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.