Suplatov Dmitry, Panin Nikolay, Kirilin Evgeny, Shcherbakova Tatyana, Kudryavtsev Pavel, Svedas Vytas
Lomonosov Moscow State University, Belozersky Institute of Physicochemical Biology and Faculty of Bioengineering and Bioinformatics, Moscow, Russia.
PLoS One. 2014 Jun 24;9(6):e100643. doi: 10.1371/journal.pone.0100643. eCollection 2014.
Protein stability provides advantageous development of novel properties and can be crucial in affording tolerance to mutations that introduce functionally preferential phenotypes. Consequently, understanding the determining factors for protein stability is important for the study of structure-function relationship and design of novel protein functions. Thermal stability has been extensively studied in connection with practical application of biocatalysts. However, little work has been done to explore the mechanism of pH-dependent inactivation. In this study, bioinformatic analysis of the Ntn-hydrolase superfamily was performed to identify functionally important subfamily-specific positions in protein structures. Furthermore, the involvement of these positions in pH-induced inactivation was studied. The conformational mobility of penicillin acylase in Escherichia coli was analyzed through molecular modeling in neutral and alkaline conditions. Two functionally important subfamily-specific residues, Gluβ482 and Aspβ484, were found. Ionization of these residues at alkaline pH promoted the collapse of a buried network of stabilizing interactions that consequently disrupted the functional protein conformation. The subfamily-specific position Aspβ484 was selected as a hotspot for mutation to engineer enzyme variant tolerant to alkaline medium. The corresponding Dβ484N mutant was produced and showed 9-fold increase in stability at alkaline conditions. Bioinformatic analysis of subfamily-specific positions can be further explored to study mechanisms of protein inactivation and to design more stable variants for the engineering of homologous Ntn-hydrolases with improved catalytic properties.
蛋白质稳定性有利于新特性的发展，并且对于耐受引入功能优先表型的突变可能至关重要。因此，了解蛋白质稳定性的决定因素对于研究结构-功能关系和设计新的蛋白质功能很重要。热稳定性已结合生物催化剂的实际应用进行了广泛研究。然而，关于探索pH依赖性失活机制的工作做得很少。在本研究中，对Ntn水解酶超家族进行了生物信息学分析，以确定蛋白质结构中功能重要的亚家族特异性位点。此外，还研究了这些位点在pH诱导失活中的作用。通过在中性和碱性条件下的分子建模分析了大肠杆菌中青霉素酰化酶的构象流动性。发现了两个功能重要的亚家族特异性残基，即Gluβ482和Aspβ484。这些残基在碱性pH下的离子化促进了稳定相互作用的埋藏网络的崩溃，从而破坏了功能性蛋白质构象。亚家族特异性位点Aspβ484被选为突变热点，以改造耐受碱性介质的酶变体。产生了相应的Dβ484N突变体，其在碱性条件下的稳定性提高了9倍。可以进一步探索亚家族特异性位点的生物信息学分析，以研究蛋白质失活机制，并设计更稳定的变体，用于工程改造具有改进催化特性的同源Ntn水解酶。
