Structure and dynamics of full-length M2 protein of influenza A virus from solid-state NMR
Name
1023627844-MIT.pdf
Description
Full printable version
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26.01 MB
Format
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Checksum (MD5)
aa4cb886cea13d03adf7200e4e3a8c5a
Author(s)
Liao, Shu-Yu, Ph. D. Massachusetts Institute of Technology
Advisor(s)
Mei Hong.
Date Issued
2017
Publisher
Massachusetts Institute of Technology
Abstract
Solid-state nuclear magnetic resonance (SSNMR) has been frequently used to elucidate the structure and dynamics of membrane proteins and fibrils that are difficult to characterize by Xray crystallography or solution NMR. This thesis focuses on the structure determination and the proton conduction mechanism of the full-length matrix protein 2 (M2) of influenza A virus. The M2 membrane protein can be separated into three domains: an N-terminal ectodomain (1-2 1), an cc-helical transmembrane domain (TM) (22-46) connected to an amphipathic helix (AH) and a Cterminal cytoplasmic tail (63-97). The TM domain of M2 is responsible for proton conduction ant the ectodomain has been the target for vaccine development. The cytoplasmic tail has been implicated in M2 interaction with other viral proteins from mutagenesis studies. Given the importance of both N- and C-termini, it is essential to determine the structure and the dynamics of M2FL. Furthermore, we are interested in how the cytoplasmic tail affects proton conduction and the interaction of the anti-viral drug amantadine with M2 in the presence of the C-terminus. Using uniformly ¹³C, ¹⁵N-labeled M2FL, our water-selected 2D ¹³C-¹³C correlation experiment indicated that N- and C- termini are on the surface of the lipid bilayer moreover combining with chemical shift prediction, we determined that these two domains are mostly disordered. Deleting the ectodomain of M2FL (M2(21-97)) proved that a small [beta]-strand is located at the N-terminus only in the DMPC-bound state. The M2 conformation is found to be cholesterol-dependent since [beta]-strand is not found in cholesterol-rich membranes. M2(21-97) shows cationic histidine at higher pH, in contrast to M2TM, indicating that the cytoplasmic tail shifts the His37 pKa equilibria. Quantification of the ¹⁵N intensities revealed two pKa's as opposed to of four in M2TM suggesting cooperative proton binding. A possible explanation is that the large number of positively charged residues in the cytoplasmic tail facilitates proton conduction. The cytoplasmic tail was also found to restore drug-binding as amantadine no longer binds to M2(21-61) a in virus-mimetic membrane. These results have extended our understanding of the influence of the cytoplasmic domain on the structure and proton conduction of M2.
Description
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Subjects
Chemistry.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
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