Worldwide epidemics of the flu caused by the Influenza A virus lead to thousands of deaths every year, making medical research on drug discovery and vaccine development indispensable. Accordingly, research on the viral machinery and its inhibition is on-going and remains highly relevant. One Influenza A protein that has been of great research interest is the small proton channel matrix protein 2 (M2), which is responsible for acidifying the viral interior during virus entry. Understanding the molecular mechanism of M2 is, on the one hand, essential for antiviral drug development, since inhibitors of the protein have already been identified. On the other hand, it also positions M2 as a model protein for small channel proteins with similar function and structure found in many human-pathogenic viruses, the family of viroporins. The aim of this thesis is to investigate the proton channel mechanism of M2, specifically its opening motion and the influence of carboxylate protonation on this process, by combining infrared spectroscopic and computational methods. To quantify the opening motion of the channel, a combined surface-enhanced infrared absorption (SEIRA) and density functional theory (DFT) approach was applied. To assess the role of carboxylate protonation, pH-dependent SEIRA experiments were complemented by DFT calculations and molecular dynamics (MD) simulations, revealing mutation-induced structural changes and their effect on the opening mechanism. MD simulations also enabled spectral predictions of M2 at different protonation states, allowing comparison with experimental spectra. The combined SEIRA und DFT approach revealed that M2 opens by 17 ± 2° with a three-step transition in a pH range of pH 8 to around pH 4. Interestingly, carboxylate protonation events were found to contribute to these transitions. Mutations of Asp24 and also Asp44 led to changes in both structure and opening mechanism of the protein, where pKA values of the transition were either shifted or completely absent in the variants. This indicates that the role of these aspartates in the mechanism has been underestimated in previous studies. MD simulations showed that structural heterogeneity might play a role in the mechanism as it is influenced by amino acid protonation and also by Asp24 and Asp44 mutation. Furthermore, MD-derived spectra were compared to experimental spectral features in their wavenumber positions, validating the MD as a complementary approach to DFT-based spectroscopy to study viroporins. Finally, cell-free expression was explored for M2 as a model system to establish a method applicable to structurally similar, human-pathogenic viral proteins, which often require high biosafety levels for in-cell expression. Cell-free expression experiments showed promise in expressing and inserting M2 into membranes, but further condition screening is needed to achieve fully active proteins routinely. The results of this work demonstrate that a combined computational and spectroscopic approach can reveal detailed insights into the opening mechanism of the M2 proton channel. The findings suggest that the opening motion is functionally relevant and potentially less symmetrical than previously assumed. It was also shown that the aspartates, and especially Asp24, play a more significant role in the channel mechanism than previously thought. Understanding this mechanism and establishing methods to study it may pave the way for future medical research on influenza A and other human-pathogenic viruses with similar proton channels.
