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Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy.

Authors
  • Bottorf, Lauren1
  • Sahu, Indra D1
  • McCarrick, Robert M1
  • Lorigan, Gary A2
  • 1 Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, United States. , (United States)
  • 2 Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, United States. Electronic address: [email protected] , (United States)
Type
Published Article
Journal
Biochimica et Biophysica Acta (BBA) - Biomembranes
Publisher
Elsevier
Publication Date
Jul 01, 2018
Volume
1860
Issue
7
Pages
1447–1451
Identifiers
DOI: 10.1016/j.bbamem.2018.04.001
PMID: 29694834
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Electron spin echo envelope modulation (ESEEM) spectroscopy in combination with site-directed spin labeling (SDSL) has been established as a valuable biophysical technique to provide site-specific local secondary structure of membrane proteins. This pulsed electron paramagnetic resonance (EPR) method can successfully distinguish between α-helices, β-sheets, and 310-helices by strategically using 2H-labeled amino acids and SDSL. In this study, we have explored the use of 13C-labeled residues as the NMR active nuclei for this approach for the first time. 13C-labeled d5-valine (Val) or 13C-labeled d6-leucine (Leu) were substituted at a specific Val or Leu residue (i), and a nitroxide spin label was positioned 2 or 3 residues away (denoted i-2 and i-3) on the acetylcholine receptor M2δ (AChR M2δ) in a lipid bilayer. The 13C ESEEM peaks in the FT frequency domain data were observed for the i-3 samples, and no 13C peaks were observed in the i-2 samples. The resulting spectra were indicative of the α-helical local secondary structure of AChR M2δ in bicelles. This study provides more versatility and alternative options when using this ESEEM approach to study the more challenging recombinant membrane protein secondary structures. Copyright © 2018 Elsevier B.V. All rights reserved.

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