Constant-pH Molecular Dynamics and Applications

QC 2024-05-16

Abstract

Although it has been long known that pH plays an important role in biology, it was only in the 20th century that people first realized that pH can greatly affect the structure and function of proteins on a molecular level. A commonly encountered problem when studying the effect of pH in proteins is that experimental techniques such as X-ray crystallography and cryogenic electron microscopy often lack the resolution required to determine the protonation states of titratable residues. Even with neutron diffraction methods, which can theoretically resolve protons, the electrostatic environment in a crystal or grid does not necessarily correspond to the cellular environment. Where experimental methods fall short, in silico approaches such as molecular dynamics (MD) simulations can provide additional insight. However, pH is not typically captured dynamically in classical MD simulations. Rather, the protonation states of titratable sites such as aspartic and glutamic acid are usually set at the start of the simulation and remain constant throughout the run. Despite significant efforts in theMDfield to model protonation more realistically, a comprehensive and efficient constant-pH implementation in GROMACS has so far been lacking.

The aim of this work was to implement a continuous constant-pH method in GROMACS, to improve the ease of use of such methodology for the GROMACS user community, and to apply such methodology to study pH-gated ion channels. To this end, the work presented in this thesis can be divided into three parts. First, in papers I and II, I discuss the implementation of a λ-dynamics-based constant-pH algorithm in GROMACS, including several important methodological aspects. Next, in paper III, I present phbuilder, a Python-based simulation builder tool that automates the often complicated, tedious, and error-prone set up process of constant-pH simulations in GROMACS. Finally, in papers IV and V, I investigate how the constant-pH method can be used to better understand the pH gating mechanism in the bacterial GLIC and sTeLIC ligand-gated ion channels.

The work presented in this thesis has contributed to both the methodological and application sides in the field of MD. I have helped to develop and implement a λ-dynamics based constant-pH method in GROMACS, and I have made such methodology available to the broader GROMACS user community by providing a tool that automates its setup process. On the application side, I have demonstrated the feasibility of the constant-pH implementation for studying large and complex protein systems, such as ion channels, and furthered our understanding of pH-mediated gating in GLIC and sTeLIC.

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