Entropy controls activity

Proteins are inherently dynamic, sampling an ensemble of conformations. How these different conformations contribute to protein activity is not easily quantifiable and is poorly understood. To address this issue, Tzeng and Kalodimos examined DNA binding to several variants of catabolite activator protein (CAP). cAMP binding to the cAMP-binding domain of CAP allosterically switches CAP's DNA-binding domains (DBDs) from inactive states to active states capable of binding DNA. The authors used NMR to examine allosteric mutants of CAP in the absence and presence of cAMP and found that the variants differentially occupied the active DBD state, varying from 2% to 100% occupancy. Unexpectedly, they found no correlation between DNA affinity and increased population of the active state, which suggests that determinants in addition to protein structure played a part in controlling binding affinity. ITC revealed that the variants used different thermodynamic strategies to bind to DNA, with some having favorable entropic contributions and some having favorable enthalpic contributions. Because the DNA-binding surfaces of these mutants were not altered, solvation-energy contributions were similar; however, binding entropies still varied substantially. To examine this further, the authors monitored amplitude changes to fast (picosecond to nanosecond) internal protein motions upon DNA binding. Interestingly, they found that the residues of wild-type CAP-cAMP₂ became more rigid when bound to DNA, giving rise to an unfavorable change in conformational entropy. In contrast, residues of two CAP variants that bind with similar affinity as wild type but populate the active state <7% of the time became more flexible upon DNA binding. The authors suggest that this favorable conformational entropic contribution leads to strong binding by these variants and that, in general, the relationship between fast and slow protein motions can regulate protein activity in ways not anticipated from examining a protein's most stable, lowest-energy structure. (Nature doi:10.1038/nature11271, published online 11 July 2012)