Chantarasiri Aiya, Meevootisom Vithaya, Isarangkul Duangnate, Wiyakrutta Suthep
Department of Microbiology, Mahidol University, Bangkok, Thailand.
J Mol Microbiol Biotechnol. 2012;22(3):147-55. doi: 10.1159/000339833. Epub 2012 Jul 24.
Structure-guided genetic engineering of D-phenylglycine aminotransferase (D-PhgAT) aimed at increasing protein solubility was attempted. In silico analyses predicted the Asn439 and Gln444 as highly solvent-exposed β-turn residues involved with protein crystal contact (CC) potential candidates for solubility-improving mutations. They were replaced with Asp and Glu creating the N439D and Q444E single mutants, and N439D/Q444E double mutant with 2.5-, 3.3- and 5.9-fold increases in solubility, respectively. The protein CC prevention effect rather than the net charge effect accounted for the dramatically improved solubility since the N439D, Q444E and N439D/Q444E mutations altered the isoelectric point of D-PhgAT by only 0.1, 0.1 and 0.3 units, respectively. Examination of the D-PhgAT structural model revealed that the N439D mutation weakened the CC attraction force and the Q444E mutation created electrostatic repulsion at the CC point. Analysis of circular dichroism spectra, melting temperature, and D-PhgAT-specific activity showed that the mutations posed no unfavorable effect on the conformational stability and catalytic performance of the enzyme. The protein solubility-improving strategy employed on D-PhgAT in this study was successful with minimal protein structure modification required. It should be applicable with a high chance of success for other proteins, especially those with 3-D structural models available.
旨在提高蛋白质溶解度的D-苯甘氨酸转氨酶(D-PhgAT)的结构导向基因工程研究得以开展。计算机分析预测,Asn439和Gln444是高度暴露于溶剂中的β-转角残基,参与蛋白质晶体接触(CC),是改善溶解度突变的潜在候选位点。将它们分别替换为Asp和Glu,构建了N439D和Q444E单突变体以及N439D/Q444E双突变体,其溶解度分别提高了2.5倍、3.3倍和5.9倍。蛋白质CC预防效应而非净电荷效应导致了溶解度的显著提高,因为N439D、Q444E和N439D/Q444E突变分别仅使D-PhgAT的等电点改变了0.1、0.1和0.3个单位。对D-PhgAT结构模型的研究表明,N439D突变减弱了CC吸引力,Q444E突变在CC位点产生了静电排斥。圆二色光谱、解链温度和D-PhgAT特异性活性分析表明,这些突变对酶的构象稳定性和催化性能没有不利影响。本研究中应用于D-PhgAT的蛋白质溶解度提高策略成功实现,且所需的蛋白质结构修饰最小。该策略很有可能成功应用于其他蛋白质,尤其是那些有三维结构模型的蛋白质。