Protein crystallization by rational mutagenesis of surface residues: Lys to Ala mutations promote crystallization of RhoGDI
Protein crystallization by rational mutagenesis of surface residues: Lys to Ala mutations promote crystallization of RhoGDI
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DOI：
10.1107/s0907444901003122
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发表时间：
2001-05-01
影响因子：
2.2
通讯作者：
Derewenda, ZS
中科院分区：
文献类型：
--
作者：
Longenecker, KL;Garrard, SM;Derewenda, ZS
结晶是一个以熵为代价的独特过程，包括表面残基的构象熵，这些残基在晶体接触形成过程中在晶格中变得有序。因此可以认为，不含高构象熵的氨基酸的表位在热力学上更有利于晶体的形成。对于难以结晶的蛋白质，这种表面氨基酸突变为没有构象熵的残基可能会导致结晶增强。本文报道了一种重要的gtp酶细胞质调节因子——人RhoGDI的实验结果，其中赖氨酸残基系统地突变为丙氨酸。将单个和多个突变引入到两个不同的RhoGDI变体N Delta 23和N Delta 66中，分别通过重组方法去除前23个和66个残基。总共制备了13个单突变体和多突变体，并对其结晶进行了评估，使用Hampton Research Crystal Screens I和II显示所有突变体都结晶了，而野生型N Delta 23和N Delta 66 RhoGDI则没有结晶。四种晶体结构（三突变体N Delta 23:K135,138,141A和N Delta 66:K135,138,141A，以及两个单突变体N Delta 66:K113A和N Delta 66:K141A）被解决，在三种情况下，新晶格的晶体接触精确地发现在突变位点。这些结果支持这样一种观点，即原则上，合理设计突变系统地增强蛋白质的结晶能力是可能的。
Crystallization is a unique process that occurs at the expense of entropy, including the conformational entropy of surface residues, which become ordered in crystal lattices during formation of crystal contacts. It could therefore be argued that epitopes free of amino acids with high conformational entropy are more thermodynamically favorable for crystal formation. For a protein recalcitrant to crystallization, mutation of such surface amino acids to residues with no conformational entropy might lead to enhancement of crystallization. This paper reports the results of experiments with an important cytosolic regulator of GTPases, human RhoGDI, in which lysine residues were systematically mutated to alanines. Single and multiple mutations were introduced into two different variants of RhoGDI, N Delta 23 and N Delta 66, in which the first 23 and 66 residues, respectively, were removed by recombinant methods. In total, 13 single and multiple mutants were prepared and assessed for crystallization and all were shown to crystallize using the Hampton Research Crystal Screens I and II, in contrast to wild-type N Delta 23 and N Delta 66 RhoGDI which did not crystallize. Four crystal structures were solved (the triple mutants N Delta 23:K135,138,141A and N Delta 66:K135,138,141A, and two single mutants N Delta 66:K113A and N Delta 66:K141A) and in three cases the crystal contacts of the new lattices were found precisely at the sites of mutations. These results support the notion that it is, in principle, possible to rationally design mutations which systematically enhance proteins' ability to crystallize.