Hydrogen/Deuterium Exchange Mass Spectrometry for Weak Binders.

This note describes theoretical and experimental considerations to observe perturbation of a protein upon binding to a ligand with weak affinity by hydrogen/deuterium exchange mass spectrometry (HDX-MS). The most popular application of HDX-MS is to determine the binding site of a drug or drug lead in a protein target. However, when the affinity of a ligand is weak, driving the equilibrium to the formation of a complex is difficult, and thus, observing the perturbation upon binding is also challenging. Theoretical consideration indicates that the original concentration of a ligand over the dissociation constant ([L0]/KD) is roughly equal to the maximum protection factor expected for the experiment when the original concentration of a ligand is significantly larger than the original concentration of a protein and the dissociation constant ([L0] ≫ [P0] and [L0] ≫ KD). When HDX-MS analysis of a protein with a ligand of low affinity and low solubility is carried out, it may be challenging to achieve high enough ligand concentration to drive the equilibrium in favor of the complex due to the low solubility. There are two methods to alleviate this issue: (i) spiking a low affinity/low solubility ligand to exchange buffer to lower the required ligand concentration in aqueous protein stock solution and (ii) mixing a 1:1 ratio of aqueous protein-ligand stock solution and deuterated buffer to initiate the exchange reaction instead of the commonly used 1:9 ratio.

Optimization of Feasibility Stage for Hydrogen/Deuterium Exchange Mass Spectrometry.

The practice of HDX-MS remains somewhat difficult, not only for newcomers but also for veterans, despite its increasing popularity. While a typical HDX-MS project starts with a feasibility stage where the experimental conditions are optimized and the peptide map is generated prior to the HDX study stage, the literature usually reports only the HDX study stage. In this protocol, we describe a few considerations for the initial feasibility stage, more specifically, how to optimize quench conditions, how to tackle the carryover issue, and how to apply the pepsin specificity rule. Two sets of quench conditions are described depending on the presence of disulfide bonds to facilitate the quench condition optimization process. Four protocols are outlined to minimize carryover during the feasibility stage: (1) addition of a detergent to the quench buffer, (2) injection of a detergent or chaotrope to the protease column after each sample injection, (3) back-flushing of the trap column and the analytical column with a new plumbing configuration, and (4) use of PEEK (or PEEK coated) frits instead of stainless steel frits for the columns. The application of the pepsin specificity rule after peptide map generation and not before peptide map generation is suggested. The rule can be used not only to remove falsely identified peptides, but also to check the sample purity. A well-optimized HDX-MS feasibility stage makes subsequent HDX study stage smoother and the resulting HDX data more reliable. Graphical Abstract ᅟ.

Characterization of the N370S mutant of glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry.

An asparagine-to-serine substitution at residue 370 (N370S) in glucocerebrosidase (GCase) is the most prevalent mutation leading to Gaucher's disease, the most common lysosomal storage disorder. Two types of hydrogen/deuterium exchange experiment coupled with proteolysis and liquid chromatography-mass spectrometry (HDX-MS) were used to investigate the dynamic properties and unfolding stability of wt, R495H, and N370S GCases in the presence and absence of ligands. R495H GCase is used for enzyme replacement therapy and is considered to be a wt surrogate, whereas N370S is the most prevalent mutation leading to Gaucher's disease. Time-course HDX experiments of the GCases were performed under near-physiological conditions to detect the protein's local unfolding motions at a submolecular level. In guanidine-titration experiments, HDX reactions were performed with various concentrations of a chemical denaturant to provide the global stability of the proteins. The two types of experiment showed that all three purified GCases, wt, R495H, and N370S, have virtually identical local unfolding motions and global stabilities in solution. Combined with previous X-ray crystallographic studies, which showed indistinguishable backbone conformations for N370S and R495H GCase mutants and very similar melting temperatures for the wt, R495H, and N370S mutants, all three GCases are likely to have virtually identical structural and dynamic properties in solution. The guanidine-titration experiments revealed that the pharmacological chaperone, isofagomine (IFG), interacts more weakly with the N370S mutant than with the R495H GCase; this is consistent with the higher IC(50) value of IFG against N370S than against R495H. The time-course experiments showed that IFG restricts the local unfolding motions of N370S in the same way as those of R495H when the ligand saturates the proteins.

Mapping of discontinuous conformational epitopes by amide hydrogen/deuterium exchange mass spectrometry and computational docking.

Understanding antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory, and intellectual property reasons, especially with increasing demand for monoclonal antibody therapeutic agents. Although various techniques are available for the determination of an epitope, there is no widely applicable, high-resolution, and reliable method available. Here, a combination approach using amide hydrogen/deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS) and computational docking was applied to investigate antigen-antibody interactions. HDX-MS is a widely applicable, medium-resolution, medium-throughput technology that can be applied to epitope identification. First, the epitopes of cytochrome c-E8, IL-13-CNTO607, and IL-17A-CAT-2200 interactions identified using the HDX-MS method were compared with those identified by X-ray co-crystal structures. The identified epitopes are in good agreement with those identified using high-resolution X-ray crystallography. Second, the HDX-MS data were used as constraints for computational docking. More specifically, the non-epitope residues of an antigen identified using HDX-MS were designated as binding ineligible during computational docking. This approach, termed HDX-DOCK, gave more tightly clustered docking poses than stand-alone docking for all antigen-antibody interactions examined and improved docking results significantly for the cytochrome c-E8 interaction.

Expansion of time window for mass spectrometric measurement of amide hydrogen/deuterium exchange reactions.

Backbone amide hydrogen exchange rates can be used to describe the dynamic properties of a protein. Amide hydrogen exchange rates in a native protein may vary from milliseconds (ms) to several years. Ideally, the rates of all amide hydrogens of the analyte protein can be determined individually. To achieve this goal, monitoring of a wider time window is critical, in addition to high sequence coverage and high sequence resolution. Significant improvements have been made to hydrogen/deuterium exchange mass spectrometry methods in the past decade for better sequence coverage and higher sequence resolution. On the other hand, little effort has been made to expand the experimental time window to accurately determine exchange rates of amide hydrogens. Many fast exchanging amide hydrogens are completely exchanged before completion of a typical short exchange time point (10-30 s) and many slow exchanging amide hydrogens do not start exchanging before a typical long exchanging time point (1-3 h). Here various experimental conditions, as well as a quenched-flow apparatus, are utilized to monitor cytochrome c amide hydrogen exchange behaviors over more than eight orders of magnitude (0.0044-1 000 000 s), when converted into the standard exchange condition (pH 7 and 23°C).

Local conformational stability of HIV-1 gp120 in unliganded and CD4-bound states as defined by amide hydrogen/deuterium exchange.

The binding reaction of the HIV-1 gp120 envelope glycoprotein to the CD4 receptor involves exceptional changes in enthalpy and entropy. Crystal structures of gp120 in unliganded and various ligand-bound states, meanwhile, reveal an inner domain able to fold into diverse conformations, a structurally invariant outer domain, and, in the CD4-bound state, a bridging sheet minidomain. These studies, however, provide only hints as to the flexibility of each state. Here we use amide hydrogen/deuterium exchange coupled to mass spectrometry to provide quantifications of local conformational stability for HIV-1 gp120 in unliganded and CD4-bound states. On average, unliganded core gp120 displayed >10,000-fold slower exchange of backbone-amide hydrogens than a theoretically unstructured protein of the same composition, with binding by CD4 reducing the rate of gp120 amide exchange a further 10-fold. For the structurally constant CD4, alterations in exchange correlated well with alterations in binding surface (P value = 0.0004). For the structurally variable gp120, however, reductions in flexibility extended outside the binding surface, and regions of expected high structural diversity (inner domain/bridging sheet) displayed roughly 20-fold more rapid exchange in the unliganded state than regions of low diversity (outer domain). Thus, despite an extraordinary reduction in entropy, neither unliganded gp120 nor free CD4 was substantially unstructured, suggesting that most of the diverse conformations that make up the gp120 unliganded state are reasonably ordered. The results provide a framework for understanding how local conformational stability influences entropic change, conformational diversity, and structural rearrangements in the gp120-CD4 binding reaction.

Structure of IL-17A in complex with a potent, fully human neutralizing antibody.

IL-17A is a pro-inflammatory cytokine produced by the newly identified Th17 subset of T-cells. We have isolated a human monoclonal antibody to IL-17A (CAT-2200) that can potently neutralize the effects of recombinant and native human IL-17A. We determined the crystal structure of IL-17A in complex with the CAT-2200 Fab at 2.6 A resolution in order to provide a definitive characterization of the epitope and paratope regions. Approximately a third of the IL-17A dimer is disordered in this crystal structure. The disorder occurs in both independent copies of the complex in the asymmetric unit and does not appear to be influenced by crystal packing. The complex contains one IL-17A dimer sandwiched between two CAT-2200 Fab fragments. The IL-17A is a disulfide-linked homodimer that is similar in structure to IL-17F, adopting a cystine-knot fold. The structure is not inconsistent with the previous prediction of a receptor binding cavity on IL-17 family members. The epitope recognized by CAT-2200 is shown to involve 12 amino acid residues from the quaternary structure of IL-17A, with each Fab contacting both monomers in the dimer. All complementarity-determining regions (CDRs) in the Fab contribute to a total of 16 amino acid residues in the antibody paratope. In vitro affinity optimization was used to generate CAT-2200 from a parental lead antibody using random mutagenesis of CDR3 loops. This resulted in seven amino acid changes (three in VL-CDR3 and four in VH-CDR3) and gave an approximate 30-fold increase in potency in a cell-based neutralization assay. Two of the seven amino acids form part of the CAT-2200 paratope. The observed interaction site between CAT-2200 and IL-17A is consistent with data from hydrogen/deuterium exchange mass spectrometry and mutagenesis approaches.

A common property of amyotrophic lateral sclerosis-associated variants: destabilization of the copper/zinc superoxide dismutase electrostatic loop.

At least 119 mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis by an unidentified toxic gain of function. We compared the dynamic properties of 13 as-isolated, partially metallated, SOD1 variant enzymes using hydrogen-deuterium exchange. We identified a shared property of these familial amyotrophic lateral sclerosis-related SOD1 variants, namely structural and dynamic change affecting the electrostatic loop (loop VII) of SOD1. Furthermore, SOD1 variants that have severely compromised metal binding affinities demonstrated additional structural and dynamic changes to the zinc-binding loop (loop IV) of SOD1. Although the biological consequences of increased loop VII mobility are not fully understood, this common property is consistent with the hypotheses that SOD1 mutations exert toxicity via aggregation or aberrant association with other cellular constituents.

Epitope mapping by amide hydrogen/deuterium exchange coupled with immobilization of antibody, on-line proteolysis, liquid chromatography and mass spectrometry.

The epitope of horse cytochrome c against monoclonal antibody E8 was determined using amide hydrogen/deuterium (H/D) exchange combined with immobilized antibody, on-line pepsin proteolysis, liquid chromatography (LC), and mass spectrometry (MS). The results were generally in good agreement with contact residues identified by an X-ray co-crystal structure of the E8-cytochrome c complex and results obtained by H/D exchange with nuclear magnetic resonance (NMR) spectrometry. The H/D exchange reaction of cytochrome c was carried out in the presence or absence of immobilized E8 antibody. Regions that gained less deuterium in the presence of the antibody than in its absence are defined as the epitope by the H/D exchange MS method. Control experiments were carefully designed to help identify the epitope with high confidence.

Identification of pharmacological chaperones for Gaucher disease and characterization of their effects on beta-glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry.

Point mutations in beta-glucocerebrosidase (GCase) can result in a deficiency of both GCase activity and protein in lysosomes thereby causing Gaucher Disease (GD). Enzyme inhibitors such as isofagomine, acting as pharmacological chaperones (PCs), increase these levels by binding and stabilizing the native form of the enzyme in the endoplasmic reticulum (ER), and allow increased lysosomal transport of the enzyme. A high-throughput screen of the 50,000-compound Maybridge library identified two, non-carbohydrate-based inhibitory molecules, a 2,4-diamino-5-substituted quinazoline (IC(50) 5 microM) and a 5-substituted pyridinyl-2-furamide (IC(50) 8 microM). They raised the levels of functional GCase 1.5-2.5-fold in N370S or F213I GD fibroblasts. Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of GCase in their ER and increased levels in lysosomes. Changes in protein dynamics, monitored by hydrogen/deuterium-exchange mass spectrometry, identified a domain III active-site loop (residues 243-249) as being significantly stabilized upon binding of isofagomine or either of these two new compounds; this suggests a common mechanism for PC enhancement of intracellular transport.

Isofagomine induced stabilization of glucocerebrosidase.

Structurally destabilizing mutations in acid beta-glucosidase (GCase) can result in Gaucher disease (GD). The iminosugar isofagomine (IFG), a competitive inhibitor and a potential pharmacological chaperone of GCase, is currently undergoing clinical evaluation for the treatment of GD. An X-ray crystallographic study of the GCase-IFG complex revealed a hydrogen bonding network between IFG and certain active site residues. It was suggested that this network may translate into greater global stability. Here it is demonstrated that IFG does increase the global stability of wild-type GCase, shifting its melting curve by approximately 15 degrees C and that it enhances mutant GCase activity in pre-treated N370S/N370S and F213I/L444P patient fibroblasts. Additionally, amide hydrogen/deuterium exchange mass spectroscopy (H/D-Ex) was employed to identify regions within GCase that undergo stabilization upon IFG-binding. H/D-Ex data indicate that the binding of IFG not only restricts the local protein dynamics of the active site, but also propagates this effect into surrounding regions.

A simple test to detect hydrogen/deuterium scrambling during gas-phase peptide fragmentation.

Amide hydrogen/deuterium (H/D) exchange coupled with mass spectrometry has become a powerful tool to study protein dynamics. Addition of a proteolysis step between the exchange reaction and mass analysis can be used to localize the positions of deuterium and improve overall resolution. The resolution can be further enhanced by the fragmentation of digested peptides in the gas phase if scrambling of exchangeable hydrogens and deuteriums on the peptides does not occur. Although some laboratories reported successful localization of deuteriums by gas-phase fragmentations, others described total scrambling. Here we propose a simple method to detect the presence or absence of scrambling using a commercially available small peptide, neurotensin (9-13; RPYIL). All exchangeable hydrogens on this pentapeptide are first deuterated by dissolving it in deuterium oxide. The deuterated peptide is loaded onto a reversed-phase column, and then washed with copious amounts of cold acidic aqueous buffer. This washing exchanges all deuteriums on both the terminals and the side chains back to hydrogens. Now only three deuteriums are attached on the pentapeptide, one on each of the amide nitrogens of Y, I, and L. After the partially deuterated peptide is eluted from the column with 95% acidic acetonitrile, collision-induced dissociation (CID) generates a series of b ions, which are analyzed by mass spectrometer. In the absence of scrambling, no deuterium should be observed in the b 2 ion, as neither R nor P have amide hydrogens. On the other hand, in the event of scrambling, b 2 should carry about half of the deuteriums of the parent pentapeptide. In theory, complete scrambling should distribute deuteriums equally among all of the exchangeable hydrogens. The b 2 portion of neurotensin (9-13) has 6 exchangeable hydrogens, whereas the +1 charge state of neurotensin (9-13) has 12 exchangeable hydrogens. We demonstrated that CID caused complete scrambling of hydrogens and deuteriums with an LCQ (a ion trap machine).

Dynamic structural changes during complement C3 activation analyzed by hydrogen/deuterium exchange mass spectrometry.

Proteolytic cleavage of component C3 to C3b is a central step in the activation of complement. Whereas C3 is largely biologically inactive, C3b is directly involved in various complement activities. While the recently described crystal structures of C3 and C3b provide a molecular basis of complement activation, they do not reflect the dynamic changes that occur in solution. In addition, the available C3b structures diverge in some important aspects. Here we have utilized hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) to investigate relative changes in the solution-phase structures of C3 and C3b. By combining two forms of mass spectrometry we could maximize the primary sequence coverage of C3b and demonstrate the feasibility of this method for large plasma proteins. While the majority of the 82 peptides that could be followed over time showed only minor alterations in HDX, we observed clear changes in solvent accessibility for 16 peptides, primarily in the alpha-chain (alpha'NT, MG6-8, CUB, TED, C345C domains). Most of these peptides could be directly linked to the structural transitions visible in the crystal structures and revealed additional information about the probability of the structural variants of C3b. In addition, a discontinuous cluster of seven peptides in the MG3, MG6, LNK and alpha'NT domains showed a decreased accessibility after activation to C3b. Although no gross conformational changes are detected in the crystal structure, this area may reflect a structurally flexible region in solution that contributes to C3 activation and function.

Conformation-dependent ligand regulation of ATP hydrolysis by human KSP: activation of basal hydrolysis and inhibition of microtubule-stimulated hydrolysis by a single, small molecule modulator.

Human kinesin spindle protein (KSP)/hsEg5, a member of the kinesin-5 family, is essential for mitotic spindle assembly in dividing human cells and is required for cell cycle progression through mitosis. Inhibition of the ATPase activity of KSP leads to cell cycle arrest during mitosis and subsequent cell death. Ispinesib (SB-715992), a potent and selective inhibitor of KSP, is currently in phase II clinical trials for the treatment of multiple tumor types. Mutations that attenuate Ispinesib binding to KSP in vitro have been identified, highlighting the need for inhibitors that target different binding sites and inhibit KSP activity by novel mechanisms. We report here a small-molecule modulator, KSPA-1, that activates KSP-catalyzed ATP hydrolysis in the absence of microtubules yet inhibits microtubule-stimulated ATP hydrolysis by KSP. KSPA-1 inhibits cell proliferation and induces monopolar-spindle formation in tumor cells. Results from kinetic analyses, microtubule (MT) binding competition assays, and hydrogen/deuterium-exchange studies show that KSPA-1 does not compete directly for microtubule binding. Rather, this compound acts by driving a conformational change in the KSP motor domain and disrupts productive ATP turnover stimulated by MT. These findings provide a novel mechanism for targeting KSP and perhaps other mitotic kinesins.

Effects of electrospray capillary temperature on amide hydrogen exchange.

Amide hydrogen/deuterium (H/D) exchange coupled with proteolysis, high-perfeomance liquid chromatographic (HPLC) separation and mass spectrometry (MS) has become a powerful tool to study protein dynamics in solution. Prior to the execution of H/D exchange experiments, various experimental parameters have to be set, including proteolysis, HPLC, and MS conditions. Here we investigate the effects of electrospray capillary temperature on deuterium retention in backbone amides of various pepsin-generated cytochrome c peptides. Lower capillary temperature generally helps retain more deuterium than higher capillary temperature. When the capillary temperature was 150 degrees C, on average 26% more deuterium was retained than when the capillary temperature was set at 250 degrees C. The effects of capillary temperature varied depending on the ions monitored. There was little difference in deuterium retention among different charge state species of the same peptide at 150 degrees C. However, a lower charge state ion loses more deuterium atoms going from 150 degrees C to 250 degrees C than the corresponding higher charge state species. These results indicate that the capillary temperature should be optimized not only to maximize the signal-to-noise of each ion followed in H/D exchange experiments, but also to minimize the deuterium loss of the ions. Also the loss of deuterium in several ions, especially lower charge state ones, should be monitored in the optimization, as the temperature effects vary among ions and are more significant for lower charge state ions.

Specificity of immobilized porcine pepsin in H/D exchange compatible conditions.

Statistical analysis of data from 39 proteins (13 766 amino acid residues) digested with immobilized porcine pepsin under conditions compatible with hydrogen/deuterium (H/D) exchange (<1 degrees C, <30 s) was performed to examine pepsin cleavage specificity. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe and Leu are favored residues each with a cleavage probability greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. Pro also cannot be at position P2 (cleavage probability <0.3%). Occupation of the P3 position by His, Lys, or Arg, or occupation of the P2' position by Pro, also leads to very little cleavage (cleavage probability <1.7%). The average cleavage probability over the entire data set was 13.6%, which is slightly lower than the value previously obtained by Powers et al. (14.8%). This is due, in part, to the larger protein sizes used in the current study. While the specificity of pepsin was similar to that previously observed, higher selectivity was observed in the present study due to less experimental variation in the conditions used to generate our database.

Hydrogen-deuterium exchange mass spectrometry for investigation of backbone dynamics of oxidized and reduced cytochrome P450cam.

Backbone dynamics of the camphor monoxygenase cytochrome P450(cam) (CYP101) as a function of oxidation/ligation state of the heme iron were investigated via hydrogen/deuterium exchange (H/D exchange) as monitored by mass spectrometry. Main chain amide NH hydrogens can exchange readily with solvent and the rate of this exchange depends upon, among other things, dynamic fluctuations in local structural elements. A fluxional region of the polypeptide will exchange more quickly with solvent than one that is more constrained. In most regions of the enzyme, exchange rates were similar between oxidized high-spin camphor-bound and reduced camphor- and CO-bound CYP101 (CYP-S and CYP-S-CO, respectively). However, in regions of the protein that have previously been implicated in substrate access by structural and molecular dynamics investigations, the reduced enzyme shows significantly slower exchange rates than the oxidized CYP-S. This observation corresponds to increased flexibility of the oxidized enzyme relative to the reduced form. Structural features previously found to be perturbed in CYP-S-CO upon binding of the biologically relevant effector and reductant putidaredoxin (Pdx) as determined by nuclear magnetic resonance are also more protected from exchange in the reduced state. To our knowledge, this study represents the first experimental investigation of backbone dynamics within the P450 family using this methodology.

Hydrogen/deuterium-exchange (H/D-Ex) of PPARgamma LBD in the presence of various modulators.

A nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), is a ligand-dependent transcription factor involved in glucose homeostasis and adipocyte differentiation. PPARgamma is the molecular target of various natural and synthetic molecules, including anti-diabetic agents such as rosiglitazone. Amide hydrogen/deuterium-exchange (H/D-Ex), coupled with proteolysis and mass spectrometry, was applied to study the dynamics of the PPARgamma ligand binding domain (LBD) with or without molecules that modulate PPARgamma activity. The H/D-Ex patterns of ligand-free PPARgamma LBD show that the ligand binding pocket of LBD is significantly more dynamic than the rest of the LBD. Presumably, the binding pocket is intrinsically disordered in order to accommodate different ligands. The presence of two full agonists (rosiglitazone and GW1929), a partial agonist (nTZDpa), and a covalent antagonist (GW9662), changed the dynamics/conformation of PPARgamma LBD and slowed the H/D exchange rate in various regions of the protein. The full agonists slowed the H/D exchange more globally and to a greater extent than the partial agonist or the antagonist, indicating that the full agonist stabilizes the PPARgamma LBD more than the partial agonist or the antagonist. One interesting observation is that the two full agonists significantly stabilized helix 12 while the partial agonist and the antagonist did not perturb the H/D exchange of this region. The results showed that the change in protein dynamics induced by ligand binding may be an important factor for the activation of genes and that H/D-Ex is a useful method for analyzing the biological activity of drug leads.

The role of protein dynamics in increasing binding affinity for an engineered protein-protein interaction established by H/D exchange mass spectrometry.

It is generally accepted that protein and solvation dynamics play fundamental roles in the mechanisms of protein-protein binding; however, assessing their contribution meaningfully has not been straightforward. Here, hydrogen/deuterium exchange mass spectrometry (H/D-Ex) was employed to assess the role of dynamics for a high-affinity human growth hormone variant (hGHv) and the wild-type growth hormone (wt-hGH) each binding to the extracellular domain of their receptor (hGHbp). Comparative analysis of the transient fluctuations in the bound and unbound states revealed that helix-1 of hGHv undergoes significant transient unfolding in its unbound state, a characteristic that was not found in wt-hGH or apparent in the temperature factor data from the X-ray analysis of the unbound hGHv structure. In addition, upon hormone binding, an overall increase in stability was observed for the beta-sheet structure of hGHbp which included sites distant from the binding interface. On the basis of the stability, binding kinetics, and thermodynamic data presented, the increase in the binding free energy of hGHv is primarily generated by factors that appear to increase the energy of the unbound state relative to the free energy of the bound complex. This implies that an alternate route to engineer new interactions aiming to increase protein-protein association energies may be achieved by introducing certain mutations that destabilize one of the interacting molecules without destabilizing the resulting bound complex. Importantly, although the hGHv molecule is less stable than its wt-hGH counterpart, its resulting active ternary complex with two copies of hGHbp has comparable stability to the wt complex.

Rapid analysis of protein structure and dynamics by hydrogen/deuterium exchange mass spectrometry.

An automated approach for the rapid analysis of protein structure has been developed and used to study acid-induced conformational changes in human growth hormone. The labeling approach involves hydrogen/deuterium exchange (H/D-Ex) of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry (MS). Briefly, the protein is incubated for defined intervals in a deuterated environment. After rapid quenching of the exchange reaction, the partially deuterated protein is enzymatically digested and the resulting peptide fragments are analyzed by liquid chromatography mass spectrometry (LC-MS). The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. Additional analyses allow mapping of the free energy of folding on localized segments along the protein sequence affording unique dynamic and structural information. While amide H/D-Ex coupled with MS is recognized as a powerful technique for studying protein structure and protein-ligand interactions, it has remained a labor-intensive task. The improvements in the amide H/D-Ex methodology described here include solid phase proteolysis, automated liquid handling and sample preparation, and integrated data reduction software that together improve sequence coverage and resolution, while achieving a sample throughput nearly 10-fold higher than the commonly used manual methods.