Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy
Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy
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DOI：
10.1529/biophysj.106.103515
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发表时间：
2007-06-01
影响因子：
3.4
通讯作者：
Gekko, Kunihiko
中科院分区：
文献类型：
--
作者：
Matsuo, Koichi;Sakurada, Yoshie;Gekko, Kunihiko
为了阐明变性蛋白质的结构，我们测量的真空紫外圆二色性（VUVCD）光谱从260至172 nm的三种蛋白质（高铁肌红蛋白，葡萄球菌核酸酶，硫氧还蛋白）在本机和酸，冷，热变性状态，使用同步辐射VUVCD分光光度计。盐酸胍（GdnHCl）完全展开的蛋白质的圆二色光谱也被测量到197 nm进行比较。这些变性蛋白质表现出特征性的VUVCD光谱，反映了相当数量的残留二级结构。使用SELCON 3程序，结合蛋白质数据库数据和我们以前研究中报道的31个参考蛋白质的VUVCD光谱，估计了三种蛋白质每种变性状态的α-螺旋，β-链，转角，聚-L-脯氨酸II型（PPII）和无序结构的含量。在此基础上，讨论了每种蛋白质的四种变性类型的特点。在所有类型的变性中，α-螺旋的减少伴随着β-链、PPII和无序结构的增加。约20%的β-链存在，即使在蛋白质完全展开的盐酸钆，其中β-折叠应该被打破。从这些结果中，我们提出，变性蛋白质构成的残余α-螺旋和β-片，部分未折叠（或扭曲）的α-螺旋和β-链，PPII，和无序结构的合奏。
To elucidate the structure of denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra from 260 to 172 nm of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and the acid-, cold-, and heat-denatured states, using a synchrotron-radiation VUVCD spectrophotometer. The circular dichroism spectra of proteins fully unfolded by guanidine hydrochloride (GdnHCl) were also measured down to 197 nm for comparison. These denatured proteins exhibited characteristic VUVCD spectra that reflected a considerable amount of residual secondary structures. The contents of alpha-helices, beta-strands, turns, poly-L-proline type II (PPII), and unordered structures were estimated for each denatured state of the three proteins using the SELCON3 program with Protein Data Bank data and the VUVCD spectra of 31 reference proteins reported in our previous study. Based on these contents, the characteristics of the four types of denaturation were discussed for each protein. In all types of denaturation, a decrease in alpha-helices was accompanied by increases in beta-strands, PPII, and unordered structures. About 20% beta-strands were present even in the proteins fully unfolded by GdnHCl in which beta-sheets should be broken. From these results, we propose that denatured proteins constitute an ensemble of residual alpha-helices and beta-sheets, partly unfolded (or distorted) alpha-helices and beta-strands, PPII, and unordered structures.