Side-chain Solvent Accessibility Research Articles

Residue-Specific Epitope Mapping of the PD-1/Nivolumab Interaction Using X-ray Footprinting Mass Spectrometry

X-ray footprinting coupled with mass spectrometry (XFMS) presents a novel approach in structural biology, offering insights into protein conformation and dynamics in the solution state. The interaction of the cancer-immunotherapy monoclonal antibody nivolumab with its antigen target PD-1 was used to showcase the utility of XFMS against the previously published crystal structure of the complex. Changes in side-chain solvent accessibility, as determined by the oxidative footprint of free PD-1 versus PD-1 bound to nivolumab, agree with the binding interface side-chain interactions reported from the crystal structure of the complex. The N-linked glycosylation sites of PD-1 were confirmed through an LC-MS/MS-based deglycosylation analysis of asparagine deamidation. In addition, subtle changes in side-chain solvent accessibility were observed in the C′D loop region of PD-1 upon complex formation with nivolumab.

Fast photochemical oxidation of proteins coupled with mass spectrometry

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical footprinting approach whereby radicals, produced by UV laser photolysis of hydrogen peroxide, induce oxidation of amino acid side-chains. Mass Spectrometry (MS) is employed to locate and quantify the resulting irreversible, covalent oxidations to use as a surrogate for side-chain solvent accessibility. Modulation of oxidation levels under different conditions allows for the characterisation of protein conformation, dynamics and binding epitopes. FPOP has been applied to structurally diverse and biopharmaceutically relevant systems from small, monomeric aggregation-prone proteins to proteome-wide analysis of whole organisms. This review evaluates the current state of FPOP, the progress needed to address data analysis bottlenecks, particularly for residue-level analysis, and highlights significant developments of the FPOP platform that have enabled its versatility and complementarity to other structural biology techniques.

Crystal structure of a human MUC16 SEA domain reveals insight into the nature of the CA125 tumor marker

MUC16 is a membrane bound glycoprotein involved in the progression and metastasis of pancreatic and ovarian cancer. The protein is shed into the serum and the resulting cancer antigen 125 (CA125) can be detected by immunoassays. The CA125 epitope is used for monitoring ovarian cancer treatment progression, and has emerged as a potential target for antibody mediated immunotherapy. The extracellular tandem repeat domain of the protein is composed of repeating segments of heavily glycosylated sequence intermixed with homologous SEA (Sperm protein, Enterokinase and Agrin) domains. Here we report the purification and the first X-ray structure of a human MUC16 SEA domain. The structure was solved by molecular replacement using a Rosetta generated structure as a search model. The SEA domain reacted with three different MUC16 therapeutic antibodies, confirming that the CA125 epitope is localized to the SEA domain. The structure revealed a canonical ferredoxin-like fold, and contained a conserved disulfide bond. Analysis of the relative solvent accessibility of side chains within the SEA domain clarified the assignment of N-linked and O-linked glycosylation sites within the domain. A model of the glycosylated SEA domain revealed two major accessible faces, which likely represent the binding sites of CA125 specific antibodies. The results presented here will serve to accelerate future work to understand the functional role of MUC16 SEA domains and antibody recognition of the CA125 epitope.

Measuring Cysteine Exposure in Unfolded Proteins with Tetraphenylethene Maleimide and its Analogs.

When proteostasis is challenged and becomes unbalanced, unfolded proteins can accumulate in the cells. Protein unfolding causes conformational changes and subsequent differentials in side-chain solvent accessibility and reactivity. In particular, when protein unfolds, non-disulfide-bonded cysteines that are usually buried in the native state can become surface exposed and thus accessible. A series of fluorogenic dyes including tetraphenylethene maleimide (TPE-MI) and its analogs were developed to capture cysteine exposure in unfolded proteins as a measure of unfolded protein load and proteostasis capacity in cells. These dyes are inherently non-fluorescent but show fluorescence turn-on effect when conjugated to unfolded proteins via reacting with exposed cysteines on the protein. Reacting with small biothiols such as glutathione does not induce fluorescence of these dyes. Here we describe the routine workflow to characterize unfolded proteins in vitro or unfolded proteomes in cells by TPE-MIs.

Surface charged amino acid-based strategy for rational engineering of kinetic stability and specific activity of enzymes: Linking experiments with computational modeling

A rational workflow for engineering kinetically stable enzymes with good specific activity by surface charged amino acids engineering was proposed based on systematically analyzing the results of mutating 44 negatively charged surface amino acids of a thermophilic β-mannanase (ManAK). Computational data, combined with experimental results indicated that percentage side-chain solvent accessibility (PSSA), changes in Gibbs free energy of unfolding (∆∆Gmut) and root-mean-square fluctuations (RMSF) could be suitable for screening kinetically stable mutants. A combinational standard (∆∆Gmut < −0.5 kJ/mol and RMSF >0.68 Å) resulted a decrease in the proportion of destabilizing mutants to 12.5%. The perturbations of substrate affinity and specific activity caused by mutation were weakened as the shortest distance from Cα of mutated site to Cα of catalytic sites (DsCα-Cα) increased. Results indicated that hotspot zones contributing to the local stability and integrity of catalytic motif at elevated temperatures might be widely distributed across spatial structure of the protein, while the mutation perturbation on enzyme specific activity demonstrated a gradually weakening trend from the catalytic core to the protein surface. These findings further our understanding of the structural-functional relationships of protein and highlight a deduced workflow to engineering industrially useful enzymes.

Mapping the Interactions of PKNB with Small Molecule Inhibitors using Plasma Induced Modifications of Biomolecules (PLIMB)

Newly advanced PLIMB (Plasma Induced Modifications of Biomolecules) technology provides a uniquely powerful method for mapping protein-protein and protein-ligand interactions. PLIMB is a mass spectrometry (MS)-based protein footprinting technique that generates μs bursts of hydroxyl radicals from water, to measure changes in protein structure via altered solvent accessibility of amino acid side chains. Here, we utilized PLIMB to characterize the interactions of PknB, a mycobacterial Ser/Thr kinase with two small molecule inhibitors, GSK690693 and GW779439X.

Optimizing Hydroxyl Radical Footprinting Analysis of Biotherapeutics Using Internal Standard Dosimetry

Hydroxyl radical footprinting-mass spectrometry (HRF-MS) is a powerful technique for measuring protein structure by quantitating the solvent accessibility of amino acid side-chains; and when used in comparative analysis, HRF-MS data can provide detailed information on changes in protein structure. However, consistently controlling the amount of hydroxyl radical labeling of a protein requires the precise understanding of both the amount of radicals generated and half-life of the radicals in solution. The latter is particularly important for applications such as protein-protein and protein-ligand interactions, which may have different characteristics such as intrinsic reactivity and buffer components, and can cause differences in radical scavenging (herein termed "scavenging potential") between samples. To address this inherent challenge with HRF-MS analysis, we describe the comprehensive implementation of an internal standard (IS) dosimeter peptide leucine enkephalin (LeuEnk) for measuring the scavenging potential of pharmaceutically relevant proteins and formulation components. This further enabled evaluation of the critical method parameters affecting the scavenging potential of samples subjected to HRF-MS using fast photochemical oxidation of proteins. We demonstrate a direct correlation between the oxidation of the IS peptide and biotherapeutic target proteins, and show the oxidation of the IS can be used as a guide for ensuring equivalent scavenging potentials when comparing multiple samples. Establishing this strategy enables optimization of sample parameters, a system suitability approach, normalization of data, and comparison/harmonization of HRF-MS analysis across different laboratories.

Structural and mechanistic insights into Hsp104 function revealed by synchrotron X-ray footprinting

Hsp104 is a hexameric AAA+ ring translocase, which drives protein disaggregation in nonmetazoan eukaryotes. Cryo-EM structures of Hsp104 have suggested potential mechanisms of substrate translocation, but precisely how Hsp104 hexamers disaggregate proteins remains incompletely understood. Here, we employed synchrotron X-ray footprinting to probe the solution-state structures of Hsp104 monomers in the absence of nucleotide and Hsp104 hexamers in the presence of ADP or ATPγS (adenosine 5'-O-(thiotriphosphate)). Comparing side-chain solvent accessibilities between these three states illuminated aspects of Hsp104 structure and guided design of Hsp104 variants to probe the disaggregase mechanism in vitro and in vivo We established that Hsp104 hexamers switch from a more-solvated state in ADP to a less-solvated state in ATPγS, consistent with switching from an open spiral to a closed ring visualized by cryo-EM. We pinpointed critical N-terminal domain (NTD), NTD-nucleotide-binding domain 1 (NBD1) linker, NBD1, and middle domain (MD) residues that enable intrinsic disaggregase activity and Hsp70 collaboration. We uncovered NTD residues in the loop between helices A1 and A2 that can be substituted to enhance disaggregase activity. We elucidated a novel potentiated Hsp104 MD variant, Hsp104-RYD, which suppresses α-synuclein, fused in sarcoma (FUS), and TDP-43 toxicity. We disambiguated a secondary pore-loop in NBD1, which collaborates with the NTD and NBD1 tyrosine-bearing pore-loop to drive protein disaggregation. Finally, we defined Leu-601 in NBD2 as crucial for Hsp104 hexamerization. Collectively, our findings unveil new facets of Hsp104 structure and mechanism. They also connect regions undergoing large changes in solvation to functionality, which could have profound implications for protein engineering.

High-Resolution Hydroxyl Radical Protein Footprinting: Biophysics Tool for Drug Discovery.

Hydroxyl radical footprinting (HRF) of proteins with mass spectrometry (MS) is a widespread approach for assessing protein structure. Hydroxyl radicals react with a wide variety of protein side chains, and the ease with which radicals can be generated (by radiolysis or photolysis) has made the approach popular with many laboratories. As some side chains are less reactive and thus cannot be probed, additional specific and nonspecific labeling reagents have been introduced to extend the approach. At the same time, advances in liquid chromatography and MS approaches permit an examination of the labeling of individual residues, transforming the approach to high resolution. Lastly, advances in understanding of the chemistry of the approach have led to the determination of absolute protein topologies from HRF data. Overall, the technology can provide precise and accurate measures of side-chain solvent accessibility in a wide range of interesting and useful contexts for the study of protein structure and dynamics in both academia and industry.

Quantitative Hydroxyl Radical Footprinting Study Reveals Structural Details of the Disorder-to-Order Transition in Amyloid-Beta (1-42) Oligomerization

This work presents a novel integrated analysis approach to characterize the intrinsically disordered 42 amino acid long β-amyloid peptide (Aβ42) and to elucidate mechanisms driving its misfolding and aggregation into neurotoxic oligomers. We describe this approach, which uses synchrotron based hydroxyl radical footprinting (HRF) results to determine residue specific solvent accessibilities for each of seventeen individual amino acids in monomer, multimer (2-4mer), and oligomer (∼150 kDa) Aβ42 sample preparations. Results complement published solid state NMR constraints with solution state structural details for the oligomers, and provide much needed experimental support for molecular dynamics (MD) computational studies of the monomer and 2-4mer species, where solid state NMR cannot be used. The pattern of protection from hydroxyl radical attack in the Aβ42 monomer (relative to the same residues in the fragmented unstructured peptide) shows partial folding in the monomer. The sequence location of this partial folding is in agreement with MD predictions of transient β-strand formation in the hydrophobic core and C-terminal regions. Sequence-specific protection (i.e. reduced side chain solvent accessibility) upon further assembly into the 2-4mer and oligomer species suggest specific conformers that are selected from the intrinsically disordered monomer ensemble to drive oligomerization. Results of the current study are also consistent with general models developed from previously published solid state NMR oligomer studies from this and other laboratories.

Plasma-Generated OH Radical Production for Analyzing Three-Dimensional Structure in Protein Therapeutics

Protein three-dimensional structure dynamically changes in solution depending on the presence of ligands and interacting proteins. Methods for detecting these changes in protein conformation include ‘protein footprinting,’ using mass spectrometry. We describe herein a new technique, PLIMB (Plasma Induced Modification of Biomolecules), that generates µs bursts of hydroxyl radicals from water, to measure changes in protein structure via altered solvent accessibility of amino acid side chains. PLIMB was first benchmarked with model compounds, and then applied to a biological problem, i.e., ligand (EGF) induced changes in the conformation of the external (ecto) domain of Epidermal Growth Factor Receptor (EGFR). Regions in which oxidation decreased upon adding EGF fall along the dimerization interface, consistent with models derived from crystal structures. These results demonstrate that plasma-generated hydroxyl radicals from water can be used to map protein conformational changes, and provide a readily accessible means of studying protein structure in solution.

Amyloid-β(1-42)Oligomer Models Developed using Combined Solid State NMR and Sequence Specific Hydroxyl Radical Footprinting Data

Increasing evidence suggests that soluble aggregates of amyloid-β (Aβ) are the pathogenic species in Alzheimer's disease (AD). However, detailed structural information on these species remains scarce due to low levels of endogenous Aβ oligomers and uncertainties surrounding current in vitro model systems. Herein, we describe a hydroxyl radical footprinting (HRF) study of Aβ42 monomers, dimers, and specially prepared stable and homogeneous oligomers. Specific side chain solvent accessibilities of individual residues in the folded and aggregated forms of Aβ42 are measured with respect to the same residues of Aβ42 in a fully exposed reference state. These data provide residue specific side chain solvent accessibility protection factors and are used in complement with biophysical characterizations and ss-NMR analyses of the same systems. Results are discussed in the context of proposed NMR models of Aβ oligomers with implications towards further development of therapeutic and diagnostic strategies.

Quantitative Mapping of Protein Structure by Hydroxyl Radical Footprinting-Mediated Structural Mass Spectrometry: A Protection Factor Analysis

Measurements from hydroxyl radical footprinting (HRF) provide rich information about the solvent accessibility of amino acid side chains of a protein. Traditional HRF data analyses focus on comparing the difference in the modification/footprinting rate of a specific site to infer structural changes across two protein states, e.g., between a free and ligand-bound state. However, the rate information itself is not fully used for the purpose of comparing different protein sites within a protein on an absolute scale. To provide such a cross-site comparison, we present a new, to our knowledge, data analysis algorithm to convert the measured footprinting rate constant to a protection factor (PF) by taking into account the known intrinsic reactivity of amino acid side chain. To examine the extent to which PFs can be used for structural interpretation, this PF analysis is applied to three model systems where radiolytic footprinting data are reported in the literature. By visualizing structures colored with the PF values for individual peptides, a rational view of the structural features of various protein sites regarding their solvent accessibility is revealed, where high-PF regions are buried and low-PF regions are more exposed to the solvent. Furthermore, a detailed analysis correlating solvent accessibility and local structural contacts for gelsolin shows a statistically significant agreement between PF values and various structure measures, demonstrating that the PFs derived from this PF analysis readily explain fundamental HRF rate measurements. We also tested this PF analysis on alternative, chemical-based HRF data, showing improved correlations of structural properties of a model protein barstar compared to examining HRF rate data alone. Together, this PF analysis not only permits a novel, to our knowledge, approach of mapping protein structures by using footprinting data, but also elevates the use of HRF measurements from a qualitative, cross-state comparison to a quantitative, cross-site assessment of protein structures in the context of individual conformational states of interest.

Sequence Specific Radiolytic Footprinting Study of Monomer, Oligomeric and Fibrillar Amyloid Beta (1-42)

Increasing evidence suggests that soluble aggregates of amyloid-β (Aβ) are the pathogenic species in Alzheimer's disease (AD). However, detailed structural information on these species remains scarce due to low levels of endogenous Aβ oligomers and uncertainties surrounding current in vitro model systems. Herein, we describe a hydroxyl radical footprinting (HRF) study of Aβ42 fibrils and a stable and homogeneous preparation of Aβ42 oligomers. Specific side chain solvent accessibilities of individual residues in the aggregated and fibril forms of Aβ42 are measured with respect to the same residues of Aβ42 in a fully exposed reference state. These data provide residue specific side chain solvent accessibility protection factors and are used in complement with biophysical characterizations and ss-NMR analyses of these systems. Results are discussed in the context of proposed NMR models of Aβ oligomers and fibrils with implications towards further development of therapeutic and diagnostic strategies.

Analysis of conformational changes in rhodopsin by histidine hydrogen-deuterium exchange.

Hydrogen-deuterium exchange (HDX) is a technique that measures the exchange of protein hydrogens for deuteriums in a D2O-containing buffer, providing readout of the structural dynamics. Histidine hydrogen-deuterium exchange mass spectrometry (His-HDX-MS) is a variation of this technique that measures the slow HDX of imidazole C2 hydrogens of histidines. This measurement, when accompanied by pH titration, provides both pK as and half-lives (t 1/2) of the HDX reaction for individual histidine residues in proteins. The pK a and t 1/2 values indicate the electrostatic environment and the degree of side-chain solvent accessibility of the histidine residues, respectively. Herein we describe an experimental protocol to characterize rhodopsin by His-HDX-MS. This technique can be used to monitor different states of rhodopsin and might be useful for monitoring longtime scale events in other GPCRs.

Tri-peptide reference structures for the calculation of relative solvent accessible surface area in protein amino acid residues

Relative amino acid residue solvent accessibility values allow the quantitative comparison of atomic solvent-accessible surface areas in different residue types and physical environments in proteins and in protein structural alignments. Geometry-optimised tri-peptide structures in extended solvent-exposed reference conformations have been obtained for 43 amino acid residue types at a high level of quantum chemical theory. Significant increases in side-chain solvent accessibility, offset by reductions in main-chain atom solvent exposure, were observed for standard residue types in partially geometry-optimised structures when compared to non-minimised models built from identical sets of proper dihedral angles abstracted from the literature. Optimisation of proper dihedral angles led most notably to marked increases of up to 54% in proline main-chain atom solvent accessibility compared to literature values. Similar effects were observed for fully-optimised tri-peptides in implicit solvent. The relief of internal strain energy was associated with systematic variation in N, Cα and Cβ atom solvent accessibility across all standard residue types. The results underline the importance of optimisation of ‘hard’ degrees of freedom (bond lengths and valence bond angles) and improper dihedral angle values from force field or other context-independent reference values, and impact on the use of standardised fixed internal co-ordinate geometry in sampling approaches to the determination of absolute values of protein amino acid residue solvent accessibility. Quantum chemical methods provide a useful and accurate alternative to molecular mechanics methods to perform energy minimisation of peptides containing non-standard (chemically modified) amino acid residues frequently present in experimental protein structure data sets, for which force field parameters may not be available. Reference tri-peptide atomic co-ordinate sets including hydrogen atoms are made freely available.

Oxidative Footprinting of Fibrillar and Prefibrillar Oligomer Forms of Amyloid Beta

The mechanism by which amyloid-β (Aβ) plaque accumulation contributes to neurodegeneration in Alzheimer's disease (AD) remains poorly understood. With biophysical, thermodynamic and kinetic characterization of the various Aβ structures involved in plaque development, we aim to identify connections between polymerization cascade events and AD pathogenesis. Herein, we present oxidative footprinting with mass spectrometry to probe the solvent accessibility of specific amino acid side chains in Aβ40 fibrils and oligomeric forms of Aβ40. These accessibilities are compared to those of a fully exposed reference state using hydroxyl radicals (∗OH) generated either by water radiolysis or by Fe(II)-EDTA reaction with peroxide. Using this information we distinguish topological relationships within the fibril to allow selection of the relevant tertiary structural model of fibrillar Aβ from those suggested by NMR and those by cryogenic electron microscopy. This work provides important steps towards correlating structure and morphology in Aβ fibrils -- essential for understanding the molecular pathogenesis of AD.

Biological insights from topology independent comparison of protein 3D structures

Comparing and classifying the three-dimensional (3D) structures of proteins is of crucial importance to molecular biology, from helping to determine the function of a protein to determining its evolutionary relationships. Traditionally, 3D structures are classified into groups of families that closely resemble the grouping according to their primary sequence. However, significant structural similarities exist at multiple levels between proteins that belong to these different structural families. In this study, we propose a new algorithm, CLICK, to capture such similarities. The method optimally superimposes a pair of protein structures independent of topology. Amino acid residues are represented by the Cartesian coordinates of a representative point (usually the Cα atom), side chain solvent accessibility, and secondary structure. Structural comparison is effected by matching cliques of points. CLICK was extensively benchmarked for alignment accuracy on four different sets: (i) 9537 pair-wise alignments between two structures with the same topology; (ii) 64 alignments from set (i) that were considered to constitute difficult alignment cases; (iii) 199 pair-wise alignments between proteins with similar structure but different topology; and (iv) 1275 pair-wise alignments of RNA structures. The accuracy of CLICK alignments was measured by the average structure overlap score and compared with other alignment methods, including HOMSTRAD, MUSTANG, Geometric Hashing, SALIGN, DALI, GANGSTA+, FATCAT, ARTS and SARA. On average, CLICK produces pair-wise alignments that are either comparable or statistically significantly more accurate than all of these other methods. We have used CLICK to uncover relationships between (previously) unrelated proteins. These new biological insights include: (i) detecting hinge regions in proteins where domain or sub-domains show flexibility; (ii) discovering similar small molecule binding sites from proteins of different folds and (iii) discovering topological variants of known structural/sequence motifs. Our method can generally be applied to compare any pair of molecular structures represented in Cartesian coordinates as exemplified by the RNA structure superimposition benchmark.

Structure of the M2 Transmembrane Segment of GLIC, a Prokaryotic Cys Loop Receptor Homologue from Gloeobacter violaceus, Probed by Substituted Cysteine Accessibility

GLIC is a homopentameric proton-gated, prokaryotic homologue of the Cys loop receptor family of neurotransmitter-gated ion channels. Recently, crystal structures of GLIC hypothesized to represent an open channel state were published. To explore the channel structure in functional GLIC channels, we tested the ability of p-chloromercuribenzenesulfonate to react with 30 individual cysteine substitution mutants in and flanking the M2 channel-lining segment in the closed state (pH 7.5) and in a submaximally activated state (pH 5.0). Nine mutants did not tolerate cysteine substitution and were not functional. From positions 10' to 27', p-chloromercuribenzenesulfonate significantly modified the currents at pH 7.5 and 5.0 in all mutants except H234C (11'), I235C (12'), V241C (18'), T243C (20'), L245C (22'), and Y250C (27'), which were not functional, except for 12'. Currents for P246C (23') and K247C (24') were only significantly altered at pH 5.0. The reaction rates were all >1000 m(-1) s(-1). The reactive residues were more accessible in the activated than in the resting state. We infer that M2 is tightly associated with the adjacent transmembrane helices at the intracellular end but is more loosely packed from 10' to the extracellular end than the x-ray structures suggest. We infer that the charge selectivity filter is in the cytoplasmic half of the channel. We also show that below pH 5.0, GLIC desensitizes on a time scale of minutes and infer that the crystal structures may represent a desensitized state.

Structure of Apolipoprotein A-I N Terminus on Nascent High Density Lipoproteins

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6-98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6-98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major α-helical domains were localized to residues 6-34 and 50-98. We identified an unstructured segment (residues 35-39) and a β-strand (residues 40-49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6-98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.