Abstract
The denaturant dependence of hydrogen–deuterium exchange (HDX) is a powerful measurement to identify the breaking of individual H-bonds and map the free energy surface (FES) of a protein including the very rare states. Molecular dynamics (MD) can identify each partial unfolding event with atomic-level resolution. Hence, their combination provides a great opportunity to test the accuracy of simulations and to verify the interpretation of HDX data. For this comparison, we use Upside, our new and extremely fast MD package that is capable of folding proteins with an accuracy comparable to that of all-atom methods. The FESs of two naturally occurring and two designed proteins are so generated and compared to our NMR/HDX data. We find that Upside’s accuracy is considerably improved upon modifying the energy function using a new machine-learning procedure that trains for proper protein behavior including realistic denatured states in addition to stable native states. The resulting increase in cooperativity is critical for replicating the HDX data and protein stability, indicating that we have properly encoded the underlying physiochemical interactions into an MD package. We did observe some mismatch, however, underscoring the ongoing challenges faced by simulations in calculating accurate FESs. Nevertheless, our ensembles can identify the properties of the fluctuations that lead to HDX, whether they be small-, medium-, or large-scale openings, and can speak to the breadth of the native ensemble that has been a matter of debate.
Highlights
Proteins populate high-energy states as determined by their free energy surfaces
All-atom molecular dynamics (MD) simulations in principle are well suited to generate Boltzmann ensembles for hydrogen−deuterium exchange (HDX) calculations.[9−14] Previously, we examined DESRES folding simulations for NuG2b, a small α/β protein, and found that the denatured state ensemble (DSE) possessed a near-native radius of gyration (Rg) and native-like H-bonding levels
To predict the larger openings and their denaturant dependence, we find it necessary to train our force field in a manner that reduces the amount of residual structure in the DSE and increases folding cooperativity
Summary
Proteins populate high-energy states as determined by their free energy surfaces. These states are often relevant in folding, catalysis, binding, conformational selection, aggregation, and allostery.[1] An ongoing challenge is to accurately calculate the free energy surface, including the generation of the Boltzmann ensemble of all major species. By providing the free energies for the breaking of individual hydrogen bonds, ΔGHX, HDX and its denaturant dependence is an excellent method to identify excited states and test the veracity of a simulated free energy surface. HDX occurs when an amide proton (NH) normally participating in an H-bond becomes exposed to solvent in a transient “open state” (Figure 1). The other, less stable H-bonds have a reduced sensitivity to denaturant, indicating that the structural opening involves only a portion of the protein
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