No abstract

To examine the relationship between protein structural dynamics and measurable hydrogen exchange (HX) data, the detailed exchange behavior of most of the backbone amide hydrogens of Staphylococcal nuclease was compared with that of their neighbors, with their structural environment, and with other information. Results show that H-bonded hydrogens are protected from exchange, with HX rate effectively zero, even when they are directly adjacent to solvent. The transition to exchange competence requires a dynamic structural excursion that removes H-bond protection and allows exposure to solvent HX catalyst. The detailed data often make clear the nature of the dynamic excursion required. These range from whole molecule unfolding, through smaller cooperative unfolding reactions of secondary structural elements, and down to local fluctuations that involve as little as a single peptide group or side chain or water molecule. The particular motion that dominates the exchange of any hydrogen is the one that allows the fastest HX rate. The motion and the rate it produces are determined by surrounding structure and not by nearness to solvent or the strength of the protecting H-bond itself or its acceptor type (main chain, side chain, structurally bound water). Many of these motions occur over time scales that are appropriate for biochemical function.

To investigate the determinants of protein hydrogen exchange (HX), HX rates of most of the backbone amide hydrogens of Staphylococcal nuclease were measured by NMR methods. A modified analysis was used to improve accuracy for the faster hydrogens. HX rates of both near surface and well buried hydrogens are spread over more than 7 orders of magnitude. These results were compared with previous hypotheses for HX rate determination. Contrary to a common assumption, proximity to the surface of the native protein does not usually produce fast exchange. The slow HX rates for unprotected surface hydrogens are not well explained by local electrostatic field. The ability of buried hydrogens to exchange is not explained by a solvent penetration mechanism. The exchange rates of structurally protected hydrogens are not well predicted by algorithms that depend only on local interactions or only on transient unfolding reactions. These observations identify some of the present difficulties of HX rate prediction and suggest the need for returning to a detailed hydrogen by hydrogen analysis to examine the bases of structure-rate relationships, as described in the companion paper (Skinner et al., Protein Sci 2012;21:996-1005).

A new method for analyzing the dynamics of proteins is developed and tested. The method, pump-probe molecular dynamics, excites selected atoms or residues with a set of oscillating forces, and the transmission of the impulse to other parts of the protein is probed using Fourier transform of the atomic motions. From this analysis, a coupling profile can be determined which quantifies the degree of interaction between pump and probe residues. Various physical properties of the method such as reciprocity and speed of transmission are examined to establish the soundness of the method. The coupling strength can be used to address questions such as the degree of interaction between different residues at the level of dynamics, and identify propagation of influence of one part of the protein on another via "pathways" through the protein. The method is illustrated by analysis of coupling between different secondary structure elements in the allosteric protein calmodulin, and by analysis of pathways of residue-residue interaction in the PDZ domain protein previously elucidated by genomics and mutational studies.

Long-time molecular dynamics (MD) simulations are now able to fold small proteins reversibly to their native structures [LindorffLarsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517-520]. These results indicate that modern force fields can reproduce the energy surface near the native structure. To test how well the force fields recapitulate the other regions of the energy surface, MD trajectories for a variant of protein G are compared with data from site-resolved hydrogen exchange (HX) and other biophysical measurements. Because HX monitors the breaking of individual H-bonds, this experimental technique identifies the stability and H-bond content of excited states, thus enabling quantitative comparison with the simulations. Contrary to experimental findings of a cooperative, all-or-none unfolding process, the simulated denatured state ensemble, on average, is highly collapsed with some transient or persistent native 2°structure. The MD trajectories of this protein G variant and other small proteins exhibit excessive intramolecular H-bonding even for the most expanded conformations, suggesting that the force fields require improvements in describing H-bonding and backbone hydration. Moreover, these comparisons provide a general protocol for validating the ability of simulations to accurately capture rare structural fluctuations. M olecular dynamics (MD) simulations can now probe protein dynamics on millisecond timescales and thereby enable investigation of a variety of biological problems, including binding, conformational changes, and folding. A landmark example is the all-atom simulations by Shaw and coworkers where multiple folding and unfolding events were observed in long time trajectories (1, 2). In addition to predicting or matching observed folding rates with a single set of parameters, these simulations produced native-like models for 12 small, fast-folding proteins. Equally impressive is their observation of multiple discrete folding and unfolding transitions, which indicates that folding proceeds on an energy landscape with two major states separated by a free energy barrier. This barrier-limited folding behavior replicates that observed for many proteins. Not surprisingly, these remarkable simulations are being extensively analyzed (3-5).The applicability of MD for many situations is limited by the extent to which the entire landscape is recapitulated. An accurate representation of native-like states does not imply a correct representation of other states (e.g., intermediates and unfolded structures). Proper validation requires a comparison with experiments that probe lowly populated conformations. NMR measurements probe subsecond dynamics with single residue resolution, although with a limitation to states with populations exceeding 0.5% (6). Fluorescence, CD, FRET, and small angle X-ray scattering (SAXS) measurements are well adapted to kinetic studies but provide limited spatial resolution.Hydrogen exchange (HX) data report on the H-bond patterns and populations of extremely rare state...

logical and reporting problems were widespread, including failure to report full results, missing population details, multiple testing, and over-reliance on subgroup analysis. In summary, the complex phenotypes of OA and SDD may have made it difficult for researchers to focus their efforts. The field is dominated by isolated analyses of disparate potential associations, a problem that is amplified by the frequent analysis of different polymorphisms within individual genes. Flaws in study methodology and interpretation undoubtedly increase the risk of publication bias. Closer adherence to published recommendations (in particular those produced by HuGENet) will help to ensure that future studies are well-designed and build on current understanding, rather than simply adding to the growing bank of potential associations.

Hydrogen−deuterium exchange mass spectrometry (HDX MS) has become an important technique for the analysis of protein structure and dynamics. Data analysis remains a bottleneck in the workflow. Sophisticated computer analysis is required to scan through the voluminous MS output in order to find, identify, and validate many partially deuterated peptides, elicit the HDX information, and extend the results to higher structural resolution. We previously made available two software suites, ExMS for identification and analysis of peptide isotopic envelopes in the HDX MS raw data and HDsite for residue-level resolution. Further experience has led to advances in the usability and performance of both programs. Also, newly added modules deal with ETD/ECD analysis, multimodal mass spectra analysis, and presentation options. These advances have been integrated into a stand-alone software solution named ExMS2. The package has been successfully tested by many workers in fine scale epitope mapping, in protein folding studies, and in dissecting structure and structure change of large protein complexes. A description and tutorial for this major upgrade are given here.

Hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (HDXMS) is a rapid and effective method for localizing and determining protein stability and dynamics. Localization is routinely limited to a peptide resolution of 5 to 20 amino acid residues. HDXMS data can contain information beyond that needed for defining protein stability at single amide resolution. Here we present a method for extracting this information from an HDX dataset to generate a HDXMS protein stability fingerprint. High resolution (HR)-HDXMS was applied to the analysis of a model protein of a spectrin tandem repeat that exemplified an intuitive stability profile based on the linkage of two triple helical repeats connected by a helical linker. The fingerprint recapitulated expected stability maximums and minimums with interesting structural features that corroborate proposed mechanisms of spectrin flexibility and elasticity. HR-HDXMS provides the unprecedented ability to accurately assess protein stability at the resolution of a single amino acid. The determination of HDX stability fingerprints may be broadly applicable in many applications for understanding protein structure and function as well as protein ligand interactions.Rapid characterization of protein conformation, dynamics, and mapping of protein-ligand interface sites is increasingly being performed by employing hydrogen-deuterium exchange mass spectrometry (HDXMS) 1 . This methodology is advantageous in terms of sensitivity, speed and capability of analyzing large proteins and protein-ligand complexes 2 . HDX is pH-and temperature-dependent 3, 4 and involves the reversible exchange of backbone amide hydrogens in a protein with deuterium (D) from the solvent. HDX rates of proteins are determined by labeling of backbone amides with deuterium oxide (D 2 O) and are measured by liquid chromatography mass spectrometry (LCMS). In order for HDX to occur, the amide hydrogen must physically contact the D from the solvent through a local or global unfolding mechanism that may necessitate disruption of any hydrogen bonding.The relationship of HDX and protein structure dynamics has been well-characterized 5,6 . It is an invaluable method for interrogating protein structure fluctuations and representing localized dynamics 6 . The HDX rate of protein amides is greatly impacted by their solvent accessibility in the context of the protein structure and may vary as many as 9 orders of magnitude on the time scale [7][8][9][10] . This wide range of HDX rates represents enormous differences in the protein stability spectrum and might also be expressed as the free energy of exchange (ΔG ex ) 8 . Consequently, the stability fingerprint of a protein structure may then also be defined as the ΔG ex versus the number of amino acids.The ultimate advantages of HDXMS in many areas of life sciences 10-15 has led to a rapid expansion of this technology 1 and fostered the development of software packages for high-throughput analysis 16,17 . The HDXMS