STRUCTURE AND ORIENTATION OF THE TRANSMEMBRANE DOMAIN OF GLYCOPHORIN-A IN LIPID BILAYERS

Rotational resonance (RR) NMR, circular dichroism (CD), and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy are used to establish the secondary structure and orientation of peptides corresponding to the transmembrane domain of human glycophorin A in dimyristoylphosphatidylcholine bilayers. An amide I vibrational frequency of 1650 cm(-1) and negative CD absorption bands at 208 and 222 nm indicate that the peptide is largely alpha-helical, while an order parameter of 0.35-0.50 in the ATR-FTIR measurements indicates that the peptide orientation is generally perpendicular to the bilayer plane. High-resolution structural data on the glycophorin A transmembrane (GPA-TM) peptides were obtained by measuring the rate of magnetization exchange between pairs of specific C-13 labels using RR NMR. The exchange rates are translated into internuclear distances with a resolution on the order of 0.3 Angstrom. These experiments are similar in design to previous experiments on crystalline peptides where the C-13 labels were incorporated into amino acids separated by 2-3 residues in the peptide sequence but close together in space due to a helical peptide geometry [Peersen, O. B., Yoshimura, S., Hojo, H., Aimoto, S., and Smith, S. O. (1992) J. Am. Chem. Sec. 114, 4332-4335]. In the GPA-TM peptides, magnetization exchange rates measured between [1-C-13]V-80 and [2-C-13]G(83) and between [1-C-13]M(81) and [2-C-13]G(83) in the middle of the transmembrane sequence correspond to internuclear distances of similar to 4.5 Angstrom and are consistent with a helical peptide structure. RR NMR measurements between [1-C-13]I-91 and [2-C-13]G(94) also indicate a helical geometry; however, longer distances are observed between [1-C-13]I-95 and [2-C-13]G(98) which argue that the transmembrane helix unravels at the membrane interface. These data demonstrate that high-resolution measurements of local protein structure can be made in lipid bilayers.