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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

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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.

Keywords: Amide I; Amide III; Calsequestrin; Curve analysis; FT-IR secondary structure; Proteins; Spectroscopy

Document Type: Research Article


Affiliations: 1: Department of Chemistry, University of Massachusetts Dartmouth, N. Dartmouth, Massachusetts 02747 2: Department of Bacteriology and Biochemistry, University of Idaho, Moscow, Idaho 83844 3: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030

Publication date: November 1, 1994

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