Date of Award
Doctor of Philosophy (PhD)
Biochemistry & Molecular Biophysics
William F. DeGrado
Influenza A/M2 is a minimalistic integral membrane protein that mediates proton transport across the viral membrane and is of interest as an antiviral drug target. This protein has been extensively studied by electrophysiologists, biophysicists, structural biologists, and medicinal chemists, but a synthesis and unified extension of the knowledge bases from these fields has not been undertaken. The principal aim of this thesis is to develop a comprehensive, quantitative, structure-based mechanism that accounts for the key functional and biophysical properties of A/M2. To demonstrate the electrophysiological equivalence of the protein’s transmembrane domain (M2TM) and full length A/M2, proteoliposome flux experiments are conducted as a first step. Next, high-resolution crystals of an M2TM variant are obtained; the resulting structure and computational simulations provide a structural basis for the unusually high degree of charge stabilization inside the M2TM helical bundle, suggest a previously unseen mechanism used by Nature to stabilize charge in a membrane, and shed light on the likely pH-dependent structural transitions that the protein undergoes. Fluorescence quenching and EPR spectroscopy experiments confirm that M2 reconstituted in bilayers undergoes pH-driven changes in its conformational equilibrium that are consistent with available structures and governed by previously reported pKa values. Mechanistic models of this process are constructed and successfully fit to functional data. The fitting results show that proton transport and rectification are mediated by conformational transitions between structural ensembles with different proton affinities. Finally, the functional implications of targeted changes to the geometric and electronic properties of the key His 37 sidechain are observed, indicating that the shape of the His 37 imidazole rings is exquisitely tuned to mediate proton transport.
Polishchuk, Alexei L., "Structure-based mechanism of proton transport through the influenza A M2 protein" (2009). Publicly Accessible Penn Dissertations. 298.