Second-derivative infrared spectroscopic studies of the secondary structures of bacteriorhodopsin and calcium-ATPase

Abstract

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.

Can conformational changes be responsible for solvent and excipient effects on the catalytic behavior of subtilisin Carlsberg in organic solvents?

“…Only two major broad bands were resolved in this case by the Gaussian curve fitting, at 1683 and 1664 cm -1 (Table V). These spectral changes are typical for protein unfolding in DMSO (Jackson and Mantsch, 1992;Lee et al, 1985;Surewicz and Mantsch, 1988;Jackson and Mantsch, 1995). Quantification of the secondary structure yielded a maximum content of 3% ␣-helices and 12% ␤-sheets (Table VI).…”

The 32kDa enamelin undergoes conformational transitions upon calcium binding

“…The broad amide bands were analyzed using deconvolution and second-derivative techniques. Fourier self-deconvolution (Yang et al, 1985) and derivative calculations (Susi and Byler, 1983;Lee et al, 1985) provide accurate assessments of the number and position of component peaks. Minima peaks in the second-derivative correspond to the positive absorptions in the original difference spectrum.…”

Conformational changes of recombinant Ca2+–ATPase studied by reaction‐induced infrared difference spectroscopy

“…IR spectroscopy has been applied to investigate the structure and mechanism of SERCA1a by several groups [17][18][19][20][21][22][23][24][25]. Different intermediate states of rabbit SERCA1a can be accumulated and IR bands have been assigned to most of them [25,26].…”