Electron Paramagnetic Resonance Studies of Functionally Active, Nitroxide Spin-Labeled Peptide Analogues of the C-Terminus of a G-Protein α Subunit

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• 作者：Ned Van Eps ; Lori L. Anderson ; Oleg G. Kisselev ; Thomas J. Baranski ; Wayne L. Hubbell ; Garland R. Marshall
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：August 17, 2010
• 年：2010
• 卷：49
• 期：32
• 页码：6877-6886
• 全文大小：1043K
• 年卷期：v.49,no.32(August 17, 2010)
• ISSN：1520-4995

文摘

The C-terminal tail of the transducin α subunit, Gtα(340−350), is known to bind and stabilize the active conformation of rhodopsin upon photoactivation (R*). Five spin-labeled analogues of Gtα(340−350) demonstrated native-like activity in their ability to bind and stabilize R*. The spin-label 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was employed at interior sites within the peptide, whereas a Proxyl (3-carboxyl-2,2,5,5-tetramethyl-pyrrolidinyloxy) spin-label was employed at the amino terminus of the peptide. Upon binding to R*, the electron paramagnetic resonance spectrum of TOAC³⁴³-Gtα(340−350) revealed greater immobilization of the nitroxide when compared to that of the N-terminally modified Proxyl-Gtα(340−350) analogue. A doubly labeled Proxyl/TOAC³⁴⁸-Gtα(340−350) was examined by DEER spectrocopy to determine the distribution of distances between the two nitroxides in the peptides when in solution and when bound to R*. TOAC and Proxyl spin-labels in this GPCR−G-protein α-peptide system provide unique biophysical probes that can be used to explore the structure and conformational changes at the rhodopsin−G-protein interface.