荧光光谱法揭示盐酸胍对嗜热栖热放线菌脂肪酶结构的稳态和时间分辨效应。
Steady state and time resolved effects of guanidine hydrochloride on the structure of Humicola lanuginosa lipase revealed by fluorescence spectroscopy.
作者信息
Zhu K, Jutila A, Kinnunen P K
机构信息
University of Helsinki, Institute of Biomedicine, Department of Medical Chemistry, Finland.
出版信息
Protein Sci. 2000 Mar;9(3):598-609. doi: 10.1110/ps.9.3.598.
Effects of guanidine hydrochloride (GdnHCl) on the structure and dynamics of wild-type Humicola lanuginosa lipase (HLL) and its two mutants were studied. The latter were S146A (with the active site Ser replaced by Ala) and the single Trp mutant W89m, with substitutions W117F, W221H, and W260H. Steady-state, stopped-flow, and time-resolved laser-induced fluorescence spectroscopy were carried out as a function of [GdnHCl]. The maximum emission wavelength and fluorescence lifetimes revealed the microenvironment of the tryptophan(s) in these lipases to become more polar upon increasing [GdnHCl]. However, significant extent of tertiary structure in GdnHCl is suggested by the observation that both wild-type HLL and W89m remain catalytically active at rather high GdnHCl concentrations of >6 and 4.0 M, respectively. Changes in steady-state emission anisotropy, as well as variation in rotational correlation times and residual anisotropy values, demonstrate that upon increasing [GdnHCl] the structure of the lipases became more loose, with increasing amplitude of structural fluctuations. Finally, intermediate states in the course of exposure of the proteins to GdnHCl were revealed by stopped-flow fluorescence measurements.
研究了盐酸胍（GdnHCl）对野生型毛霉脂肪酶（HLL）及其两个突变体结构和动力学的影响。后者分别是S146A（活性位点的丝氨酸被丙氨酸取代）和单色氨酸突变体W89m，其中W89m包含W117F、W221H和W260H取代。作为[GdnHCl]的函数，进行了稳态、停流和时间分辨激光诱导荧光光谱分析。最大发射波长和荧光寿命表明，随着[GdnHCl]的增加，这些脂肪酶中色氨酸的微环境变得更加极性。然而，野生型HLL和W89m分别在>6和4.0 M的相当高的GdnHCl浓度下仍保持催化活性，这一观察结果表明GdnHCl中存在显著程度的三级结构。稳态发射各向异性的变化以及旋转相关时间和残余各向异性值的变化表明，随着[GdnHCl]的增加，脂肪酶的结构变得更加松散，结构波动幅度增大。最后，通过停流荧光测量揭示了蛋白质在暴露于GdnHCl过程中的中间状态。
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