ABSTRACT
Electron spin echo envelope modulation (ESEEM) spectroscopy was used to distinguish between the local secondary structures of an α-helix and a 310-helix. Previously, we have shown that ESEEM spectroscopy in combination with site-directed spin labeling (SDSL) and 2H-labeled amino acids (i) can probe the local secondary structure of α-helices, resulting in an obvious deuterium modulation pattern, where i+4 positions generally show larger 2H ESEEM peak intensities than i+3 positions. Here, we have hypothesized that due to the unique turn periodicities of an α-helix (3.6 residues per turn with a pitch of 5.4 Å) and a 310-helix (3.1 residues per turn with a pitch of 5.8-6.0 Å), the opposite deuterium modulation pattern would be observed for a 310-helix. In this study, 2H-labeled d10-leucine (Leu) was substituted at a specific Leu residue (i) and a nitroxide spin label was positioned 2, 3, and 4 residues away (denoted i+2 to i+4) on an amphipathic model peptide, LRL8. When LRL8 is solubilized in trifluoroethanol (TFE), the peptide adopts an α-helical structure, and alternatively, forms a 310-helical secondary structure when incorporated into liposomes. Larger 2H ESEEM peaks in the FT frequency domain data were observed for the i+4 samples when compared to the i+3 samples for the α-helix whereas the opposite pattern was revealed for the 310-helix. These unique patterns provide pertinent local secondary structural information to distinguish between the α-helical and 310-helical structural motifs for the first time using this ESEEM spectroscopic approach with short data acquisition times (â¼30 min) and small sample concentrations (â¼100 µM) as well as providing more site-specific secondary structural information compared to other common biophysical approaches, such as CD.
