(746j) Replica-Exchange on-the-Fly Parameterization:  Application of a High-Precision Free-Energy Method to Understanding the Roles of the M129V/D178N Polymorphisms in the Conformational Thermodynamics of Human Prion Protein

Barriers in the sampling of â??hiddenâ? variables constitute the most important limitations on accuracy and precision of free-energy landscapes computed using collective-variable (CV) biasing approaches, such as thermodynamic integration, umbrella sampling, Metadynamics, adaptive-biasing force calculations, and others. So-called orthogonal-space sampling variants of these methods attack this issue using additional biasing, but they arenâ??t always guaranteed to find all relevant hidden variables. This problem is particularly acute in situations where one wishes to understand changes in protein conformational thermodynamics induced by substitutions of amino acid side-chains that evidently do not result in changes to any specific interactions (no net loss or gain of H-bonds, salt-bridges, dipole-dipole interactions, etc.). A prominent example is the M/V polymorphism at position 129 of the human prion protein (hPrP), which has recently been shown to be a major determinant of a variety of histologically and clinically distinct phenotypes of transmissible spongiform encephalopathies (TSEs). We present here a new free-energy method termed Replica-Exchange On-the-Fly Parameterization (REOTFP), which combines the advantages of history-free CV-biasing of temperature-accelerated MD with the general hyperthermal enhanced sampling of replica-exchange MD, to overcome the hidden-variables problem. We show that REOTFP can produce free-energy profiles precise enough to rationalize effects of the M/V-129 polymorphism of hPrP. In particular, we use REOTFP to measure the thermodynamic stability of the N-terminal β-sheet of native hPrP and show that V129 causes loss of a low-stability metastable state that may correlate to a phenotype protective against TSE acquisition. Additionally, in the presence of the heritable-TSE-associated mutation D178N, REOTFP predicts that V129 causes acquisition of this metastable intermediate, which may underlie the distinct natures of inherited Creutzfeldt-Jakob disease and Fatal Familial Insomnia, two different TSEâ??s that seem to owe their divergence solely to the M/V polymorphism at position 129. We compare the performance of REOTFP to both serial OTFP and well-tempered Metadynamics and explain the basis of REOTFPâ??s superiority in terms of specific hidden variables that it efficiently samples with no additional bias. On a per-unit-error basis, REOTFP is vastly less expensive than virtually any serial free-energy method to date, and should therefore open new pathways of inquiry previously off-limits to existing free-energy methods.