Probing the Secondary Structure of Membrane Peptides Using H-2-Labeled d(10)-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 H-2 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 H-2-labeled, such as H-2-labeled d(10)-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 H-2-labeled d(10)-Leu, four Leu residues within the AChR M2 delta peptide were fully mapped out using this ESEEM method. Unique H-2-ESEEM patterns were observed with the H-2-labeled d(10)-Leu for the AChR M2 delta alpha-helical model peptide. For proteins and peptides with an alpha-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 H-2 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 alpha-helix from a pi-helix or 3(10)-helix. Moreover, H-2 modulation depth for ESEEM samples on Leul0 were significantly enhanced which was consistent with a kinked or curved structural model of the AChR M2 delta peptide as suggested by previous MD simulations and NMR experiments.