Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Protein-protein interactions are essential for life. Yet, our understanding of the general principles governing binding is not complete. In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules. The boundaries between modules are defined by clustering the contact map of the interface. We show that mutations in one module do not affect residues located in a neighboring module. As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small. To the contrary, within their module, mutations cause complex energetic and structural consequences. Experimentally, this phenomenon is shown on the interaction between TEM1-␤-lactamase and ␤-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography. Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface. The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved.cluster analysis ͉ binding energy ͉ protein-protein interaction ͉ structure T he evolution of protein-protein binding sites is a result of optimization with three boundary conditions: the definition of the complex structure, optimization of the binding affinity for a given task, and the generation of specificity. Thermodynamic, kinetic, and structural studies of protein-protein interactions have taught us much of how proteins interact rapidly, tightly, and in a specific manner. These results have been translated into the development of in silico tools to calculate binding free energies, to weigh the energetic consequence of mutations, and for protein design (1-4). However, from the modest degree of success of the aforementioned methods, it is clear that our understanding of protein-protein interactions is still lacking (5).The most popular approach for studying the energetic contribution of an amino acid to the free energy of binding (or stability) of proteins is to introduce a mutation and then measure the subsequent change in free energy (6). However, accurate interpretation of the results of such experiments is limited by the fact that the properties of a protein are a function of the entire system, and not necessarily the sum of its parts (7). As a result, the energetic impact of any given residue on binding is not a simple additive quantity (8). A more elaborate approach to study the contributions of amino acids to binding and stability involves the use of double and higher-order mutant cycles, where interactions between amino acids are treated within their native contexts (7, 9). Such cycles reveal whether the contributions from a pair of residues are additive, or whether the effects of mutations are coupled (10, 11).Cha...

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SUMMARY The redox-regulated chaperone Hsp33 protects organisms against oxidative stress that leads to protein unfolding. Activation of Hsp33 is triggered by the oxidative unfolding of its own redox-sensor domain, making Hsp33 a member of a recently discovered class of chaperones that require partial unfolding for full chaperone activity. Here we address the long-standing question of how chaperones recognize client proteins. We show that Hsp33 uses its own intrinsically disordered regions to discriminate between unfolded and partially structured folding intermediates. Binding to secondary structure elements in client proteins stabilizes Hsp33’s intrinsically disordered regions, and this stabilization appears to mediate Hsp33’s high affinity for structured folding intermediates. Return to nonstress conditions reduces Hsp33’s disulfide bonds, which then significantly destabilizes the bound client proteins and in doing so converts them into less-structured, folding-competent client proteins of ATP-dependent foldases. We propose a model in which energy-independent chaperones use internal order-to-disorder transitions to control substrate binding and release.

Some of the most challenging stress conditions that organisms encounter during their lifetime involve the transient accumulation of reactive oxygen and chlorine species. Extremely reactive to amino acid side chains, these oxidants cause widespread protein unfolding and aggregation. It is therefore not surprising that cells draw on a variety of different strategies to counteract the damage and maintain a healthy proteome. Orchestrated largely by direct changes in the thiol oxidation status of key proteins, the response strategies involve all layers of protein protection. Reprogramming of basic biological functions helps decrease nascent protein synthesis and restore redox homeostasis. Mobilization of oxidative stress-activated chaperones and production of stress-resistant non-proteinaceous chaperones prevent irreversible protein aggregation. Finally, redox-controlled increase in proteasome activity removes any irreversibly damaged proteins. Together, these systems pave the way to restore protein homeostasis and enable organisms to survive stress conditions that are inevitable when living an aerobic lifestyle.