Date of Award
© 2017 D. Stuart Arbuckle Jr. All rights reserved. This work is freely available courtesy of the author. It may only be used for non-commercial, educational, and research purposes. For all other uses, including reproduction and distribution, please contact the copyright holder.
Bachelor of Arts
Chemistry & Biochemistry
Kathleen P. Howard
Matrix 2 (M2) is a homotetrameric integral membrane protein of the influenza A virus demonstrated to be required for generation of membrane curvature resulting in viral budding. The C-terminal amphipathic helix of M2 has been shown to generate negative Gaussian membrane curvature, required at the neck of a budding virion, dependent on membrane composition. In this thesis, electron paramagnetic resonance (EPR) spectroscopy is used to investigate the conformational dynamics of M2's amphipathic helix in liposomes consisting of DOPE and DOPC, lipids of varying intrinsic curvature propensity, as well as cholesterol enrichment. The amphipathic helix is demonstrated to populate two conformations, one of which is stabilized by lipids of high intrinsic curvature propensity or cholesterol. This conformation affects the structure of the amphipathic helix and the transmembrane domain, and is hypothesized to be budding-relevant. Additionally, mutations that alter the budding function of M2 by reducing the hydrophobicity of the amphipathic helix are investigated. Alterations to the helix abolish conformational exchange in response to membrane intrinsic curvature, but not cholesterol, consistent with a role for the amphipathic helix in sensing and responding to membrane curvature properties and binding cholesterol. Changes to the preferred structure as a result of amphipathic helix mutations may explain the recent finding that M2's transmembrane domain is capable of limited curvature generation. The mutated amphipathic helix's conformational dynamics are consistent with a canonical curvature generating/sensing quasi-ALPS motif. The membrane-protein interaction is discussed in the context of the flexible surface model and the spontaneous curvature frustration mechanism, which point to a hydrophobic insertion role for the amphipathic helix that explains lipid-dependent curvature generation.
Arbuckle, D. Stuart Jr, '17, "EPR Studies of Curvature-Dependent Conformational Equilibrium in Full-Length Influenza A M2 Protein" (2017). Senior Theses, Projects, and Awards. 224.