Reversible trifluoroacetic acid-induced conformational changes in glycophorin A as detected by proton nuclear magnetic resonance spectroscopy.

High-field (270 MHz) 1H-NMR has been employed to study the solution conformation of glycophorin A, a sialoglycoprotein which spans the human erythrocyte membrane. Glycophorin A is one of the most fully characterized integral membrane proteins known, making it an excellent model for the study of membrane-bound proteins. This protein consists of three distinct domains: a glycosylated extracellular N-terminus, a hydrophobic intramembranous segment, and a polar cytoplasmic C-terminus. These domains contain aromatic residues which serve as convenient 1H-NMR conformational probes. The aromatic region of the NMR spectrum of glycophorin A in 2H2O shows single, well-resolved His and Tyr resonances. No resonances are observed, however, for the Phe residues which are located in or near the hydrophobic domain. These observations suggest that considerable heterogeneity with respect to segmental motions exists within the protein. This is consistent with circular dichroism data showing the intramembranous segment to be completely helical with the extremities of the protein being predominantly random coils. The helix of the hydrophrobic domain is remarkably resistant to conventional denaturing conditions including variations in pH, and temperature, and treatment with guanidine hydrochloride. However, in trifluoroacetic acid, which strongly solvates peptide backbones, there is extensive reversible unfolding of the helical structure as evidenced by the appearance of Phe resonances. Solvent titration experiments indicate that approximately a 1 : 1 volume ratio of trifluoroacetic acid to 2H2O is required to initiate unfolding of the helix.