Selection pressures on evolution of ribonuclease H explored with rigorous free–energy–based design

Abstract

Understanding natural protein evolution and designing novel proteins are motivating interest in development of high-throughput methods to explore large sequence spaces. In this work, we demonstrate the application of multisite λ dynamics (MSλD), a rigorous free energy simulation method, and chemical denaturation experiments to quantify evolutionary selection pressure from sequence–stability relationships and to address questions of design. This study examines a mesophilic phylogenetic clade of ribonuclease H (RNase H), furthering its extensive characterization in earlier studies, focusing on E. coli RNase H (ecRNH) and a more stable consensus sequence (AncCcons) differing at 15 positions. The stabilities of 32,768 chimeras between these two sequences were computed using the MSλD framework. The most stable and least stable chimeras were predicted and tested along with several other sequences, revealing a designed chimera with approximately the same stability increase as AncCcons, but requiring only half the mutations. Comparing the computed stabilities with experiment for 12 sequences reveals a Pearson correlation of 0.86 and root mean squared error of 1.18 kcal/mol, an unprecedented level of accuracy well beyond less rigorous computational design methods. We then quantified selection pressure using a simple evolutionary model in which sequences are selected according to the Boltzmann factor of their stability. Selection temperatures from 110 to 168 K are estimated in three ways by comparing experimental and computational results to evolutionary models. These estimates indicate selection pressure is high, which has implications for evolutionary dynamics and for the accuracy required for design, and suggests accurate high-throughput computational methods like MSλD may enable more effective protein design.

How to Sample Dozens of Substitutions per Site with λ Dynamics

“…62 For neutral boxes, the discrete solvent correction is the dominant term, so we apply it to PME calculations, following previous work. 10,37,40 Comparing results obtained from 21 pairwise simulations run without θ biases to results obtained from the full 22 substituent simulations with θ biases reveals very close agreement (Figures 6A & S4A & Table 4). The centered root mean squared differences are quite small for ΔG in both the folded and unfolded ensembles and for the stability ΔΔG fold .…”

“…Experimental studies of all possible mutations at four interacting sites are common, 64,65 and larger numbers of sites would be of interest if they were accessible. Previous studies with λ dynamics have focused on making robust free energy predictions for large numbers of sites, 37,40 and together with the present work, they enable exploration of all possible amino acids at each of those sites simultaneously.…”

“…Several mutations include charge changes, and correction terms for charge changes with PME electrostatics have been developed . For neutral boxes, the discrete solvent correction is the dominant term, so we apply it to PME calculations, following previous work. ,, …”

Massively parallel free energy calculations forin silicoaffinity maturation of designed miniproteins

“…An assessment of the quality of free energy methods such as EE for large-scale mutational prediction is timely, as many new methods are being developed to examine protein fitness landscapes through the combination of free energy approaches and machine learning. [11, 13, 45]…”

“…Meanwhile, alchemical free energy approaches utilizing all-atom simulations have shown remarkable success in predicting relative binding affinities. [5][6][7][8][9]Recent benchmarks from free energy perturbation (FEP), [10,11] non-equilibrium work, [12] and multisite λ dynamics [13] approaches suggest that relative binding free energies (RBFE) for single-residue mutations in protein-protein interfaces can be predicted within an accuracy of 2 kcal/mol. Can all-atom free energy approaches more accurately predict SSMs?…”