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

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 ?-helices, ?-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 M2? (AChR M2?) 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 ?-helical local secondary structure of AChR M2? 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.

SUBMITTER: Bottorf L 

PROVIDER: S-EPMC5957090 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature