How the interplay between protein structure and internal dynamics regulates protein function is poorly understood. Often, ligand binding, post-translational modifications and mutations modify protein activity in a manner that is not possible to rationalize solely on the basis of structural data. It is likely that changes in the internal motions of proteins have a major role in regulating protein activity, but the nature of their contributions remains elusive, especially in quantitative terms. Here we show that changes in conformational entropy can determine whether protein-ligand interactions will occur, even among protein complexes with identical binding interfaces. We have used NMR spectroscopy to determine the changes in structure and internal dynamics that are elicited by the binding of DNA to several variants of the catabolite activator protein (CAP) that differentially populate the inactive and active DNA-binding domain states. We found that the CAP variants have markedly different affinities for DNA, despite the CAPĝ̂'DNA-binding interfaces being essentially identical in the various complexes. Combined with thermodynamic data, the results show that conformational entropy changes can inhibit the binding of CAP variants that are structurally poised for optimal DNA binding or can stimulate the binding activity of CAP variants that only transiently populate the DNA-binding-domain active state. Collectively, the data show how changes in fast internal dynamics (conformational entropy) and slow internal dynamics (energetically excited conformational states) can regulate binding activity in a way that cannot be predicted on the basis of the protein's ground-state structure.
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Acknowledgements We are grateful to L. Kay for providing the methyl relaxation pulse sequences, D. Korzhnev for his help with the relaxation dispersion data analysis, and S. Kim and M.-T. Pai for their help with protein preparation and NMR experiments. This work was supported by National Science Foundation (NSF) grant MCB1121896 to C.G.K.
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