Cai S, Singh B R
Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth 02747, USA.
Biophys Chem. 1999 Jul 19;80(1):7-20. doi: 10.1016/s0301-4622(99)00060-5.
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 alpha-helix and beta-sheets (Fu, F.-N., et al. (1994) Appl. Spectrosc. 48, 1432-1441), bands corresponding to beta-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 beta-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, alpha-helix; 1295-1270 cm-1, beta-turns; 1270-1250 cm-1, random coils; and 1250-1220 cm-1, beta-sheets. The estimations of secondary structural elements by the above assignments correlated quite well with secondary structure estimations from X-ray crystallography data.
傅里叶变换红外光谱法正日益成为测定肽和蛋白质二级结构的重要方法。在酰胺键的耦合和非耦合伸缩及弯曲模式产生的光谱区域中，酰胺I和酰胺III光谱带被发现对二级结构折叠的变化最为敏感。酰胺I光谱区域（1700 - 1600 cm⁻¹），尽管因其信号强而最常被使用，但存在一些局限性，包括来自水振动带的强烈干扰、相对无结构的光谱轮廓以及对应于各种二级结构的旋转带的重叠。相比之下，酰胺III光谱区域（1350 - 1200 cm⁻¹），尽管信号相对较弱，但没有上述局限性。易于分辨且定义更好的酰胺III带非常适合蛋白质二级结构的定量分析。虽然酰胺III区域已成功用于测定α - 螺旋和β - 折叠（傅，F.-N.等人（1994年）《应用光谱学》48卷，1432 - 1441页），但到目前为止，对应于β - 转角和无规卷曲的谱带尚未被识别。在本文中，我们首次描述了通过用变性试剂处理选择性增强无规卷曲来识别对应于β - 转角和无规卷曲的酰胺III谱带，以及使用这些谱带归属对几种蛋白质进行二级结构估计。光谱带的归属如下：1330 - 1295 cm⁻¹，α - 螺旋；1295 - 1270 cm⁻¹，β - 转角；1270 - 1250 cm⁻¹，无规卷曲；1250 - 1220 cm⁻¹，β - 折叠。通过上述归属对二级结构元件的估计与基于X射线晶体学数据的二级结构估计相关性很好。
