Abstract

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 (~ 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 (α-helix, 1328-1289 cm^-1; unordered, 1288-1256 cm^-1; and β-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 α-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.

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