STUDIES ON ENGINEERING CRYSTALLIZABILITY BY MUTATION OF SURFACE RESIDUES OF HUMAN THYMIDYLATE SYNTHASE
STUDIES ON ENGINEERING CRYSTALLIZABILITY BY MUTATION OF SURFACE RESIDUES OF HUMAN THYMIDYLATE SYNTHASE
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DOI：
10.1016/0022-0248(92)90255-h
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发表时间：
1992-08-01
影响因子：
1.8
通讯作者：
VILLAFRANCA, JE
中科院分区：
文献类型：
--
作者：
MCELROY, HE;SISSON, GW;VILLAFRANCA, JE
进行研究以确定改变蛋白质的内在溶剂化性质以改善其结晶性质的功效。为了改变其溶解性，制备了12个胸苷酸合成酶（TS）突变体，改变蛋白质表面11个不同位置上的单个氨基酸。突变改变了野生型氨基酸的电荷或极性。使野生型TS和每种突变体经受不同pH、沉淀剂和盐的结晶条件的基质。两周后，检查每次结晶尝试并对蛋白质溶解度和结晶进行评分。因此，调整每种条件的参数，然后重复，以驱使蛋白质趋于饱和，而不引起非特异性聚集。结果发现，TS表面上的单个氨基酸变化可以对溶解度产生显著影响，同时不降低稳定性。此外，发现一些突变TS的晶体在野生型TS不结晶的条件下发生，并且一些突变TS显示出增强的结晶性。确定了在独特条件下发现的或具有独特形态的所得晶体的空间群。几个突变晶体与野生型TS的空间群不同。
A study was made to determine the efficacy of altering a protein's intrinsic solvation properties to improve its crystallization properties. In order to change its solubility properties, twelve mutants of thymidylate synthase (TS) were made altering single amino acids at eleven different positions on the protein surface. The mutations changed either the charge or polarity of the wild-type amino acid. Wild-type TS and each of the mutants were subjected to a matrix of crystallization conditions varying pH, precipitant, and salt. After two weeks, each crystallization attempt was examined and scored for protein solubility and crystallization. Accordingly, the parameters of each condition were adjusted then repeated to drive the protein toward saturation without precipitating nonspecific aggregation. It was found that single amino acid changes on the surface of TS could have a dramatic effect on solubility while not decreasing stability. Furthermore, crystals of some mutant TSs were found to occur in conditions where wildtype TS did not crystallize and some mutant TSs showed enhanced crystallizability. The space groups of resulting crystals found in unique conditions or having unique morphologies were determined. Several of the mutant crystals were of different space groups than wild-type TS.