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 α-helical, while an order parameter of0.35–0.50 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 ¹³C labels using RR NMR. The exchange rates are translated into internuclear distances with a resolution on the order of 0.3 A. These experiments are similar in design to previous experiments on crystalline peptides where the ¹³C 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., & Smith, S. O. (1992) /. Am. Chem. Soc. 114, 4332–4335]. In the gpA–TM peptides, magnetization exchange rates measured between [1-¹³C]V₈₀ and [2-¹³C]G₈₃ and between [1-¹³C]M₈₁ and [2-¹³C]G₈₃ in the middle of the transmembrane sequence correspond to internuclear distances of ∼4.5 A and are consistent with a helical peptide structure. RR NMR measurements between [1-¹³C]I₉₁ and [2-¹³C]-G₉₄ also indicate a helical geometry; however, longer distances are observed between [1-¹³C]I₉₅ and [2-¹³C]-G₉₈ 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.