STABILITY AND FUNCTION - 2 CONSTRAINTS IN THE EVOLUTION OF BARSTAR AND OTHER PROTEINS

Background: Barstar is the intracellular inhibitor of barnase, an extr acellular RNAse of Bacillus amylolique-faciens. The dissociation const ant of the barnase-barstar complex is 10(-14) M with an association ra te constant between barnase and barstar of 3.7x10(8) s(-1) M(-1). The rapid association arises in part from the clustering of four acidic re sidues (Asp35, Asp39, Glu76 and Glu80) on the barnase-binding surface of barstar. The negatively charged barnase-binding surface of barstar effectively 'steers' the inhibitor towards the positively charged acti ve site of barnase. Results: Mutating any one of the four acidic side chains of barstar to an alanine results in an approximately two-fold d ecrease in the association rate constant, while the dissociation rate constant increases from five orders of magnitude for Asp39-->Ala, to n o significant change for Glu80-->Ala. The stability of barstar is incr eased by all four mutations, the increase ranging from 0.3 kcal mol(-1 ) for Asp35-->Ala or Asp39-->Ala, to 2.1 kcal mol(-1) for Glu80-->Ala. Conclusions: The evolutionary pressure on barstar for rapid binding o f barnase is so strong that glutamate is preferred over alanine at pos ition 80, even though it does not directly interact with barnase in th e complex and significantly destabilizes the inhibitor structure. This , and other examples from the literature, suggest that proteins evolve primarily to optimize their function in vivo, with relatively little evolutionary pressure to increase stability above a certain threshold, thus allowing greater latitude in the evolution of enzyme activity.