Abstract
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.
Highlights
To perform their functions, most proteins form compact native structures that are stabilized by complex networks of covalent bonds, non-covalent hydrophobic, electrostatic, van der Waals interactions and hydrogen bonds [1,2,3]
Molecular modeling of the pH-induced destabilization Penicillin acylase (EC 3.5.1.11) from Escherichia coli (EcPA) is a heterodimer that contains two monomer chains referred to as a and b, consisting of 209 and 557 amino acid residues, respectively
The subfamily-specific positions Asnb20, Glub482, and Aspb484 in EcPA and in the related enzymes were shown to stabilize the interactions between two antiparallel b-sheets of the abba-core domain. Such interactions typically involve a dense hydrogen bond network with buried solvent molecules that can have different localization in the structure. These results suggest that the outlined hydrogen bond network that involves subfamilyspecific positions is highly important for the stability of all Ntnhydrolases
Summary
Most proteins form compact native structures that are stabilized by complex networks of covalent bonds, non-covalent hydrophobic, electrostatic, van der Waals interactions and hydrogen bonds [1,2,3]. Structural stability, is largely necessary for the maintenance of functional conformations under adverse environmental conditions (temperature, pressure, pH, presence of solvents, and salts, etc.). Stability is a fundamental property that affects the structure and function of macromolecules and determines biological fitness. Retention of the native fold is, in general, a prerequisite for the evolution of new functions [4,5]. Exploring the mechanisms of protein stability appears to be important for studying enzyme evolution and understanding structure-function relationship, and for the engineering of novel enzymes
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