Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability.
Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability.
复制标题
DOI：
10.1006/jmbi.1999.3102
复制
发表时间：
1999-10
影响因子：
5.6
通讯作者：
J. W. Wray;W. Baase;J. Lindstrom;L. Weaver;A. Poteete;Brian W. Matthews
中科院分区：
文献类型：
--
作者：
J. W. Wray;W. Baase;J. Lindstrom;L. Weaver;A. Poteete;Brian W. Matthews
T4溶菌酶中的突变Glu 108-->瓦尔（E108 V）先前被分离为第二位点回复突变体，其特异性补偿与去稳定化取代Leu 99-->Gly（L99 G）相关的功能丧失。令人惊讶的是，这两个位点相距11 A，Leu 99位于蛋白质的核心，Glu 108位于蛋白质的表面。为了更好地理解这一结果，我们对这些突变溶菌酶以及具有Leu 99->Ala取代的相关变体进行了详细的热力学、酶促和结构分析。发现E108 V确实增加了L99 G的稳定性，但它也以基本上相等的量增加了野生型蛋白和L99 A的稳定性。E108 V对酶活性的影响较为复杂。该突变略微降低了野生型、L99 G和L99 A的细胞壁水解的最大速率。同时，L99 G是一种不稳定的蛋白质，在测定过程中会迅速失去活性，特别是在20 ℃以上的温度下。因此，即使双突变体L99 G/E108 V具有比L99 G略低的最大速率，在20-30分钟的时间内，它水解更多的底物。这种热失活率的降低似乎是E108 V作为L99 G的第二位点回复突变体的作用的基础。突变体L99 A产生体积为149 A的空腔（3）。突变体L99 G没有扩大该空腔，而是导致螺旋F的一部分（残基108-113）的4-5 A置换，产生溶剂可接近的斜面。在双突变体L99 G/E108 V中，该螺旋返回到类似于野生型的位置，导致体积为203 A的空腔（3）。无论突变Glu 108-->瓦尔是否掺入野生型溶菌酶或L99 A或L99 G中，它都会导致晶体学热因子的降低，特别是在包括残基99和108的螺旋中。这种刚性的增加，这似乎是由于增加的疏水稳定性加上构象波动的限制的组合，提供了热稳定性增加的结构基础。
The mutation Glu108-->Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99-->Gly (L99G). Surprisingly, the two sites are 11 A apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99-->Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20-30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 A(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 A displacement of part of helix F (residues 108-113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 A(3). Whether the mutation Glu108-->Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability.