Probing the Secondary Structure of Membrane Peptides Using 2H-Labeled d10-Leucine via Site-Directed Spin-Labeling and Electron Spin Echo Envelope Modulation Spectroscopy

摘要

Previously, we reported an electron spin echo envelope modulation (ESEEM) spectroscopic approach for probing the local secondary structure of membrane proteins and peptides utilizing ²H isotopic labeling and site-directed spin-labeling (SDSL). In order to probe the secondary structure of a peptide sequence, an amino acid residue (i) side chain was ²H-labeled, such as ²H-labeled d10-Leucine, and a cysteine residue was strategically placed at a subsequent nearby position (denoted as i + 1 to i + 4) to which a nitroxide spin label was attached. In order to fully access and demonstrate the feasibility of this new ESEEM approach with ²H-labeled d10-Leu, four Leu residues within the AChR M2δ peptide were fully mapped out using this ESEEM method. Unique ²H-ESEEM patterns were observed with the ²H-labeled d10-Leu for the AChR M2δ α-helical model peptide. For proteins and peptides with an α-helical secondary structure, deuterium modulation can be clearly observed for i ± 3 and i ± 4 samples, but not for i ± 2 samples. Also, a deuterium peak centered at the ²H Larmor frequency of each i ± 4 sample always had a significantly higher intensity than the corresponding i + 3 sample. This unique feature can be potentially used to distinguish an α-helix from a π-helix or 310-helix. Moreover, ²H modulation depth for ESEEM samples on Leu10 were significantly enhanced which was consistent with a kinked or curved structural model of the AChR M2δ peptide as suggested by previous MD simulations and NMR experiments.