Identification of β-turn and random coil amide III infrared bands for secondary structure estimation of proteins

Fourier transform infrared spectroscopy is increasingly becoming an important method to determine secondary structure of peptides and proteins. Among the spectral regions arising out of coupled and uncoupled stretching and bending modes of amide bonds, amide I and amide III spectral bands have been found to be the most sensitive to the variations in secondary structure folding. Amide I spectral region (1700–1600 cm−1), although most commonly used primarily because of its strong signal, suffers from several limitations, including a strong interference from water vibrational band, relatively unstructured spectral contour, and overlap of revolved bands correspondingly to various secondary structures. In contrast, amide III spectral region (1350–1200 cm−1), albeit relatively weak in signals, does not have the above limitations. Easily resolved and better defined amide III bands are quite suitable for quantitative analysis of protein secondary structure. While amide III region has been successfully used for determination of α-helix and β-sheets (Fu, F.-N., et al. (1994) Appl. Spectrosc. 48, 1432–1441), bands corresponding to β-turns and random coils have not been identified, so far. In this paper, we describe, for the first time, identification of amide III bands corresponding to β-turns and random coils by selectively enhancing random coils by treatment with a denaturing reagent, and secondary structure estimation of several proteins by using the band assignments. The assignments of spectral bands were as follows: 1330–1295 cm−1, α-helix; 1295–1270 cm−1, β-turns; 1270–1250 cm−1, random coils; and 1250–1220 cm−1, β-sheets. The estimations of secondary structural elements by the above assignments correlated quite well with secondary structure estimations from X-ray crystallography data.