Proton Transport Mechanisms in the Influenza A M2 Channel and De Novo Designed Proteins

Proton transport (PT) in biomolecular systems is the controlled movement of hydrogen ions across a cellular membrane, frequently through a channel or transporter, and is essential for sustaining life. PT is necessary for a variety of functions including maintaining pH gradients, driving ATP synthesis, and facilitating the co- or anti-transport of other small molecules, among many others. Because of their crucial role, protein channels and transporters are often chosen as drug targets in bacteria or viruses that cause disease. Thus, beyond elucidating PT mechanisms to understand how a specific channel or transporter works, studying the detailed interactions that facilitate PT is critical as it can inform drug design efforts to help treat disease, and it can reveal fundamental principles of PT that can then be used to control this function in designed systems. In this thesis, I use computer simulation to study processes of PT in two systems, the influenza A M2 channel and two de novo designed proteins. Through three different projects focused on M2, I provide a detailed view on PT in M2, deducing important interactions and providing insight into inhibitor binding. First, using extensive Multiscale Reactive Molecular Dynamics (MS-RMD) simulations with explicit Grotthuss-shuttling hydrated excess protons, I show how a hydrated excess proton strongly influences both the protein and water hydrogen-bonding network throughout the channel, providing further insight into the channel’s acid-activation mechanism and rectification behavior. Second, I extend this work to focus on inhibitor binding. In this work, I illuminate a dynamic understanding of the mechanism of drug inhibition in M2, grounded in the fundamental properties that enable the channel to transport and stabilize excess protons, with critical implications for future drug design efforts. Third, I use combined MS-RMD and quantum mechanics/molecular mechanics simulations to calculate the potential of mean force (PMF) of PT in a prominent mutant of M2, showing how the D44N mutation alters the free energy and mechanism of PT. Finally, I use these methods to study two de novo designed proteins in collaboration with experimental experts in protein design. In this work, our collaborators find that introducing a single polar residue into an otherwise hydrophobic channel is sufficient to induce proton conduction. Using MS-RMD simulations, I calculate 2D PMFs of PT for two systems, one with the mutation and one without, to understand how the mutation facilitates PT. This work reveals fundamental characteristics of PT and provides insight into how it can be harnessed in future protein design. Altogether, the work in this thesis presents a dynamic picture of proton transport in proteins and reveals key characteristics that are critical for understanding PT in biological systems overall, with implications for future drug design efforts and de novo protein design.