Cumulative Effect of Intragenic Amino Acid Replacements on Thermostability of a Protein
The marginal net stability of a folded protein is considered to rest upon a small difference among large compensating, individual forces. Therefore, the net free energy of stabilization of proteins is unexpectedly small, ~40 kJ mol−1. Meanwhile, the contribution of individual forces such as hydrogen bonds and salt bridges to the stabilization are evaluated as 4 to 12 kJ mol−1, and several additional forces are thought to be sufficient to account for the extra thermostability of thermophilic proteins. The native conformation of a protein is determined by the totality of interatomic interactions and hence, by the amino acid sequence. If a few amino acid residues which individually contribute to the stabilization could be implemented concurrently into the sequence, the multiple replacement would enhance an overall stability of the protein molecule. Herein a report on concrete evidence in favor of this argument will be presented. Thermal inactivation kinetics and proteolytic resistance for mutants of a kanamycin nucleotidyltransferase revealed that a few intragenic amino acid replacements stabilized the protein cumulatively. This work demonstrates not only the feasibility of elevating a protein’s thermostability but also a way to better understanding of forces and interactions that are responsible for the protein stability.