Determination of Membrane Peptide Orientation by 1H-Detected 2H NMR Spectroscopy

✍ Scribed by Satoru Yamaguchi; Mei Hong

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 144 KB
• Volume: 155
• Category: Article
• ISSN: 1090-7807
• DOI: 10.1006/jmre.2002.2517

✦ Synopsis

We demonstrate the application of the proton inverse detected deuteron (PRIDE) NMR technique to the measurement of the orientation of membrane-bound peptides with enhanced sensitivity. Gramicidin D, a transmembrane peptide, and ovispirin, a surfacebound peptide, were used as model systems. The peptides were 2 Hlabeled by 1 H/ 2 H exchange and oriented uniaxially on glass plates. The directly detected 2 H spectra of both peptides showed only a strong D 2 O signal and no large quadrupolar splittings. In contrast, the PRIDE spectrum of gramicidin exhibited quadrupolar splittings as large as 281 kHz, consistent with its transmembrane orientation. Moreover, the large D 2 O signal in the directly detected 2 H spectra was cleanly suppressed in the PRIDE spectrum. For ovispirin, the 1 H indirectly detected 2 H spectrum revealed a 104 kHz splitting and a zero-frequency peak. The former reflects the in-plane orientation of most of the helix axis, while the latter results from residues with a magic-angle orientation of the N-D bonds. These are consistent with previous 15 N NMR results on ovispirin. The combination of PRIDE and exchange labeling provides an economical and sensitive method of studying membrane peptide orientations in lipid bilayers without the influence of D 2 O and with the ability to detect N-D bonds at the magic angle from the bilayer normal.