Molecular Dynamics-Assisted Optimization of Protein NMR Relaxation Analysis

Abstract

NMR relaxation analysis of the mobile residues in globular proteins is sensitive to the form of the experimentally fitted internal autocorrelation function, which is used to represent that motion. Different order parameter representations can precisely fit the same set of 15 N R 1 , R 2 , and heteronuclear NOE measurements while yielding significantly divergent predictions of the underlying autocorrelation functions, indicating the insufficiency of these experimental relaxation data for assessing which order parameter representation provides the most physically realistic predictions. Molecular dynamics simulations offer an unparalleled capability for discriminating among different order parameter representations to assess which representation can most accurately model a wide range of physically realistic autocorrelation functions. Six currently utilized AMBER and CHARMM force fields were applied to calculate autocorrelation functions for the backbone H–N bond vectors of ubiquitin as an operational test set. An optimized time constant-constrained triexponential (TCCT) representation was shown to markedly outperform the widely used ( S f 2 ,τ s , S 2 ) extended Lipari–Szabo representation and the more closely related ( S f 2 , S H 2 , S N 2 ) Larmor frequency-selective representation. Optimization of the TCCT representation at both 600 and 900 MHz 1 H converged to the same parameterization. The higher magnetic field yielded systematically larger deviations in the back-prediction of the autocorrelation functions for the mobile amides, indicating little added benefit from multiple field measurements in analyzing amides that lack slower (∼ms) exchange line-broadening effects. Experimental 15 N relaxation data efficiently distinguished among the different force fields with regard to their prediction of ubiquitin backbone conformational dynamics in the ps–ns time frame. While the earlier AMBER 99SB and CHARMM27 force fields underestimate the scale of backbone dynamics, which occur in this time frame, AMBER 14SB provided the most consistent predictions for the well-averaged highly mobile C-terminal residues of ubiquitin.

Novel conformational filter allows unambiguous identification of specific conformational ensembles for the large NS3pro/NS2B Dengue-associated protease; Implications for the appropriate choice of protein conformations in drug discovery

“…There is still an ongoing discussion about how the applied force field and the used computational protocols can influence the results of back calculated relaxation parameters [35][36][37][38][39][40] . We consider this discussion as outside the scope of this publication.…”

“…Although MD simulations provided information on the motions of all atoms in NS3pro/NS2B in complex with inhibitors [31][32][33] , it should be noted that the results strongly depend on the applied force field and the computational protocols 34 . It is well established that the validation of such MD simulations by experimental results is critically important [35][36][37][38][39][40] . Based on previous results 39,41 , we have here developed and employed a novel protocol for conformation filtering in which MD simulation results were compared with NMR relaxation data.…”

Parsing Dynamics of Protein Backbone NH and Side-Chain Methyl Groups using Molecular Dynamics Simulations

“…Although NMR spectroscopy allows for detailed characterization of the extent and time scales of conformational fluctuations, NMR spectroscopy rarely defines unique atomistic mechanisms needed for the full understanding of such processes. , In addition, NMR probes are limited by the isotopic labeling patterns that can be prepared efficiently (and economically), limiting the sites that can be investigated. , Finally, the time scale of overall rotational diffusion, typically tens of nanoseconds, limits the range of time scales for intramolecular conformational processes accessible to NMR spin relaxation measurements in solution. The same time scales accessible to NMR spin relaxation measurements are accessible to modern molecular dynamics (MD) simulations with trajectories lasting hundreds of nanoseconds or longer, and detailed atomistic mechanistic information is accessible because of the high resolution in both space and time of the resulting trajectories. − However, MD simulations are limited by the accuracy of molecular mechanics force fields. − Thus, discrepancies continue to persist between MD simulations and NMR measurements of protein conformational dynamics. , Despite these qualifications, recent work has used NMR spin relaxation data to modify MD force field parameters , and begun to merge analysis of NMR spin relaxation data with MD simulations. − …”