| Autor: |
Kim SS; Department of Chemistry and Biochemistry, Swarthmore College , Swarthmore, Pennsylvania 19081, United States., Upshur MA; Department of Chemistry and Biochemistry, Swarthmore College , Swarthmore, Pennsylvania 19081, United States., Saotome K; Department of Chemistry and Biochemistry, Swarthmore College , Swarthmore, Pennsylvania 19081, United States., Sahu ID; Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States., McCarrick RM; Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States., Feix JB; Department of Biophysics, National Biomedical EPR Center, Medical College of Wisconsin , Milwaukee, Wisconsin 53226, United States., Lorigan GA; Department of Chemistry and Biochemistry, Miami University , Oxford, Ohio 45056, United States., Howard KP; Department of Chemistry and Biochemistry, Swarthmore College , Swarthmore, Pennsylvania 19081, United States. |
| Abstrakt: |
The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol-dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry-related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol-dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all of our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding. |
