Influence of urea and guanidine hydrochloride on lysozyme stability and thermal denaturation; a correlation between activity, protein dynamics and conformational changes
Influence of urea and guanidine hydrochloride on lysozyme stability and thermal denaturation; a correlation between activity, protein dynamics and conformational changes
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DOI：
10.1039/c0cp00602e
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发表时间：
2010-01-01
影响因子：
3.3
通讯作者：
Siepmann, Juergen
中科院分区：
文献类型：
--
作者：
Hedoux, Alain;Krenzlin, Stefanie;Siepmann, Juergen
本文用活性测定、微量热法和拉曼光谱研究了尿素和盐酸胍对溶菌酶稳定性的影响。溶解在H2O和D2 O中的溶菌酶在高达10 M的变性剂的存在下的拉曼调查已经揭示了蛋白质和两种变性剂之间的直接结合。在酰胺I区的同位素交换的分析允许识别的结合位点作为亲水性和疏水性基团，分别为尿素和盐酸胍。在尿素存在下溶菌酶活性的弱损失（类似于最大15%）主要归因于对应于熔融球状态的三级结构的转化，而没有α-螺旋结构的展开，与GuHCl相反，GuHCl明显诱导构象变化，与较大的活性损失（最大40%）相关。尿素和盐酸胍对溶菌酶的变性力与溶剂动力学和蛋白质动力学有关，反映了变性剂与蛋白质之间的直接相互作用。显然，溶剂动力学控制蛋白质动力学，和显着硬化的动力学盐酸胍水溶液被认为是负责其重要的变性能力。在不同类型的添加剂（尿素，盐酸胍，海藻糖）的存在和不存在的情况下，溶剂和溶菌酶水溶液的低频光谱之间的比较揭示了水合水动力学的拉曼签名。该比较指出了蛋白质表面周围的海藻糖的排除。
The effect of urea and guanidine hydrochloride (GuHCl) on lysozyme stability has been investigated using activity measurements, microcalorimetry and Raman spectroscopy in the low-frequency and amide I regions. Raman investigations on lysozyme dissolved in H2O and D2O in the presence of up to 10 M denaturants have revealed direct binding between the protein and both denaturants. The analysis of isotopic exchanges in the amide I region allows the identification of binding sites as hydrophilic and hydrophobic groups, respectively, for urea and GuHCl. The weak loss of activity of lysozyme in the presence of urea (similar to 15% maximum) is mainly assigned to a transformation of the tertiary structure corresponding to a molten globule state without unfolding of alpha-helix structures, in contrast to GuHCl which clearly induces conformational changes, associated with a larger loss of activity (40% maximum). The denaturing power of urea and guanidine hydrochloride on lysozyme has been related to the solvent and protein dynamics, reflecting direct interaction between denaturants and protein. It clearly appears that solvent dynamics control protein dynamics, and the significant hardening of the dynamics of GuHCl aqueous solutions is considered responsible for its important denaturing power. The comparison between the low-frequency spectra of solvents and lysozyme aqueous solutions in the absence and presence of different types of additives (urea, GuHCl, trehalose) reveals the Raman signature of the hydration water dynamics. This comparison points out the exclusion of trehalose around the protein surface.