Molecular Dynamics-Assisted Optimization of Protein NMR Relaxation Analysis

NMR relaxation analysis of the mobile residues inglobular proteins is sensitive to the form of the experimentallyfitted internal autocorrelation function, which is used to representthat motion. Different order parameter representations canpreciselyfit the same set of15NR1,R2, and heteronuclear NOEmeasurements while yielding significantly divergent predictions ofthe underlying autocorrelation functions, indicating the insuffi-ciency of these experimental relaxation data for assessing whichorder parameter representation provides the most physicallyrealistic predictions. Molecular dynamics simulations offer anunparalleled capability for discriminating among different orderparameter representations to assess which representation can mostaccurately model a wide range of physically realistic autocorrelation functions. Six currently utilized AMBER and CHARMM forcefields 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(Sf2,Tau s,S2) extended Lipari-Szabo representation and the more closely related (Sf2,SH2,SN2) Larmor frequency-selective representation.Optimization of the TCCT representation at both 600 and 900 MHz1H converged to the same parameterization. The highermagneticfield yielded systematically larger deviations in the back-prediction of the autocorrelation functions for the mobile amides,indicating little added benefit from multiplefield measurements in analyzing amides that lack slower (similar to ms) exchange line-broadening effects. Experimental15N relaxation data efficiently distinguished among the different forcefields with regard to theirprediction of ubiquitin backbone conformational dynamics in the ps-ns time frame. While the earlier AMBER 99SB andCHARMM27 forcefields underestimate the scale of backbone dynamics, which occur in this time frame, AMBER 14SB provided themost consistent predictions for the well-averaged highly mobile C-terminal residues of ubiquitin.