Measuring hydrogen exchange rates in invisible protein excited states

Abstract

Hydrogen exchange rates have become a valuable probe for studying the relationship between dynamics and structure and for dissecting the mechanism by which proteins fold to their native conformation. Typically measured rates correspond to averages over all protein states from which hydrogen exchange can occur. Here we describe a new NMR experiment based on chemical exchange saturation transfer that provides an avenue for obtaining uncontaminated, per-residue amide hydrogen exchange rates for interconverting native and invisible states so long as they can be separated on the basis of distinct 15 N chemical shifts. The approach is applied to the folding reaction of the Fyn SH3 domain that exchanges between a highly populated, NMR-visible native state and a conformationally excited, NMR-invisible state, corresponding to the unfolded ensemble. Excellent agreement between experimentally derived hydrogen exchange rates of the excited state at a pair of pHs is obtained, taking into account the expected dependence of exchange on pH. Extracted rates for the unfolded ensemble have been used to test hydrogen exchange predictions based on the primary protein sequence that are used in many analyses of solvent exchange rates, with a Pearson correlation coefficient of 0.84 obtained.amide exchange with solvent | conformationally excited protein states | protein folding T he energy landscape of a protein is a multidimensional surface composed of many local minima in addition to the global minimum that is the native conformation (1, 2). An understanding of the relation between protein structure, dynamics, and function is predicated, therefore, on an analysis of the various conformational states that populate the minima on the landscape. This requires detailed structural and dynamics studies of each of the conformers and quantification of their relative energies as well as the kinetics of exchange between them. Biophysical techniques such as X-ray diffraction and NMR spectroscopy are available for obtaining detailed structural information on the molecules populating the lowest-energy regions of the landscape, providing insight into the structure-function paradigm for a great number of proteins. However, it is becoming increasingly well understood that studies of the ground states of proteins are not sufficient. Additional states that can be sparsely populated and transiently formed, referred to as excited states in what follows, are often important for processes that include molecular recognition, ligand binding, enzyme catalysis, and folding (3-9). Detailed studies of such excited states are, however, challenging because they are not "visible" to standard biophysical methods and as a consequence atomic resolution information is lacking.Recent developments in solution NMR spectroscopy are changing this paradigm by providing an avenue for quantifying excited states at a level of detail that has typically only been possible in studies of highly populated native protein conformers (10). Backbone 1 H, 13 C, and 15 N chemical shifts fo...

Benchmarking all-atom simulations using hydrogen exchange

“…This mild difference could reflect residual structure, although it is at the level of our statistical error and also could result from minor errors in pH and T, or the inaccuracy of the linear extrapolation. Recently, the same energy difference was observed for the Fyn SH3 domain by Kay and coworkers (28), who likewise interpreted HX as occurring from an unstructured state because the sites exhibited random coil chemical shifts in the DSE.…”

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“…The exchange timescale accessible to R 1 ρ is broader than CPMG (~60 s −1 < k ex < ~100,000 s −1 ) (Palmer & Massi, 2006) and for slow-intermediate exchange, the sign of excited state chemical shift sign can deduced at a single magnetic field strength (Trott et al, 2002). For processes occurring at even slower timescales (~20 s −1 < k ex < ~300 s −1 ) chemical-exchange saturation transfer (CEST) experiments employing weak RF spin-lock fields have recently been shown to be a robust approach to characterize lowly populated conformational states in both proteins and nucleic acids (Fawzi et al, 2011; Vallurupalli et al, 2012; Long et al, 2014; Zhao et al, 2014). …”

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“…Methods have also been introduced to directly obtain structural constraints on the ES including orientational constraints from measurements of residual dipolar couplings (RDCs) 23–25 , residual chemical shift anisotropies (RCSAs) 26,27 , and distance-based constraints from paramagnetic relaxation enhancements (PRE) 28,29 . Methods have also been developed to gain insights into the dynamic properties of protein ESs that rely on the analysis of chemical shifts 30 and H/D exchange 31 . Recently, CEST-based methods have also been introduced to measure RDCs in RNA ESs paving the way for atomic resolution structure characterization 32 .…”