Urea denaturation of staphylococcal nuclease monitored by Fourier transform infrared spectroscopy.
Urea denaturation of staphylococcal nuclease monitored by Fourier transform infrared spectroscopy.
复制标题
DOI：
10.1021/bi970620l
复制
发表时间：
1998-01
期刊：
影响因子：
2.9
通讯作者：
N. B. From;B. Bowler
中科院分区：
文献类型：
--
作者：
N. B. From;B. Bowler
傅里叶变换红外（FTIR）酰胺I光谱尚未被广泛用作研究蛋白质折叠的方法。已经进行了一些深入的研究，热展开，然而，蛋白质展开的广泛使用的变性剂盐酸胍和尿素的存在下，最近才有报道。盐酸胍和尿素在酰胺I区都有很强的吸收，H2O也是如此。在这里，我们使用氘代13 C-尿素作为化学变性剂，并监测氘交换葡萄球菌核酸酶（SNase）在D2 O中的展开过渡。这些条件避免了所有减法困难，因为D2 O和变性剂的吸收带移出酰胺I'区[Fabian，H.，Manstch，H. H.（1995）Biochemistry 34，13651-13655]。一个非常可重复的展开过渡SNase获得。13 C-尿素诱导的SNase的展开被发现不仅是以前的FTIR热展开数据相媲美，但也有一个类似于其他热变性蛋白质的变性态光谱。展开近似是两个状态。变性状态下的红外光谱显示出一些残留的β-折叠结构以及其他不属于随机结构的谱带成分的证据。
Fourier transform infrared (FTIR) amide I spectroscopy has not been widely used as a method to study protein folding. Some thorough studies of thermal unfolding have been carried out; however, protein unfolding in the presence of the widely used denaturants guanidine hydrochloride and urea has only recently been reported. Guanidine hydrochloride and urea both absorb strongly in the amide I region, as does H2O. Here, we have used deuterated 13C-urea as the chemical denaturant and monitored the unfolding transition with deuterium-exchanged staphylococcal nuclease (SNase) in D2O. These conditions circumvent all subtraction difficulties as the absorption bands of D2O and denaturant are shifted out of the amide I' region [Fabian, H., and Manstch, H. H. (1995) Biochemistry 34, 13651-13655]. A very reproducible unfolding transition is obtained for SNase. 13C-Urea-induced unfolding of SNase was found not only to be comparable to previous FTIR thermal unfolding data but also to have a denatured-state spectrum similar to those of other thermally denatured proteins. The unfolding is approximately two-state. The infrared spectra in the denatured state show evidence of some residual beta-sheet structure as well as other band components not attributable to random structure.