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The exposure of electrospray droplets to vapors of deuterating reagents during droplet desolvation in the interface of a mass spectrometer results in hydrogen/deuterium exchange (HDX) on the sub-millisecond time scale. Deuterated water is used to label ubiquitin and cytochrome c with minimal effect on the observed charge state distribution (CSD), suggesting that the protein conformation is not being altered. However, the introduction of deuterated versions of various acids (e.g., CD3COOD and DCl) and bases (ND3) induces unfolding or refolding of the protein while also labeling these newly formed conformations. The extent of HDX within a protein CSD associated with a particular conformation is essentially constant, whereas the extent of HDX can differ significantly for CSDs associated with different conformations from the same protein. In some cases, multiple HDX distributions can be observed within a given charge state (as is demonstrated with cytochrome c) suggesting that the extent of HDX and CSDs share a degree of complementarity in their sensitivities for protein conformation. The CSD is established late in the evolution of ions in electrospray whereas the HDX process presumably takes place in the bulk of the droplet throughout the electrospray process. Back exchange is also performed in which proteins are prepared in deuterated solvents prior to ionization and exposed to undeuterated vapors to exchange deuteriums for hydrogens. The degree of deuterium uptake is easily controlled by varying the identity and partial pressure of the reagent introduced into the interface. Since the exchange occurs on the sub-millisecond time scale, the use of deuterated acids or bases allows for transient species to be generated and labeled for subsequent mass analysis. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: The hydrogen isotope exchange reaction between hydrogen and water is a key process in water detritiation and heavy water production. To determine the optimum operating parameters and predict the exchange performance in a packed column, the hydrogen/deuterium exchange process was simulated, and the experiment results were verified by the simulation. The optimum exchange temperature was found to decrease with increased hydrogen feed flow rate. The deuterium concentration at the top of the column exhibited nonlinearity throughout all stages. The pressure drop per stage was also observed to induce no significant change in the hydrogen/deuterium exchange performance. With increased temperature, the change trend of calculated results was consistent with that of the experimental values. However, the calculated results are somewhat higher than the experimental ones, and the reason for the difference was discussed. [Copyright &y& Elsevier]

Monoclonal antibody (mAb) has grown to be the major asset in protein therapeutics market since its initial introduction in 1980s. To identify a suitable mAb as a drug candidate for development requires deep understanding of disease biology and attribute sciences, including epitope-paratope binding recognition. Mass spectrometry (MS) has become a critical technology platform in epitope mapping. MS-based approaches utilize chemical labeling to assess the changes of solvent accessibilities during binding interactions, and the labeling can be either reversible or irreversible. Reversible labeling is represented by hydrogen/deuterium exchange (HDX), which probes the changes via exchange between backbone amide hydrogen and deuterium in the solvent. Irreversible labeling targets amino acid residue side chains and involves chemical based labeling such as glycine ethyl ester labeling, radical based labeling such as fast photochemical oxidation of proteins (FPOP), and chemical cross-linking. All these methods have been developed extensively for characterization of binding interface within an immunocomplex. This review covers the fundamentals of these different MS-based methods and highlights recent case studies to illustrate unique capabilities of MS-based approaches in epitope mapping of protein therapeutics. • Critical role of epitope mapping in development of protein therapeutics. • Unique capabilities of mass spectrometry-based approaches in epitope mapping. • Reversible and irreversible chemical labeling in mass spectrometry-based methods. • Combination methods with orthogonal approaches in characterizing binding interface. [ABSTRACT FROM AUTHOR]

Abstract: Monoclonal antibodies (mAbs) are powerful therapeutics, and their characterization has drawn considerable attention and urgency. Unlike small-molecule drugs (150–600Da) that have rigid structures, mAbs (∼150kDa) are engineered proteins that undergo complicated folding and can exist in a number of low-energy structures, posing a challenge for traditional methods in structural biology. Mass spectrometry (MS)-based biophysical characterization approaches can provide structural information, bringing high sensitivity, fast turnaround, and small sample consumption. This review outlines various MS-based strategies for protein biophysical characterization and then reviews how these strategies provide structural information of mAbs at the protein level (intact or top-down approaches), peptide, and residue level (bottom-up approaches), affording information on higher order structure, aggregation, and the nature of antibody complexes. [Copyright &y& Elsevier]

ABSTRACT Protein-DNA interactions are essential for many biological processes. X-ray crystallography can provide high-resolution structures, but protein-DNA complexes are difficult to crystallize and typically contain only small DNA fragments. Thus, there is a need for computational methods that can provide useful predictions to give insights into mechanisms and guide the design of new experiments. We used the program DOT, which performs an exhaustive, rigid-body search between two macromolecules, to investigate four diverse protein-DNA interactions. Here, we compare our computational results with subsequent experimental data on related systems. In all cases, the experimental data strongly supported our structural hypotheses from the docking calculations: a mechanism for weak, nonsequence-specific DNA binding by a transcription factor, a large DNA-binding footprint on the surface of the DNA-repair enzyme uracil-DNA glycosylase (UNG), viral and host DNA-binding sites on the catalytic domain of HIV integrase, and a three-DNA-contact model of the linker histone bound to the nucleosome. In the case of UNG, the experimental design was based on the DNA-binding surface found by docking, rather than the much smaller surface observed in the crystallographic structure. These comparisons demonstrate that the DOT electrostatic energy gives a good representation of the distinctive electrostatic properties of DNA and DNA-binding proteins. The large, favourably ranked clusters resulting from the dockings identify active sites, map out large DNA-binding sites, and reveal multiple DNA contacts with a protein. Thus, computational docking can not only help to identify protein-DNA interactions in the absence of a crystal structure, but also expand structural understanding beyond known crystallographic structures. Proteins 2013; 81:2106-2118. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

This review outlines the application of time-resolved electrospray ionization mass spectrometry ( TRESI- MS) and hydrogen-deuterium exchange ( HDX) to study rapid, activity-linked conformational transitions in proteins. The method is implemented on a microfluidic chip which incorporates all sample-handling steps required for a 'bottom-up' HDX workflow: a capillary mixer for sub-second HDX labeling, a static mixer for HDX quenching, a microreactor for rapid protein digestion, and on-chip electrospray. By combining short HDX labeling pulses with rapid digestion, this approach provides a detailed characterization of the structural transitions that occur during protein folding, ligand binding, post-translational modification and catalytic turnover in enzymes. This broad spectrum of applications in areas largely inaccessible to conventional techniques means that microfluidics-enabled TRESI- MS/ HDX is a unique and powerful approach for investigating the dynamic basis of protein function. [ABSTRACT FROM AUTHOR]

Abstract: We studied hydrogen/deuterium-exchange reactions of peptide amide protons of GroES using two different techniques: (1) two-dimensional 1H–15N transverse-optimized NMR spectroscopy and (2) the dimethylsulfoxide-quenched hydrogen-exchange method combined with conventional 1H–15N heteronuclear single quantum coherence spectroscopy. By using these techniques together with direct heteronuclear single quantum coherence experiments, we quantitatively evaluated the exchange rates for 33 out of the 94 peptide amide protons of GroES and their protection factors, and for the remaining 61 residues, we obtained the lower limits of the exchange rates. The protection factors of the most highly protected amide protons were on the order of 106–107, and the values were comparable in magnitude to those observed in typical small globular proteins, but the number of the highly protected amide protons with a protection factor larger than 106 was only 10, significantly smaller than the numbers reported for the small globular proteins, indicating that significant portions of free heptameric GroES are flexible and natively unfolded. The highly protected amino acid residues with a protection factor larger than 105 were mainly located in three β-strands that form the hydrophobic core of GroES, while the residues in a mobile loop (residues 17–34) were not highly protected. The protection factors of the most highly protected amide protons were orders of magnitude larger than the value expected from the equilibrium unfolding parameters previously reported, strongly suggesting that the equilibrium unfolding of GroES is more complicated than a simple two-state or three-state mechanism and may involve more than a single intermediate. [Copyright &y& Elsevier]

The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.1 M NaCl control, sucrose (0.5 M) increased conformational stability ( Tm values), slowed the rate of monomer loss, reduced the formation of insoluble aggregates, and resulted in a global trend of small decreases in local flexibility across most regions of the mAb. In contrast, the addition of arginine (0.5 M) decreased the mAb's conformational stability, increased the rate of loss of monomer with elevated levels of soluble and insoluble aggregates, and led to significant increases in the local flexibility in specific regions of the mAb, most notably within the constant domain 2 of the heavy chain (CH2). These results provide new insights into the effect of sucrose and arginine on the local dynamics of IgG1 domains as well as preliminary correlations between local flexibility within specific segments of the CH2 domain (notably heavy chain 241-251) and the mAb's overall physical stability. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2136-2151, 2013 [ABSTRACT FROM AUTHOR]

The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: We generalize the concept of allostery from the traditional non-active-site control of enzymes to virus maturation. Virtually, all animal viruses transition from a procapsid noninfectious state to a mature infectious state. The procapsid contains an encoded chemical program that is executed following an environmental cue. We developed an exceptionally accessible virus system for the study of the activators of maturation and the downstream consequences that result in particle stability and infectivity. Nudaurelia capensis omega virus (NωV) is a T=4 icosahedral virus that undergoes a dramatic maturation in which the 490-Å spherical procapsid condenses to a 400-Å icosahedral-shaped capsid with associated specific auto-proteolysis and stabilization. Employing X-ray crystallography, time-resolved electron cryo-microscopy and hydrogen/deuterium exchange as well as biochemistry, it was possible to define the mechanisms of allosteric communication among the four quasi-equivalent subunits in the icosahedral asymmetric unit. These gene products undergo proteolysis at different rates, dependent on quaternary structure environment, while particle stability is conferred globally following only a few local subunit transitions. We show that there is a close similarity between the concepts of tensegrity (associated with geodesic domes and mechanical engineering) and allostery (associated with biochemical control mechanisms). [Copyright &y& Elsevier]

Mass spectrometry is now an indispensable tool in the armamentarium of molecular biophysics, where it is used for tasks ranging from protein sequencing and mapping of post-translational modifications to studies of higher order structure, conformational dynamics, and interactions of proteins with small molecule ligands and other biopolymers. This mini-review highlights several popular mass spectrometry-based tools that are now commonly used for structural studies of proteins beyond their covalent structure with a particular emphasis on hydrogen exchange and direct electrospray ionization mass spectrometry. [ABSTRACT FROM AUTHOR]

Abstract: Structural proteomics approaches are valuable tools, particularly in cases where the exact mechanisms of protein conformational changes or the structures of proteins and protein complexes cannot be elucidated by traditional structural biology techniques like X-ray crystallography or NMR methods. Each structural proteomics method can provide a different set of data, all of which can be used as structural constraints for modeling the protein. We have applied a combination of limited proteolysis, surface modification, chemical crosslinking, and hydrogen/deuterium exchange for the characterization of structural differences in prion proteins in native monomeric and in the aggregated β-oligomeric states. Data from these multiple proteomics approaches are in remarkable agreement in pointing to the rearrangement of the beta sheet 1–helix1–beta sheet 2–helix 2 (β1–H1–β2–H2) region as a major conformational change between the native and oligomeric prion protein forms. This data is also consistent with the β1–H1–β2 loop moving away from the H2–H3 core during the prion protein conversion. This is an example of how complementary data from multiple structural proteomics approaches can provide novel insights into the three-dimensional structures of proteins and protein complexes. This article is part of a Special Issue entitled: From protein structures to clinical applications. [Copyright &y& Elsevier]

Nm23-H1/NDPK-A, a tumour metastasis suppressor, is a multifunctional housekeeping enzyme with nucleoside diphosphate kinase activity. Hexameric Nm23-H1 is required for suppression of tumour metastasis and it is dissociated into dimers under oxidative conditions. Here, the crystal structure of oxidized Nm23-H1 is presented. It reveals the formation of an intramolecular disulfide bond between Cys4 and Cys145 that triggers a large conformational change that destabilizes the hexameric state. The dependence of the dissociation dynamics on the H2O2 concentration was determined using hydrogen/deuterium-exchange experiments. The quaternary conformational change provides a suitable environment for the oxidation of Cys109 to sulfonic acid, as demonstrated by peptide sequencing using nanoUPLC-ESI-q-TOF tandem MS. From these and other data, it is proposed that the molecular and cellular functions of Nm23-H1 are regulated by a series of oxidative modifications coupled to its oligomeric states and that the modified cysteines are resolvable by NADPH-dependent reduction systems. These findings broaden the understanding of the complicated enzyme-regulatory mechanisms that operate under oxidative conditions. [ABSTRACT FROM AUTHOR]

Abstract: This Feature describes the use of electron transfer dissociation (ETD) to analyze the hydrogen/deuterium exchange (HDX) of proteins at increased spatial resolution down to the level of individual residues. A practical overview of how to couple ETD to the classical bottom-up HDX-MS workflow is given and new options for method optimization are discussed and exemplified. In addition, the real-world applicability of the HDX-ETD method to pinpoint conformational changes in a large 75kDa protein complex of therapeutic interest is demonstrated. This feature highlights how the conformation and interactions of complex protein systems of biological or pharmaceutical interest can now be analyzed at a hitherto unprecedented level of structural detail using an MS-based method. [Copyright &y& Elsevier]

Differential hydrogen/deuterium exchange (H/DX) coupled with mass spectrometry (H/DX-MS) offers a rapid and sensitive characterization of changes in proteins following perturbations induced by changes in folding, ligand binding, oligomerization, and modification. The characterization of H/DX rates by software tools and automated data processing often relies on the centroid mass calculation and, thereby, the deuterium distribution in the mass spectra is neglected. Here we present an example demonstrating the clear limitation of using only a centroid approach to characterize the H/DX rate, in which the change in protein is not reflected as the difference in deuterium uptake based on centroid calculation. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: Xeroderma pigmentosum (XP) is a genetic disease affecting 1 in 10,000–100,000 and predisposes people to early-age skin cancer, a disease that is increasing. Those with XP have decreased ability to repair UV-induced DNA damage, leading to increased susceptibility of cancerous non-melanomas and melanomas. A vital, heterotrimeric protein complex is linked to the nucleotide excision repair pathway for the damaged DNA. The complex consists of XPC protein, human centrin 2, and RAD23B. One of the members, human centrin 2, is a ubiquitous, acidic, Ca2+-binding protein belonging to the calmodulin superfamily. The XPC protein contains a sequence motif specific for binding to human centrin 2. We report here the Ca2+-binding properties of human centrin 2 and its interaction with the XPC peptide motif. We utilized a region-specific H/D exchange protocol to localize the interaction of the XPC peptide with the C-terminal domain of centrin, the binding of which is different than that of calmodulin complexes. The binding dynamics of human centrin 2 to the XPC peptide in the absence and presence of Ca2+ are revealed by the observation of EX1 H/D exchange regime, indicating that a locally unfolded population exists in solution and undergoes fast H/D exchange. [Copyright &y& Elsevier]

Abstract: The gas-phase hydrogen/deuterium exchange behavior for protonated lysine and its three shorter homologs, ornithine (Orn), 2,4-diamino butanoic acid (Daba), and 2,3-diamino propanoic acid (Dapa) were studied in a modified LCQ-DECA ion trap mass spectrometer. The HDX behavior of protonated lysine has been studied previously by several groups. It undergoes five equivalent exchanges, corresponding to the hydrogen atoms attached to the protonated lysine side chain and amine terminus nitrogen, and one slower exchange corresponding to the COOH hydrogen. The shorter homologs also exchange all six labile hydrogen atoms but at different rates. Relative to protonated lysine, DapaH+ exchanges two times faster, DabaH+ exchanges slightly slower, and OrnH+ exchanges nearly 30 times more slowly. High-level hybrid density functional theory calculations attribute this difference to the increased barrier for the relay-type exchange mechanism in OrnH+, relative to the other homologs. [Copyright &y& Elsevier]

Abstract: Amyloid fibrils with an ordered cross-β structure are one form of protein aberrant aggregates. Fibrils themselves and on-pathway small aggregates are involved in many neurodegenerative diseases and amylodoses. Over the past decade, much has been learned about the conformation of amyloid fibrils by using various biochemical and biophysical approaches. Amyloid fibrils accommodate rigid core structures composed of regular intra- and intermolecular non-covalent bonds such as hydrogen bonds, and disordered flexible regions exposed to solvents. In contrast to the improved understanding of fibril structures, few studies have investigated the short-living monomeric intermediates which interact with amyloid fibrils for elongation and the self-associated intermediates in the course of amyloidogenesis at the residue level. To study static fibrillar structures and kinetic intermediates, hydrogen/deuterium exchange (HDex) coupled with solution-state NMR spectroscopy is one of the most powerful methods with a high time and atomic resolution. Here, we review studies on the structural properties of amyloid fibrils based on a combination of dimethylsulfoxide-quenched HDex and NMR spectroscopy. Recent studies on transient kinetic intermediates during fibril growth by means of pulse-labeling HDex aided by a quenched-flow apparatus and NMR spectroscopy are focused on. [Copyright &y& Elsevier]

The addition of mass spectrometry (MS) analysis to the hydrogen exchange (HX) proteolytic fragmentation experiment extends powerful HX methodology to the study of large biologically important proteins. A persistent problem is the degradation of HX information due to back exchange of deuterium label during the fragmentation-separation process needed to prepare samples for MS measurement. This paper reports a systematic analysis of the factors that influence back exchange (solution pH, ionic strength, desolvation temperature, LC column interaction, flow rates, system volume). The many peptides exhibit a range of back exchange due to intrinsic amino acid HX rate differences. Accordingly, large back exchange leads to large variability in D-recovery from one residue to another as well as one peptide to another that cannot be corrected for by reference to any single peptide-level measurement. The usual effort to limit back exchange by limiting LC time provides little gain. Shortening the LC elution gradient by 3-fold only reduced back exchange by ~2 %, while sacrificing S/N and peptide count. An unexpected dependence of back exchange on ionic strength as well as pH suggests a strategy in which solution conditions are changed during sample preparation. Higher salt should be used in the first stage of sample preparation (proteolysis and trapping) and lower salt (<20 mM) and pH in the second stage before electrospray injection. Adjustment of these and other factors together with recent advances in peptide fragment detection yields hundreds of peptide fragments with D-label recovery of 90 % ± 5 %. [ABSTRACT FROM AUTHOR]

The 1.1 Å, ultrahigh resolution neutron structure of hydrogen/deuterium (H/D) exchanged crambin is reported. Two hundred ninety-nine out of 315, or 94.9%, of the hydrogen atom positions in the protein have been experimentally derived and resolved through nuclear density maps. A number of unconventional interactions are clearly defined, including a potential O-H…π interaction between a water molecule and the aromatic ring of residue Y44, as well as a number of potential C-H…O hydrogen bonds. Hydrogen bonding networks that are ambiguous in the 0.85 Å ultrahigh resolution X-ray structure can be resolved by accurate orientation of water molecules. Furthermore, the high resolution of the reported structure has allowed for the anisotropic description of 36 deuterium atoms in the protein. The visibility of hydrogen and deuterium atoms in the nuclear density maps is discussed in relation to the resolution of the neutron data. [ABSTRACT FROM AUTHOR]

Abstract: Top-down hydrogen/deuterium exchange mass spectrometry (HDX-MS) is becoming a powerful tool for structural biology. Using gas-phase sequencing methods such as electron capture dissociation (ECD), “H/D scrambling free” top-down HDX measurements on proteins can be made with high resolution and full sequence coverage. Although it has clear advantages over traditional proteolysis- and liquid chromatography-based HDX-MS, its application has thus far been limited to small (<18kDa) proteins. The current study has extended the top-down approach to structural dynamics characterization of a nearly 30kDa protein, carbonic anhydrase II (CAII). Employing a novel “ECD-stitching” method, which combines two different ECD settings optimized for the observation of small and large fragments respectively, a total of 120 fragment ions were obtained with sufficient signal/noise for top-down HDX-MS analysis. These ions cover the whole sequence of the protein and provide an average resolution of 2 residues. The results have shown that the most protected region in CAII is the β-sheet motif located in the center of the enzyme, while α-helices located at the outer surface exhibit much less protection. A high degree of protection was also found in loops where solvent access is restricted. These observations are compatible with the structural features of CAII as provided by X-ray crystallographic studies. In addition, the drug-induced conformational transitions of CAII were also characterized. The ECD-stitching top-down approach should be readily applicable to other large proteins and protein complexes, and may give better insights into conformational changes and other solution-phase processes than can be obtained via crystallographic techniques. [Copyright &y& Elsevier]

A long lasting recombinant factor IX -Fc fusion protein (rFIX-Fc) is being developed for the treatment of hemophilia B and is currently in late stage clinical investigation. By limiting injection frequency and maintaining efficacy, rFIX-Fc shows promise as a new therapeutic option for hemophilia B patients. However, before gaining regulatory approval, rFIX-Fc must undergo rigorous analytical and biological testing, in addition to clinical trials. Included in this testing is the need to understand this protein's higher-order structure and dynamics. In this study, we investigated and compared the biophysical properties of rFIX-Fc, rFIX, and Fc using hydrogen/deuterium exchange mass spectrometry and differential scanning calorimetry. Within the limits of these techniques, our results show that structural comparability exists between rFIX and the FIX region of rFIX-Fc. In addition, changes in the structure and dynamics of both proteins, in response to calcium binding, a requirement for FIX function, are also highly comparable. In the case of Fc and Fc region of rFIX-Fc, conformational comparability is also established. These biophysical results further support the conclusion that fusing an immunoglobulin gamma 1 Fc to rFIX does not significantly alter the higher-order structure of FIX or Fc, Ca binding to FIX, or Fc functionality. [ABSTRACT FROM AUTHOR]

Abstract: The manipulation of modular regulatory domains from allosteric enzymes represents a possible mechanism to engineer allostery into non-allosteric systems. Currently, there is insufficient understanding of the structure/function relationships in modular regulatory domains to rationally implement this methodology. The LeuA dimer regulatory domain represents a well-conserved, novel fold responsible for the regulation of two enzymes involved in branched chain amino acid biosynthesis, α-isopropylmalate synthase and citramalate synthase. The LeuA dimer regulatory domain is responsible for the feedback inhibition of these enzymes by their respective downstream products. Both enzymes display multidomain architecture with a conserved N-terminal TIM barrel catalytic domain and a C-terminal (βββα)2 LeuA dimer domain joined by a flexible linker region. Due to the similarity of three-dimensional structure and catalytic mechanism combined with low sequence similarity, we propose these enzymes can be classified as members of the LeuA dimer superfamily. Despite their similarity, members of the LeuA dimer superfamily display diversity in their allosteric mechanisms. In this review, structural aspects of the LeuA dimer superfamily are discussed followed by three examples highlighting the diversity of allosteric mechanisms in the LeuA dimer superfamily. [Copyright &y& Elsevier]

Abstract: Protein structure determines function in biology, and a variety of approaches have been employed to obtain structural information about proteins. Mass spectrometry-based protein footprinting is one fast-growing approach. One labeling-based footprinting approach is the use of a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine ethyl ester (GEE) to modify solvent-accessible carboxyl groups on glutamate (E) and aspartate (D). This paper describes method development of carboxyl-group modification in protein footprinting. The modification protocol was evaluated by using the protein calmodulin as a model. Because carboxyl-group modification is a slow reaction relative to protein folding and unfolding, there is an issue that modifications at certain sites may induce protein unfolding and lead to additional modification at sites that are not solvent-accessible in the wild-type protein. We investigated this possibility by using hydrogen deuterium amide exchange (H/DX). The study demonstrated that application of carboxyl group modification in probing conformational changes in calmodulin induced by Ca2+ binding provides useful information that is not compromised by modification-induced protein unfolding. [Copyright &y& Elsevier]

Hydrogen/deuterium exchange in combination with mass spectrometry (H/D MS) is a sensitive technique for detection of changes in protein conformation and dynamics. However, wide application of H/D MS has been hindered, in part, by the lack of computational tools necessary for efficient analysis of the large data sets associated with this technique. We report a novel web-based application for automatic analysis of H/D MS experimental data. This application relies on the high resolution of mass spectrometers to extract all isotopic envelopes before correlating these envelopes with individual peptides. Although a fully automatic analysis is possible, a variety of graphical tools are included to aid in the verification of correlations and rankings of the isotopic peptide envelopes. As a demonstration, the rate constants for H/D exchange of peptides from rabbit muscle pyruvate kinase are mapped onto the structure of this protein. [ABSTRACT FROM AUTHOR]

Abstract: Eukaryotic protein kinases (EPKs) feature two coevolved structural segments, the Activation segment, which starts with the Asp-Phe-Gly (DFG) and ends with the Ala-Pro-Glu (APE) motifs, and the helical GHI subdomain that comprises αG–αH–αI helices. Eukaryotic-like kinases have a much shorter Activation segment and lack the GHI subdomain. They thus lack the conserved salt bridge interaction between the APE Glu and an Arg from the GHI subdomain, a hallmark signature of EPKs. Although the conservation of this salt bridge in EPKs is well known and its implication in diseases has been illustrated by polymorphism analysis, its function has not been carefully studied. In this work, we use murine cAMP-dependent protein kinase (protein kinase A) as the model enzyme (Glu208 and Arg280) to examine the role of these two residues. We showed that Ala replacement of either residue caused a 40- to 120-fold decrease in catalytic efficiency of the enzyme due to an increase in K m(ATP) and a decrease in k cat. Crystal structures, as well as solution studies, also demonstrate that this ion pair contributes to the hydrophobic network and stability of the enzyme. We show that mutation of either Glu or Arg to Ala renders both mutant proteins less effective substrates for upstream kinase phosphoinositide-dependent kinase 1. We propose that the Glu208–Arg280 pair serves as a center hub of connectivity between these two structurally conserved elements in EPKs. Mutations of either residue disrupt communication not only between the two segments but also within the rest of the molecule, leading to altered catalytic activity and enzyme regulation. [Copyright &y& Elsevier]

Mass spectrometry has become a powerful method to study the structure and dynamics of proteins. Hydrogen/ deuterium exchange in conjunction with mass spectrometry is now becoming more widely used to monitor conformational changes in proteins, and when combined with proteolytic digestion or gas-phase dissociation, it can provide spatial resolution of structural regions which participate in conformational change. The biggest advantage offered by mass spectrometry is that it can distinguish different conformations of a protein even when they are present together, and this has made it an indispensable tool for studying the heterogeneity inherent in protein folding and unfolding reactions. This review surveys the application of mass spectrometry to study protein folding and unfolding reactions, and describes the important insights obtained from these studies. It also briefly examines the use of mass spectrometry to study the assembly and disassembly of large multimeric protein complexes, and to obtain structural information on amyloid protofibrils and fibrils. [ABSTRACT FROM AUTHOR]

There is currently a renewed focus aimed at understanding allosteric mechanisms at atomic resolution. This current interest seeks to understand how both changes in protein conformations and changes in protein dynamics contribute to relaying an allosteric signal between two ligand binding sites on a protein (e.g., active and allosteric sites). Both nuclear magnetic resonance (NMR), by monitoring protein dynamics directly, and hydrogen/deuterium exchange, by monitoring solvent accessibility of backbone amides, offer insights into protein dynamics. Unfortunately, many allosteric proteins exceed the size limitations of standard NMR techniques. Although hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) offers an alternative evaluation method, any application of hydrogen/deuterium exchange requires that the property being measured functions in both HO and DO. Due to the promising future H/DX-MS has in the evaluation of allosteric mechanisms in large proteins, we demonstrate an evaluation of allosteric regulation in DO. Exemplified using phenylalanine inhibition of rabbit muscle pyruvate kinase, we find that binding of the inhibitor is greatly reduced in DO, but the effector continues to elicit an allosteric response. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) proteoglycans regulate a number of biological functions in many systems. Most of the functions of HS are attributed to its unique structure, consisting of sulfated and non-sulfated domains, arising from the differential presence of iduronyl and glucuronyl residues along the polysaccharide chain. A single glucuronyl C5-epimerase enzyme acts on HS precursors, converts glucuronyl residues into iduronyl residues, and modulates subsequent biosynthetic steps in vivo. Previously, the ratios of non-sulfated epimers within the polysaccharide chain have been calculated by resolving radiolabeled GlcA-Man and IdoA-Man disaccharides using a tedious paper chromatography technique. This radioactive assay, based on measuring either the release or incorporation of H at C5 carbon of uronyl residues of H-labeled HS precursor substrate, has been in use over three decades to characterize the action of HS C5-epimerase. We have developed a non-radioactive assay to estimate the epimerase activity through resolving GlcA-Man and IdoA-Man disaccharides on high-performance liquid chromatography in conjunction with hydrogen/deuterium exchange upon epimerization protocol-liquid chromatography mass spectrometry (DEEP-LC-MS). Utilizing this new, non-radioactive-based assay, DEEP-LC-MS, we were able to determine the extent of both forward and reverse reactions on the same substrate catalyzed by C5-epimerase. The results from this study also provide insights into the action of C5-epimerase and provide an opportunity to delineate snapshots of biosynthetic events that occur during the HSPG assembly in the Golgi. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The recent application of electron transfer dissociation (ETD) to measure the hydrogen exchange of proteins in solution at single-residue resolution (HX-ETD) paves the way for mass spectrometry-based analyses of biomolecular structure at an unprecedented level of detail. The approach requires that activation of polypeptide ions prior to ETD is minimal so as to prevent undesirable gas-phase randomization of the deuterium label from solution (i.e., hydrogen scrambling). Here we explore the use of ETD in a traveling wave ion guide of a quadrupole-time-of-flight (Q-TOF) mass spectrometer with a 'Z-spray' type ion source, to measure the deuterium content of individual residues in peptides. We systematically identify key parameters of the Z-spray ion source that contribute to collisional activation and define conditions that allow ETD experiments to be performed in the traveling wave ion guide without gas-phase hydrogen scrambling. We show that ETD and supplemental collisional activation in a subsequent traveling wave ion guide allows for improved extraction of residue-specific deuterium contents in peptides with low charge. Our results demonstrate the feasibility, and illustrate the advantages of performing HX-ETD experiments on a high-resolution Q-TOF instrument equipped with traveling wave ion guides. Determination of parameters of the Z-spray ion source that contribute to ion heating are similarly pertinent to a growing number of MS applications that also rely on an energetically gentle transfer of ions into the gas-phase, such as the analysis of biomolecular structure by native mass spectrometry in combination with gas-phase ion-ion/ion-neutral reactions or ion mobility spectrometry. [ABSTRACT FROM AUTHOR]

Hemoglobin (Hb) (αβ) is a tetrameric protein-protein complex. Collision cross sections, hydrogen exchange levels, and tandem mass spectrometry have been used to investigate the properties of gas-phase monomer, dimer, and tetramer ions of adult human hemoglobin (Hb A, αβ), and two variant hemoglobins: fetal hemoglobin (Hb F, αγ) and sickle hemoglobin (Hb S, αβ, E6V[β]). All three proteins give similar mass spectra. Monomers of Hb S and Hb F have similar cross sections, ca. 10% greater than those of Hb A. Cross sections of dimer ions of Hb S are 11% greater than those of Hb A and 6% greater than those of Hb F. Tetramers of Hb S are 13% larger than tetramers of Hb A or Hb F. Monomers and dimers of all three Hb have similar hydrogen-deuterium exchange (HDX) levels. Tetramers of Hb S exchange 16% more hydrogens than Hb A and Hb F. In tandem mass spectrometry, monomers of Hb S and Hb F require ca. 10% greater internal energy for heme loss than Hb A. Dimers (+11) of Hb A and Hb S dissociate to monomers with asymmetrical charge division; dimers of Hb F (+11) dissociate with nearly equal charge division. Tetramer ions dissociate to monomers and trimers, unlike solution Hb, which dissociates to dimers. The most stable dimers are from Hb S; the most stable tetramers from Hb F. The results with Hb S show that a single mutation in the β chain can change the physical properties of this gas-phase protein-protein complex. [ABSTRACT FROM AUTHOR]

Purpose: The impact of freeze-thaw (F/T) on structure integrity of protein therapeutics is poorly understood, partially due to lack of methods to detect protein structural perturbations during F/T processing in the frozen state. Methods: A new approach of hydrogen/deuterium exchange was developed to separate and distinguish the specific impact of single freezing and F/T cycling on protein structure, using lactate dehydrogenase (LDH) as model system. Results: In the freezing process, a fraction of LDH molecules that was inversely dependent on protein concentration was observed to partially denature its structure. Local structural perturbations were localized by peptide level HX analysis to the surface residues in segments 91-132, 170-237 and 288-331. In contrast, F/T cycling led to irreversible LDH aggregation with global structural unfolding. Residual solvent-protected structure was only detected in the aggregates for three segments, 13-31, 109-117 and 133-143, that were coincident with the consensus aggregation hotspots predicted by four different algorithms. Conclusions: Results indicate freezing preferentially disturbs local structure at the surface residues, consistent with ice-solution interface-mediated denaturation mechanism. F/T-induced aggregation begins as partial denaturation during freezing, but is accompanied by more comprehensive structural rearrangement during F/T cycling. [ABSTRACT FROM AUTHOR]

Therapeutic approaches for treatment of various diseases aim at the interruption of transcription or translation. Modified oligonucleotides, such as 2′- O-methyl- and methylphosphonate-derivatives, exhibit high resistance against cellular nucleases, thus rendering application for, e.g., antigene or antisense purposes possible. Other approaches are based on administration of cross-linking agents, such as cis-diamminedichloroplatinum(II) (cisplatin, DDP), which is still the most widely used anticancer drug worldwide. Due to the formation of 1,2-intrastrand cross links at adjacent guanines, replication of the double-strand is disturbed, thus resulting in significant cytotoxicity. Evidence for the gas-phase dissociation mechanism of platinated RNA is given, based on nano-electrospray ionization high-resolution multistage tandem mass spectrometry (MS). Confirmation was found by investigating the fragmentation pattern of platinated and unplatinated 2′-methoxy oligoribonucleotide hexamers and their corresponding methylphosphonate derivatives. Platinated 2′-methoxy oligoribonucleotides exhibit a similar gas-phase dissociation behavior as the corresponding DNA and RNA sequences, with the 3′-C-O bond adjacent to the vicinal guanines being cleaved preferentially, leading to w-ion formation. By examination of the corresponding platinated methylphosphonate derivatives of the 2′-methoxy oligoribonucleotides, the key role of the negatively charged phosphate oxygen atoms in direct proximity to the guanines was proven. The significant alteration of fragmentation due to platination is demonstrated by comparison of the fragment ion patterns of unplatinated and platinated 2′- O-methyl- and 2′- O-methyl methylphosphonate oligoribonucleotides, and the results obtained by H/D exchange experiments. [ABSTRACT FROM AUTHOR]

Abstract: Calmodulin (CaM), a ubiquitous intracellular sensor protein, binds Ca2+ and interacts with various targets as part of signal transduction. Using hydrogen/deuterium exchange (H/DX) and a high-resolution PLIMSTEX (protein–ligand interactions by mass spectrometry, titration, and H/D exchange) protocol, we examined five different states of calmodulin: calcium-free, calcium-loaded, and three states of calcium-loaded in the presence of either melittin, mastoparan, or skeletal myosin light-chain kinase (MLCK). When CaM binds Ca2+, the extent of H/DX decreased, consistent with the protein becoming stabilized upon binding. Furthermore, Ca2+-saturated calmodulin exhibits increased protection when bound to the peptides, forming high-affinity complexes. The protocol reveals significant changes in EF hands 1, 3, and 4 with saturating levels of Ca2+. Titration of the protein using PLIMSTEX provides the Ca2+-to-calmodulin binding affinity, which agrees well with previously reported values. The affinities of calmodulin to Ca2+ increase by factors of 300 and 1000 in the presence of melittin and mastoparan, respectively. A modified PLIMSTEX protocol whereby the protein is digested to component peptides gives a region-specific titration. The resulting titration data show a decrease in the root mean square fit of the residuals, indicating a better fit of the data. The global H/D exchange results and those obtained in a region-specific way provide new insight into the Ca2+-binding properties of this well-studied protein. [Copyright &y& Elsevier]

Abstract: Hydrogen/deuterium exchange coupled to mass spectrometry is an excellent technique to study protein structure changes and interactions, especially in cases where traditional high-resolution structural techniques are not applicable. A major drawback of this technique is the absence of universal software tools for data processing and visualization of the results. Here we describe a set of PHP scripts facilitating H/D data visualization and presentation. The application runs on a web server and is freely accessible. It comprises tools helping in preparation of H/D experiments as well as tools for turning simple tables with data on H/D exchange into different ways of representation used by the HXMS community. [Copyright &y& Elsevier]

Abstract: Protein glycosylation commonly stabilizes proteins thereby increasing protein half-lives and protecting against denaturation or proteolytic degradation. While generally beneficial, such stabilization is potentially disadvantageous in the case of inhibitory serpins. These protease inhibitors are metastable and a conformational transition to a more stable form is key to their function. Instability is therefore essential for these inhibitory serpins and mutagenesis has demonstrated that substantial stabilization results in compromised function. We have used optical spectroscopy and hydrogen/deuterium exchange and mass spectrometry to investigate the effects of glycosylation on the human serpin alpha-1 antitrypsin (α1-AT). Previous studies found that unglycosylated recombinant α1-AT populates a molten globule at low denaturant and that the ability to populate this state is correlated with efficient protease inhibition. Further, a high degree of conformational flexibility was found in several important regions. Guanidine hydrochloride denaturation monitored by circular dichroism indicates that plasma α1-AT, which is glycosylated at 3 sites, is substantially stabilized relative to the unglycosylated form. However, hydrogen exchange reveals complete loss of protection in plasma α1-AT above 1M GuHCl, similar to what is seen for the recombinant form. Sugars therefore appear to stabilize the compact denatured state of α1-AT without significant stabilization of the folded state. Native state hydrogen exchange reveals minor perturbations to native flexibility, but high flexibility in key regions such as the f helix is conserved. β-Strand 1c is stabilized in plasma α1-AT, which may confer increased resistance to forming pathogenic polymers. Overall, our results indicate that glycosylation of inhibitory serpins does not interfere with either native state flexibility or the native instability that is required for efficient function, though it may confer resistance to degradation by proteases and thus extend the half-life of circulating serpins. [Copyright &y& Elsevier]

Abstract: Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry (HDX-MS) has become a valuable tool in studies of protein dynamics and protein interactions. Isotopic exchange is typically initiated by diluting a protein solution into deuterated buffer at physiological conditions. For MS analysis, the exchange reaction is quenched by acidification and cooling and the labeled protein (or a digest thereof) is analyzed by mass spectrometry. An inevitable deuterium loss occurs during quench conditions (i.e., back-exchange). The primary structure of the peptide influences the back-exchange rate due to steric hindrance by bulky side groups and by inductive and charge effects. Here we show that the back-exchange in histidine repeats (His6) at HDX-MS quench conditions is complete within a few seconds using either acetic acid or formic acid in the quench solution, while aspartic acid repeats (Asp6) retain deuterons for several minutes using formic acid. We employ electron transfer dissociation to obtain residue-specific deuterium levels of the Asp repeat in K2D6IIKIIK using a hybrid linear ion trap-Orbitrap mass spectrometer. Our results show an unexpected uneven distribution of deuterium in the Asp repeat. The implication of the rapid back-exchange of His repeats for HDX-MS protein hydrogen exchange studies is discussed. We also discuss the implications of retained deuterons in the Asp repeat of K2D6IIKIIK when this peptide is used as a probe for the occurrence of hydrogen scrambling. [Copyright &y& Elsevier]

Abstract: A remarkable feature of prion biology is that the same prion protein can misfold into more than one infectious conformation, and these conformations in turn lead to distinct heritable prion strains with different phenotypes. The yeast prion [PSI+ ] is a powerful system for studying how changes in strain conformation affect cross-species transmission. We have previously established that a chimera of the Saccharomyces cerevisiae (SC) and Candida albicans (CA) Sup35 prion domains can cross the SC/CA species barrier in a strain-dependent manner. In vitro, the conversion of the monomeric chimera into the prion (amyloid) form can be seeded by either SC or CA Sup35 amyloid fibers, resulting in two strains: Chim[SC] and Chim[CA]. These strains have a “molecular memory” of their originating species in that Chim[SC] preferentially seeds the conversion of SC Sup35, and vice versa. To investigate how this species specificity is conformationally encoded, we used amide exchange and limited proteolysis to probe the structures of these two strains. We found that the amyloid cores of Chim[SC] and Chim[CA] are predominantly confined to the SC-derived and CA-derived residues, respectively. In addition, the chimera is able to propagate the Chim[CA] conformation even when the SC residues comprising the Chim[SC] core were deleted. Thus, the two strains have non-overlapping and modular amyloid cores that determine whether SC or CA residues are presented on the growing face of the prion seed. These observations establish how conformations determine the specificity of prion transmission and demonstrate a remarkable plasticity to amyloid misfolding. [Copyright &y& Elsevier]

Abstract: Trifluoroethanol (TFE)-induced conformational changes in two endogenous opioid peptides α-neoendorphin (α-NE; YGGFLRKYPK) and β-neoendorphin (β-NE; YGGFLRKYP) were studied using hydrogen/deuterium exchange (HX), coupled with electrospray ionization mass spectrometry (ESI-MS). HX data revealed that both peptides predominantly exist in an unfolded conformation in water and assume a compact conformation with increasing concentrations of TFE. Through tandem mass spectrometry it was identified that, because the LRKY segment in α-NE and N-terminal YG and RKY regions in β-NE contain the unexchanged hydrogens, they participate in secondary structure formation. The circular dichroism (CD) spectra of the peptides acquired in water and in mixed solvents that are comprised of varied amounts of TFE in water showed the presence of both β-turn and helical type secondary structures in these peptides. With increasing concentrations of the helical-inducing solvent, TFE, the α-helical content increases and that of β-turn decreases. [Copyright &y& Elsevier]

Abstract: RNase A forms 3D domain-swapped oligomers with novel enzymatic and biological activities. We study how crowding agents and osmolytes affect the formation and dissociation of RNase A oligomers. The crowding agents Ficoll and dextran were found to enhance oligomer formation, whereas the stabilizers sodium sulfate, glycine and trimethylammonium oxide (TMAO) do not. In contrast, TMAO significantly slows RNase A dimer dissociation, while the effect of Ficoll is small. These results lead us to propose that the mechanisms of oligomer formation and dissociation are different. In the RNase A “C-dimer”, the C-terminal β-strand is swapped between two subunits. The loop preceding this β-strand adopts a β-sheet which has been proposed to resemble amyloid structurally. Hydrogen/deuterium (H/D) exchange of the RNase A C-dimer reveal that the H-bonds formed between the swapped C-terminal β-strand and the other subunit are strong. Their rupture may be crucial for C-dimer dissociation. In contrast, H-bonds formed by Asn 113 in the novel β-sheet adopted by the hinge loop in the C-dimer are not strongly protected. Besides the fundamental insights obtained, the results represent a technical advance for obtaining increased oligomer yields and storage lifetimes. [ABSTRACT FROM AUTHOR]

Abstract: Spider dragline silk, one of the strongest polymers in nature, is composed of proteins termed major ampullate spidroin (MaSp) 1 and MaSp2. The N-terminal (NT) domain of MaSp1 produced by the nursery web spider Euprosthenops australis acts as a pH-sensitive relay, mediating spidroin assembly at around pH 6.3. Using amide hydrogen/deuterium exchange combined with mass spectrometry (MS), we detected pH-dependent changes in deuterium incorporation into the core of the NT domain, indicating global structural stabilization at low pH. The stabilizing effects were diminished or abolished at high ionic strength, or when the surface-exposed residues Asp40 and Glu84 had been exchanged with the corresponding amides. Nondenaturing electrospray ionization MS revealed the presence of dimers in the gas phase at pH values below—but not above—6.4, indicating a tight electrostatic association that is dependent on Asp40 and Glu84 at low pH. Results from analytical ultracentrifugation support these findings. Together, the data suggest a mechanism whereby lowering the pH to <6.4 results in structural changes and alteration of charge-mediated interactions between subunits, thereby locking the spidroin NT dimer into a tight entity important for aggregation and silk formation. [Copyright &y& Elsevier]

Abstract: Escherichia coli Rho is a doughnut-shaped homohexameric ATP-dependent RNA–DNA helicase that releases newly synthesized RNA molecules from transcription complexes. Rho binds 60–80 bases of RNA among six primary RNA binding sites around the inside of its N-terminal crown; the RNA then passes through the central hole of the hexamer. Here it triggers ATP hydrolysis and is moved with respect to the protein. We study protein conformation changes upon ligand binding using amide proton hydrogen/deuterium exchange and mass spectrometry. Global-exchange studies indicate net mass differences of about 15 Da after 1 h of exchange in the presence—versus in the absence—of the ligand MgATP or the RNA poly(C). Sites of ligand-dependent exchange differences were localized by mass determination of the peptic peptides of Rho. A peptide of the N-terminal domain near the known primary RNA sites (aa 56–63) was protected from amide proton exchange in the presence of poly(C), as was a novel N-terminal domain peptide that is not near RNA in the crystal structures or in NMR structures with RNA oligomers (aa 37–46). This result may further define the primary interaction site of RNA with Rho. The Q-loop-containing peptide in the central hole of the protein that interacts with RNA was also protected by RNA (aa 271–286). The exchange rate of one peptide near the ATPase active site (aa 206–218) slowed in the presence of MgATP and increased in the presence of RNA. Overall, the results show changes in a few protein segments rather than a different overall conformation. [ABSTRACT FROM AUTHOR]

Abstract: A hydrophilic interaction liquid chromatography (HILIC) method was used to separate a commonly used pharmaceutical starting material, 4-aminomethylpyridine (4-AMP), and its degradants. The structures of the major degradants were characterized and elucidated without prior isolation by accurate mass measurement, MS/MS analysis and on-line hydrogen/deuterium (H/D) exchange experiments. The mass spectra obtained from H/D exchange experiments are particularly useful to differentiate structural isomers, to elucidate the fragmentation pathways, and to aid in structure elucidation in the absence of MS/MS fragmentation information. The impact of deuterium oxide and temperature on HILIC separation has also been explored here. The integration of H/D exchange with HILIC has been described here for the first time and has been demonstrated to be a powerful structure elucidation tool via the study of degradants in 4-AMP. [Copyright &y& Elsevier]

The joint x-ray/neutron diffraction model of the Type I copper protein, amicyanin from Paracoccus denitrificans was determined at 1.8 Å resolution. The protein was crystallized using reagents prepared in D2O. About 86% of the amide hydrogen atoms are either partially or fully exchanged, which correlates well with the atomic depth of the amide nitrogen atom and the secondary structure type, but with notable exceptions. Each of the four residues that provide copper ligands is partially deuterated. The model reveals the dynamic nature of the protein, especially around the copper-binding site. A detailed analysis of the presence of deuterated water molecules near the exchange sites indicates that amide hydrogen exchange is primarily due to the flexibility of the protein. Analysis of the electron transfer path through the protein shows that residues in that region are highly dynamic, as judged by hydrogen/deuterium exchange. This could increase the rate of electron transfer by transiently shortening through-space jumps in pathways or by increasing the atomic packing density. Analysis of C-H⋯X bonding reveals previously undefined roles of these relatively weak H bonds, which, when present in sufficient number can collectively influence the structure, redox, and electron transfer properties of amicyanin. [ABSTRACT FROM AUTHOR]

Mutations of the receptor tyrosine kinase KIT are linked to certain cancers such as gastrointestinal stromal tumors (GISTs). Biophysical, biochemical, and structural studies have provided insight into the molecular basis of resistance to the KIT inhibitors, imatinib and sunitinib. Here, solution-phase hydrogen/deuterium exchange (HDX) and direct binding mass spectrometry experiments provide a link between static structure models and the dynamic equilibrium of the multiple states of KIT, supporting that sunitinib targets the autoinhibited conformation of WT-KIT. The D816H mutation shifts the KIT conformational equilibrium toward the activated state. The V560D mutant exhibits two low energy conformations: one is more flexible and resembles the D816H mutant shifted toward the activated conformation, and the other is less flexible and resembles the wild-type KIT in the autoinhibited conformation. This result correlates with the V560D mutant exhibiting a sensitivity to sunitinib that is less than for WT KIT but greater than for KIT D816H. These findings support the elucidation of the resistance mechanism for the KIT mutants. [ABSTRACT FROM AUTHOR]

Abstract: HK97 is an exceptionally amenable system for characterizing major conformational changes associated with capsid maturation in double-stranded DNA bacteriophage. HK97 undergoes a capsid expansion of ∼20%, accompanied by major subunit rearrangements during genome packaging. A previous 3.44-Å-resolution crystal structure of the mature capsid Head II and cryo-electron microscopy studies of other intermediate expansion forms of HK97 suggested that, primarily, rigid-body movements facilitated the maturation process. We recently reported a 3.65-Å-resolution structure of the preexpanded particle form Prohead II (P-II) and found that the capsid subunits undergo significant refolding and twisting of the tertiary structure to accommodate expansion. The P-II study focused on major twisting motions in the P-domain and on refolding of the spine helix during the transition. Here we extend the crystallographic comparison between P-II and Head II, characterizing the refolding events occurring in each of the four major domains of the capsid subunit and their effect on quaternary structure stabilization. In addition, hydrogen/deuterium exchange, coupled to mass spectrometry, was used to characterize the structural dynamics of three distinct capsid intermediates: P-II, Expansion Intermediate, and the nearly mature Head I. Differences in the solvent accessibilities of the seven quasi-equivalent capsid subunits, attributed to differences in secondary and quaternary structures, were observed in P-II. Nearly all differences in solvent accessibility among subunits disappear after the first transition to Expansion Intermediate. We show that most of the refolding is coupled to this transformation, an event associated with the transition from asymmetric to symmetric hexamers. [Copyright &y& Elsevier]