SECONDARY STRUCTURE ESTIMATION OF PROTEINS USING THE AMIDE-III REGION OF FOURIER-TRANSFORM INFRARED-SPECTROSCOPY - APPLICATION TO ANALYZE CALCIUM BINDING-INDUCED STRUCTURAL-CHANGES IN CALSEQUESTRIN

A Fourier transform infrared spectroscopic method has been developed to analyze protein secondary structure by employing the amide III spectral region (1350-1200 cm(-1)). Benefits of using the amide III region have been shown to be substantial. The interference from the water vibration (similar to 1640 cm(-1)) in the amide I region can be avoided when one is using the amide III band; furthermore, the amide III region also presents a more characterized spectral feature which provides easily resolved and better defined bands for quantitative analysis. Estimates of secondary structure are accomplished with the use of Fourier self-deconvolution, second derivatization, and curve-fitting on original protein spectra. The secondary structure frequency windows (alpha-helix, 1328-1289 cm(-1); unordered, 1288-1256 cm(-1); and beta-sheets, 1255-1224 cm(-1)) have been obtained, and estimates of secondary structural contents are consistent with X-ray crystallography data for model proteins and parallel results obtained with the use of the amide I region. We have further applied the analysis to the structural change of calsequestrin upon Ca2+ binding. Treatment of calsequestrin with 1 mM Ca2+ results in the formation of crystalline aggregates accompanied by a 10% increase in alpha-helical structure, which is consistent with previous results obtained by Raman spectroscopy. Thus the amide III region of protein IR spectra appears to be a valuable tool in estimating individual protein secondary structural contents.