High-resolution NMR study of the pressure-induced unfolding of lysozyme.
High-resolution NMR study of the pressure-induced unfolding of lysozyme.
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压力诱导溶菌酶展开的高分辨率核磁共振研究。
DOI：
10.1021/bi00149a005
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Jonas,J
中科院分区：
文献类型：
--
作者：
Samarasinghe,SD;Campbell,DM;Jonas,A;Jonas,J
摘要：采用高分辨率质子磁共振波谱法研究了溶菌酶压力诱导的可逆展开过程，分析了溶菌酶残基His-15d、Trp-28< 3、Leu-17{2、Cys-64”和Trp-108’3的质子谱。实验分别在pH为3.9和68.5 C，压力范围为1 bar至5 kbar的条件下，在不存在和不存在三- tv -乙酰氨基葡萄糖（tri-NAG）的情况下进行。根据压力引起的天然溶菌酶和变性溶菌酶之间平衡的变化，计算每个残留物的反应体积（AV）。发现位于蛋白质不同区域的残基在afr上存在很小但有统计学意义的差异。例如，二硫化物键合的Cys-64 '的AFs比其他残基的AFs小。特别是，tri-NAG与溶菌酶结合的效果是位于活性位点附近的trp -108* 3残基的aff从10.3±0.6 cm3/mol变化到18.1±1.7 cm3/mol。重要的是要注意，Cys-64”残基也感应底物模拟物的结合。检测溶菌酶不同区域单个残基AF的统计显著差异的能力是这些实验的主要结果。大多数关于蛋白质变性的研究都是在大气压下进行的，使用温度或介质的化学成分作为实验变量。这类实验结果的解释并不简单，因为温度的变化会同时产生体积和热能的变化，而且它们的影响很难分开。相反，在蛋白质溶液的研究中，使用压力作为实验变量，只通过改变分子间距离，就可以以连续可控的方式扰动蛋白质的环境（Weber & Drickamer, 1983）。众所周知，蛋白质在溶液中的可逆变性（展开）不仅可以由温度升高或介质组成的变化引起，而且还可以由高压的施加引起(Zipp & Kauzmann，
Revised Manuscript Received June 4, 1992 abstract: The pressure-induced reversible unfolding of lysozyme was investigated by high-resolution proton magnetic resonance spectroscopy by following the proton spectra of the following residues: His-15d, Trp-28< 3, Leu-17 {2, Cys-64 “, and Trp-108'3. The experiments were performed at pH 3.9 and 68.5 C in the pressure range from 1 bar to 5 kbar both in the absence and presence of tri-TV-acetylglucosamine (tri-NAG). From the pressure-induced changes of the equilibrium between the native and denaturated forms of lysozyme, the reaction volumes (AV) were calculated for each residue. Small but statistically significant differences in AFwere found for residues located in different regions of the protein. For example, AFfor the disulfide bonded Cys-64 “is smaller than the AFs found for the other residues. In particular, the effect of tri-NAG binding to lysozyme was a change of AFfrom-10.3±0.6 cm3/mol to-18.1±1.7 cm3/mol for theTrp-108* 3 residue which is located close to the active site. It is important to note that the Cys-64 “residue also senses the binding of the substrate analog. The ability to detect statistically significant differences for AF of individual residues located in different regions of lysozyme represents the main result of these experiments.Most studies dealing with the denaturation of proteins have been carried out at atmospheric pressure using temperature or the chemical composition of the medium as experimental variables. The interpretation of the results of such experiments is not straightforward since the change of temperature produces simultaneous changes both in volume and thermal energy, and their effects are difficult to separate. In contrast, using pressure as the experimental variable in studies of solutions of proteins allows one to perturb the environment of the protein in a continuous controlled way by changing only intermolecular distances (Weber & Drickamer, 1983). It is well known that reversible denaturation (unfolding) of a protein in solution can be caused notonly by an increase in temperature or by changes in the composition of the medium but also byapplication of high pressure (Zipp & Kauzmann,