Detection of drug-induced conformational change of a transmembrane protein in lipid bilayers using site-directed spin labeling

• Published in: Protein science Vol. 22; no. 1; pp. 65 - 73
• Main Authors: Thomaston, Jessica L, Nguyen, Phuong A, Brown, Emily C, Upshur, Mary Alice, Wang, Jun, DeGrado, William F, Howard, Kathleen P
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01-01-2013; Wiley Subscription Services, Inc., A Wiley Company
• Subjects: Adamantane - chemistry; Adamantane - pharmacology; Antiviral Agents - chemistry; Antiviral Agents - pharmacology; Binding Sites - drug effects; Electron Spin Resonance Spectroscopy; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Membranes; Protein Conformation - drug effects; Proteins; Spin Labels; Structure-Activity Relationship; Viral Matrix Proteins - chemistry; Viral Matrix Proteins - metabolism
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.2186

As a target of antiviral drugs, the influenza A M2 protein has been the focus of numerous structural studies and has been extensively explored as a model ion channel. In this study, we capitalize on the expanding body of high-resolution structural data available for the M2 protein to design and interpret site-directed spin-labeling electron paramagnetic resonance spectroscopy experiments on drug-induced conformational changes of the M2 protein embedded in lipid bilayers. We obtained data in the presence of adamantane drugs for two different M2 constructs (M2TM 22-46 and M2TMC 23-60). M2TM peptides were spin labeled at the N-terminal end of the transmembrane domain. M2TMC peptides were spin labeled site specifically at cysteine residues substituted for amino acids within the transmembrane domain (L36, I39, I42, and L43) and the C-terminal amphipathic helix (L46, F47, F48, C50, I51, Y52, R53, F54, F55, and E56). Addition of adamantane drugs brought about significant changes in measured electron paramagnetic resonance spectroscopy environmental parameters consistent with narrowing of the transmembrane channel pore and closer packing of the C-terminal amphipathic helices.