Prestrelski S J, Byler D M, Liebman M N
Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, New York 10029.
Biochemistry. 1991 Jan 8;30(1):133-43. doi: 10.1021/bi00215a020.
Fourier-transform infrared spectroscopy is a valuable method for the study of protein conformation in solution primarily because of the sensitivity to conformation of the amide I band (1700-1620 cm-1) which arises from the backbone C = O stretching vibration. Combined with resolution-enhancement techniques such as derivative spectroscopy and self-deconvolution, plus the application of iterative curve-fitting techniques, this method provides a wealth of information concerning protein secondary structure. Further extraction of conformational information from the amide I band is dependent upon discerning the correlations between specific conformational types and component bands in the amide I region. In this paper, we report spectra-structure correlations derived from conformational perturbations in bovine trypsin which arise from autolytic processing, zymogen activation, and active-site inhibition. IR spectra were collected for the single-chain (beta-trypsin) and once-cleaved, double-chain (alpha-trypsin) forms as well as at various times during the course of autolysis and also for zymogen, trypsinogen, and beta-trypsin inhibited with diisopropyl fluorophosphate. Spectral differences among the various molecular forms were interpreted in light of previous biochemical studies of autolysis and the known three-dimensional structures of the zymogen, the active enzyme, and the DIP-inhibited form. Our spectroscopic results from these proteins in D2O imply that certain loop structures may absorb in the region of 1655 cm-1. Previously, amide I' infrared bands near 1655 cm-1 have been interpreted as arising solely from alpha-helices. These new data suggest caution in interpreting this band. We have also proposed that regions of protein molecules which are known from crystallographic experiments to be disordered absorb in the 1645 cm-1 region and that type II beta-turns absorb in the region of 1672-1685 cm-1. Our results also corroborate assignment of the low-frequency component of extended strands to bands below 1636 cm-1. Additionally, the results of multiple measurements have allowed us to estimate the variability present in component band areas calculated by curve fitting the resolution-enhanced IR spectra. We estimate that this approach to data analysis and interpretation is sensitive to changes of 0.01 unit or less in the relative integrated intensities of component bands in spectra whose peaks are well resolved.
傅里叶变换红外光谱法是研究溶液中蛋白质构象的一种重要方法，主要是因为它对酰胺I带（1700 - 1620 cm⁻¹）的构象敏感，该谱带源于主链C = O伸缩振动。结合诸如导数光谱法和自去卷积等分辨率增强技术，再加上迭代曲线拟合技术的应用，这种方法能提供大量有关蛋白质二级结构的信息。从酰胺I带进一步提取构象信息取决于识别特定构象类型与酰胺I区域中各组分谱带之间的相关性。在本文中，我们报告了源自牛胰蛋白酶构象扰动的光谱 - 结构相关性，这些扰动源于自溶加工、酶原激活和活性位点抑制。我们收集了单链（β - 胰蛋白酶）和一次裂解的双链（α - 胰蛋白酶）形式的红外光谱，以及自溶过程中不同时间的光谱，还收集了酶原、胰蛋白酶原以及用氟磷酸二异丙酯抑制的β - 胰蛋白酶的光谱。根据先前关于自溶的生化研究以及酶原、活性酶和二异丙基氟磷酸酯抑制形式的已知三维结构，解释了各种分子形式之间的光谱差异。我们对这些蛋白质在重水中的光谱研究结果表明，某些环结构可能在1655 cm⁻¹区域有吸收。此前，1655 cm⁻¹附近的酰胺I'红外谱带一直被解释为仅源于α - 螺旋。这些新数据表明在解释该谱带时需谨慎。我们还提出，从晶体学实验已知为无序的蛋白质分子区域在1645 cm⁻¹区域有吸收，II型β - 转角在1672 - 1685 cm⁻¹区域有吸收。我们的结果还证实了将伸展链的低频组分归属于1636 cm⁻¹以下的谱带。此外，多次测量的结果使我们能够估计通过对分辨率增强的红外光谱进行曲线拟合计算出的各组分谱带面积中的变异性。我们估计，这种数据分析和解释方法对峰分辨率良好的光谱中各组分谱带相对积分强度变化0.01单位或更小的变化敏感。
