP-DNA complex. Amino acid side chains within the C-terminal domain of unliganded full-length TBP that are predicted to be accessible from crystal structures of the isolated domain are protected from oxidation. Residues within the N-terminal domain are also protected from oxidation in both the absence and presence of DNA. Some residues within the DNA-binding "saddle" of the C-terminal domain are protected upon formation of a TBP-DNA complex as expected, while others are protected in both the absence and presence of bound DNA. In addition, residues on the upper side of the beta-sheets undergo reactivity changes as a function of DNA binding. These data suggest that the DNA-binding saddle of monomeric unliganded yeast TBP is only partially accessible to solvent, the N-terminal domain is partially structured, and the N- and C-terminal domains form a different set of contacts in the free and DNA-bound protein. The functional implications of these results are discussed. 相似文献
10.
A variety of functionally important protein properties, such as secondary structure, transmembrane topology and solvent accessibility, can be encoded as a labeling of amino acids. Indeed, the prediction of such properties from the primary amino acid sequence is one of the core projects of computational biology. Accordingly, a panoply of approaches have been developed for predicting such properties; however, most such approaches focus on solving a single task at a time. Motivated by recent, successful work in natural language processing, we propose to use multitask learning to train a single, joint model that exploits the dependencies among these various labeling tasks. We describe a deep neural network architecture that, given a protein sequence, outputs a host of predicted local properties, including secondary structure, solvent accessibility, transmembrane topology, signal peptides and DNA-binding residues. The network is trained jointly on all these tasks in a supervised fashion, augmented with a novel form of semi-supervised learning in which the model is trained to distinguish between local patterns from natural and synthetic protein sequences. The task-independent architecture of the network obviates the need for task-specific feature engineering. We demonstrate that, for all of the tasks that we considered, our approach leads to statistically significant improvements in performance, relative to a single task neural network approach, and that the resulting model achieves state-of-the-art performance. 相似文献
11.
蔡红 《氨基酸和生物资源》1994,(3)
介绍了一种新的可及表面面积近似计算的解析方法。文中通过统计处理导出了一种可及表面面积的表达式,在该表达式中,可及表面面积作为原子对或残基对之间距离的函数。这一函数不仅可以方便地用来计算多种蛋白质可及表面面积及亚基间相互接触时所埋藏的面积,而且该函数可被求导,这就开辟了一条可及性(Accessibility)测定的新途径。 相似文献
12.
Lins L Thomas A Brasseur R 《Protein science : a publication of the Protein Society》2003,12(7):1406-1417
We analyzed the total, hydrophobic, and hydrophilic accessible surfaces (ASAs) of residues from a nonredundant bank of 587 3D structure proteins. In an extended fold, residues are classified into three families with respect to their hydrophobicity balance. As expected, residues lose part of their solvent-accessible surface with folding but the three groups remain. The decrease of accessibility is more pronounced for hydrophobic than hydrophilic residues. Amazingly, Lysine is the residue with the largest hydrophobic accessible surface in folded structures. Our analysis points out a clear difference between the mean (other studies) and median (this study) ASA values of hydrophobic residues, which should be taken into consideration for future investigations on a protein-accessible surface, in order to improve predictions requiring ASA values. The different secondary structures correspond to different accessibility of residues. Random coils, turns, and beta-structures (outside beta-sheets) are the most accessible folds, with an average of 30% accessibility. The helical residues are about 20% accessible, and the difference between the hydrophobic and the hydrophilic residues illustrates the amphipathy of many helices. Residues from beta-sheets are the most inaccessible to solvent (10% accessible). Hence, beta-sheets are the most appropriate structures to shield the hydrophobic parts of residues from water. We also show that there is an equal balance between the hydrophobic and the hydrophilic accessible surfaces of the 3D protein surfaces irrespective of the protein size. This results in a patchwork surface of hydrophobic and hydrophilic areas, which could be important for protein interactions and/or activity. 相似文献
13.
Bradley B. Stocks 《Journal of molecular biology》2010,398(2):362-158
The current work employs a novel approach for characterizing structural changes during the refolding of acid-denatured cytochrome c (cyt c). At various time points (ranging from 10 ms to 5 min) after a pH jump from 2 to 7, the protein is exposed to a microsecond hydroxyl radical (·OH) pulse that induces oxidative labeling of solvent-exposed side chains. Most of the covalent modifications appear as + 16-Da adducts that are readily detectable by mass spectrometry. The overall extent of labeling decreases as folding proceeds, reflecting dramatic changes in the accessibility of numerous residues. Peptide mapping and tandem mass spectrometry reveal that the side chains of C14, C17, H33, F46, Y48, W59, M65, Y67, Y74, M80, I81, and Y97 are among the dominant sites of oxidation. Temporal changes in the accessibility of these residues are consistent with docking of the N- and C-terminal helices as early as 10 ms. However, structural reorganization at the helix interface takes place up to at least 1 s. Initial misligation of the heme iron by H33 leads to distal crowding, giving rise to low solvent accessibility of the displaced (native) M80 ligand and the adjacent I81. W59 retains a surprisingly high level of accessibility long into the folding process, indicating the presence of packing defects in the hydrophobically collapsed core. Overall, the results of this work are consistent with previous hydrogen/deuterium exchange studies that proposed a foldon-mediated mechanism. The structural data obtained by ·OH labeling monitor the packing and burial of side chains, whereas hydrogen/deuterium exchange primarily monitors the formation of secondary structure elements. Hence, the two approaches yield complementary information. Considering the very short time scale of pulsed oxidative labeling, an extension of the approach used here to sub-millisecond folding studies should be feasible. 相似文献
14.
Patricio O. Craig Gabriela E. Gómez Julio J. Caramelo José M. Delfino 《Journal of molecular biology》2009,394(5):982-963
Each conformational state of a protein is inextricably related to a defined extent of solvent exposure that plays a key role in protein folding and protein interactions. However, accurate measurement of the solvent-accessible surface area (ASA) is difficult for any state other than the native (N) state. We address this fundamental physicochemical parameter through a new experimental approach based on the reaction of the photochemical reagent diazirine (DZN) with the polypeptide chain. By virtue of its size, DZN is a reasonable molecular mimic of aqueous solvent. Here, we structurally characterize nonnative states of the paradigmatic protein α-lactalbumin. Covalent tagging resulting from unspecific methylene (:CH2) reaction allows one to obtain a global estimate of ASA and to map out solvent accessibility along the amino acid sequence. By its mild apolar nature, DZN also reveals a hydrophobic phase in the acid-stabilized state of α-lactalbumin, in which there is clustering of core residues accessible to the solvent. In a fashion reminiscent of the N state, this acid-stabilized state also exhibits local regions where increased :CH2 labeling indicates its nonhomogenous nature, likely pointing to the existence of packing defects. By contrast, the virtual absence of a defined long-range organization brings about a featureless labeling pattern for the unfolded state. Overall, :CH2 labeling emerges as a fruitful technique that is able to quantify the ASA of the polypeptide chain, thus probing conformational features such as the outer exposed surface and inner cavities, as well as revealing the existence of noncompact apolar phases in nonnative states. 相似文献
15.
Photoaffinity labeling is used to covalently attach ligands to macromolecules to determine their spatial arrangement and structure. Benzophenone (BP) groups are widely used for covalent photoaffinity labeling and for probing protein interactions. We developed bifunctional BP photoactivatable derivatives using three different general chemical approaches. In addition to the photoaffinity reactivity of the BP, these derivatives contain an additional group: A radioactive tracer for biological studies, or an N-ethylmaleimide group as an additional crosslinker, or a biotin group to be used during purification and characterization of probe-protein complexes using the high-affinity biotin-avidin interaction. A model series of photoaffinity labeling probes was synthesized based on the arbutin ligand. These compounds can be used as probes to study the arbutin binding site of microbial beta-glucoside transporters by photolabeling residues in its vicinity. The second functionality provides additional options for studying proteins and binding sites. The probes were developed using different methodologies: (i) a diazotation reaction; (ii) protecting group methodology; and (iii) solid-phase synthesis. These procedures are general and provide a simple and versatile approach for synthesizing bifunctional BP ligands, as demonstrated here on arbutin. 相似文献
16.
Pugalenthi G Kandaswamy KK Chou KC Vivekanandan S Kolatkar P 《Protein and peptide letters》2012,19(1):50-56
Prediction of protein structure from its amino acid sequence is still a challenging problem. The complete physicochemical understanding of protein folding is essential for the accurate structure prediction. Knowledge of residue solvent accessibility gives useful insights into protein structure prediction and function prediction. In this work, we propose a random forest method, RSARF, to predict residue accessible surface area from protein sequence information. The training and testing was performed using 120 proteins containing 22006 residues. For each residue, buried and exposed state was computed using five thresholds (0%, 5%, 10%, 25%, and 50%). The prediction accuracy for 0%, 5%, 10%, 25%, and 50% thresholds are 72.9%, 78.25%, 78.12%, 77.57% and 72.07% respectively. Further, comparison of RSARF with other methods using a benchmark dataset containing 20 proteins shows that our approach is useful for prediction of residue solvent accessibility from protein sequence without using structural information. The RSARF program, datasets and supplementary data are available at http://caps.ncbs.res.in/download/pugal/RSARF/. 相似文献
17.
A method for identifying cysteine-containing peptides in proteins is presented using 2-bromoacetamido-4-nitrophenol (BNP) to introduce an easily detectable probe. The formation of a covalent bond between the protein sulfhydryl group and the acetamido moiety of BNP introduces a chromophore with an absorbance maximum at 410 nm. The modified protein can then be cleaved with appropriate proteases and the resulting peptides separated by chromatographic methods. Monitoring the effluent at a single wavelength (405 nm) provides a rapid and simple method of detecting and isolating only those peptides which contain cysteine residue(s). The nitrophenol derivative is stable under conditions required for protease cleavage. The reagent is therefore useful for locating cysteine-containing peptides in protein digests and can be used to explore the accessibility of different cysteines under a variety of conditions. The ease of modification, specificity of reaction, product stability, and simple detection of modified peptides make BNP ideal for investigation of cysteine residues. 相似文献
18.
We developed dictionaries of two-, three-, and five-residue patterns in proteins and computed the average solvent accessibility of the central residues in their native proteins. These dictionaries serve as a look-up table for making subsequent predictions of solvent accessibility of amino acid residues. We find that predictions made in this way are very close to those made using more sophisticated methods of solvent accessibility prediction. We also analyzed the effect of immediate neighbors on the solvent accessibility of residues. This helps us in understanding how the same residue type may have different accessible surface areas in different proteins and in different positions of the same protein. We observe that certain residues have a tendency to increase or decrease the solvent accessibility of their neighboring residues in C- or N-terminal positions. Interestingly, the C-terminal and N-terminal neighbor residues are found to have asymmetric roles in modifying solvent accessibility of residues. As expected, similar neighbors enhance the hydrophobic or hydrophilic character of residues. Detailed look-up tables are provided on the web at www.netasa.org/look-up/. 相似文献
19.
Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron "white" beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its "inactive" and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation. 相似文献
20.
Considerable progress in deciphering the mechanisms of protein folding has been made. However, most work in this area has focused on single-chain systems, whereas the majority of proteins are oligomers. The spontaneous assembly of intact multi-subunit systems from disordered building blocks encompasses the formation of intramolecular as well as intermolecular contacts. Both types of interaction affect the solvent accessibility of individual protein segments. This work employs pulsed hydroxyl radical (·OH) labeling for tracking time-dependent accessibility changes during folding and assembly of the S100A11 homodimer. ·OH induces covalent modifications at exposed residues. Structural snapshots are obtained by combining ·OH labeling with rapid mixing and mass spectrometry. The free subunits are found to possess a partially non-native hydrophobic core that prevents subunit association during the initial stages of the reaction. Instead, the protein forms an early (10 ms) monomeric intermediate that exhibits reduced solvent accessibility in regions distant from helices I and IV, which constitute the dimerization interface. Subunit association is complete after 800 ms, although the protein retains significant disorder in helices II and III at this point. Subsequent consolidation of these elements leads to the native state. The experimental strategy used here could become a general tool for deciphering kinetic mechanisms of biomolecular self-assembly processes. 相似文献