A Linear Correlation between the Energetics of Allosteric Communication and Protein Flexibility in the Escherichia coli Cyclic AMP Receptor Protein Revealed by Mutation-Induced Changes in Compressibility and Amide Hydrogen-Deuterium Exchange

Amino acid substitutions at distant sites in the Escherichia coli cyclic AMP receptor protein (CRP) have been shown to affect both the nature and magnitude of the energetics of cooperativity of cAMP binding, ranging from negative to positive. In addition, the binding to DNA is concomitantly affected. To correlate the effects of amino acid substitutions on the functional energetics and global structural properties in CRP, the partial specific volume (v̄°), the coefficient of adiabatic compressibility (β̄ _s°), and the rate of amide proton exchange were determined for the wild-type and eight mutant CRPs (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) by using sound velocity, density measurements, and hydrogen-deuterium exchange as monitored by Fourier transform infrared spectroscopy at 25 °C. These mutations induced large changes in v̄° (0.747-0.756 mL/g) and β̄_s° (6.89-9.68 Mbar^-1) compared to the corresponding values for wild-type CRP (v̄° = 0.750 mL/g and β̄_s° = 7.98 Mbar ^-1). These changes in global structural properties correlated with the rate of amide proton exchange. A linear correlation was established between β̄_s° and the energetics of cooperativity of binding of cAMP to the high-affinity sites, regardless of the nature of cooperativity, be it negative or positive. This linear correlation indicates that the nature and magnitude of cooperativity are a continuum. A similar linear correlation was established between compressibility and DNA binding affinity. In addition, linear correlations were also found among the dynamics of CRP and functional energetics. Double mutation (K52N/H159L) at positions 52 and 159, whose α-carbons are separated by 34.6 Å, showed nonadditive effects on v̄° and β̄_s°. These results demonstrate that a small alteration in the local structure due to amino acid substitution is dramatically magnified in the overall protein dynamics which plays an important role in modulating the allosteric behavior of CRP.