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

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Authors: Yamaguchi S.; Hong M.

Source: Journal of Magnetic Resonance, Volume 155, Number 2, April 2002 , pp. 244-250(7)

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Abstract:

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 surface-bound peptide, were used as model systems. The peptides were ²H-labeled by ¹H/²H exchange and oriented uniaxially on glass plates. The directly detected ²H spectra of both peptides showed only a strong D₂O 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 D₂O signal in the directly detected ²H spectra was cleanly suppressed in the PRIDE spectrum. For ovispirin, the ¹H indirectly detected ²H 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 ¹⁵N 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 D₂O and with the ability to detect N–D bonds at the magic angle from the bilayer normal. © 2002 Elsevier Science (USA).