Cumulative effect of intragenic amino-acid replacements on the thermostability of a protein.
The marginal net stability of a folded protein is thought to depend on a small difference between large, compensating individual forces. Therefore, the net free energy of stabilization of proteins is unexpectedly small (approximately 10 kcal mol-1). The contribution of individual forces such as hydrogen bonds and salt bridges to the stabilization is evaluated as 1-3 kcal 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 total number of interatomic interactions and hence by the amino-acid sequence. If the few amino-acid residues that individually contribute to the stabilization could be implemented concurrently into the sequence, the multiple replacement would enhance the overall stability of the protein molecule. Here we report evidence to support this argument. Thermal inactivation kinetics and proteolytic resistance for mutants of a kanamycin nucleotidyltransferase reveal that a few intragenic amino-acid replacements stabilize the protein cumulatively. Our experiments not only demonstrate the feasibility of elevating the thermostability of a protein but also lead to better understanding of the forces that are responsible for protein stability.
D004274:DNA, Recombinant; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000235:genetics*; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D004274:DNA, Recombinant; D009713:Nucleotidyltransferases; C024251:kanamycin nucleotidyltransferase; D010447:Peptide Hydrolases