Second-derivative infrared spectroscopic studies of the secondary structures of bacteriorhodopsin and Ca2+-ATPase.

The resolution of minor amide components in the infrared spectra of membrane proteins has, in the past, been limited by the small differences in frequency compared to the large half-widths of the bands that are assigned to different secondary conformations. Here, second-derivative calculations are used to resolve the relatively weak bands that are associated with the beta-sheet conformation and the vibrations of some amino acid side chains in the infrared spectra of bacteriorhodopsin and Ca2+-activated adenosine-5'-triphosphatase (Ca2+-ATPase). The spectra presented indicate that bacteriorhodopsin in the purple membrane contains an appreciable amount of beta structure in addition to the predominant alpha II-helical structure. Both sarcoplasmic reticulum and purified Ca2+-ATPase in native lipids contain alpha-helical and random coil conformations together with a small amount of beta structure. In 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) Ca2+-ATPase adopts a secondary conformation similar to that in the sarcoplasmic reticulum, and this structure is unaffected by the phospholipid phase transition. A shift to a predominantly random coil conformation is associated with solubilization of both bacteriorhodopsin and Ca2+-ATPase in 20% Triton X-100. Second-derivative analysis of the carbonyl stretching vibrations of DMPC bilayers indicates that below the phase-transition temperature (Tm) both bacteriorhodopsin and Ca2+-ATPase perturb the interface region such that the sn-2 carbonyls adopt a conformation similar to the sn-1 carbonyls. Above Tm, these integral proteins have no effect on the static order of the interface region, and the conformational inequivalence of the sn-1 and sn-2 carbonyls is similar to that found in a pure lipid bilayer.