Prediction and Validation of a Protein’s Free Energy Surface Using Hydrogen Exchange and (Importantly) Its Denaturant Dependence

Abstract

The denaturant dependence of hydrogen−deuterium exchange (HDX) is a powerful measurement to identify the breaking of individual Hbonds 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.

Folding stabilities of ribosome-bound nascent polypeptides probed by mass spectrometry

“…However, it is important to note that whereas pulse proteolysis reports on the global stabilities of proteins, the SPROX methodology provides measurements of local/sub-global stabilities (see also Experimental Procedures). Similar to what has been observed in native-state hydrogen-deuterium exchange (HDX) experiments 50 , solvent exposure of most protein regions can occur by localized unfolding events that are more energetically favorable than global unfolding. Thus, localized thermodynamic folding stabilities of most protein regions are expected to be lower and differentially impacted by the ribosome in comparison to global folding stabilities.…”

“…Where U unox and F unox are the folded and unfolded conformations of the protein harboring an unoxidized methionine residue, U ox and F ox are conformations harboring the oxidized methionine residue, and and are the oxidation rate constants from the unfolded “4 ”4 and folded states, respectively. Akin to the kinetic model that is commonly used to inerpret hydrogen-deuterium exchange (HDX) experiments 50 , the observed rate constant for oxidation can be expressed as: In a folded protein, k f >> k u , and hence: If we assume that a methionine is mostly protected from oxidation, then is negligible and: If we consider a protein that can exist in either RNC or soluble states, the ratio of oxidation rates in these two states is related to their respective folding equilibria as follows: Where ΔΔ Gfolding is the difference in folding free energies between the two states, R is the gas constant, and T is the absolute temperature in Kelvin.…”

Computational Modeling of Molecular Structures Guided by Hydrogen-Exchange Data

“…Finally, Peng et al predict protection from HDX using a model based on hydrogen bonding and backbone burial. 171 As their predictions are derived from conformational ensembles generated by their in-house coarse-grained modeling approach, called Upside, the HDX model is also defined in a coarsegrained manner. Specifically, the burial level of an amide nitrogen is assessed through contributions from backbone heavy atoms and side-chain beads.…”

Sequential Activation and Local Unfolding Control Poly(A)-Binding Protein Condensation

“…HDX of Peptide 418-442 from P-domain was slowed for half of the molecules. CTD’s helix 2 (Peptide 521-535 , part of the CTD’s peptide binding site 45 ) was unfolded for the majority of the molecules, which potentially led to a disruption of the hydrophobic core and destabilization of other CTD regions (Fig. 3a).…”

“…Data were collected and analyzed using RAW 40 and GNOM 41 with the Rg values obtained from the pair-distance distribution function, P(r), that were calculated using an indirect Fourier transform of the scattering data. The simulated conformational ensembles were created using the Upside molecular dynamics algorithm [42][43][44] .…”