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

Abstract

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 (v(o)), the coefficient of adiabatic compressibility (beta(s)(o)), 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 degrees C. These mutations induced large changes in v(o) (0.747-0.756 mL/g) and beta(s)(o) (6.89-9.68 Mbar(-1)) compared to the corresponding values for wild-type CRP (v(o)= 0.750 mL/g and beta(s)(o)= 7.98 Mbar(-1)). These changes in global structural properties correlated with the rate of amide proton exchange. A linear correlation was established between beta(s)(o) 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 alpha-carbons are separated by 34.6 A, showed nonadditive effects on v(o) and beta(s)(o). 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.

cAMP activation of PKA defines an ancient signaling mechanism

“…Our findings reported here are a successful attempt of studying CBD-A of the R-subunit by multidimensional solution NMR methods. Furthermore, the proposed model defines a mechanism that is highly conserved and thus relevant for cAMP recognition in other homologous CBDs coupled to effector proteins with diverse functions, such as transcription factors (catabolite-activator protein) (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31), guanine nucleotide exchange factors (11), and ion channel proteins (both hyperpolarization-activated cyclic nucleotide-dependent channels and cyclic nucleotide-gated channels) (32,33). This model also serves as a general paradigm for how small molecules such as cAMP allosterically control large proteinprotein interfaces.…”

Solvent effects on the molecular structures of crude gliadins as revealed by density and ultrasound velocity measurements

“…To approximate the properties of the ideal isolated protein molecules, which have no intermolecular interactions, the apparent specific volume is extrapolated to the limit of zero protein concentration and the partial specific volume of the protein molecule in the solution, o v , is obtained as equation 3 (Gekko and Noguchi, 1974;Millero et al, 1976;Gekko and Noguchi, 1979;Gekko and Hasegawa, 1986;Gekko and Hasegawa, 1989;Gekko and Yamagami, 1991;Kamiyama and Gekko, 2000;Gekko et al, 2003;Gekko et al, 2004;Chalikian et al, 1993;Blandamer et al, 2001;Taulier et al, 2005).…”

“…For macromolecules such as protein molecules To RT M β is small and can be neglected (Stillinger, 1973;Kharakoz, 1992;Chalikian, 2003). (Gekko and Noguchi, 1979;Gekko and Hasegawa, 1986;Gekko and Hasegawa, 1989;Gekko and Yamagami, 1991;Kharakoz, 1992;Chalikian, 1998;Chalikian and Breslauer, 1998;Murphy et al, 1998;Chalikian, 2001;Gekko, 2002;Valdez et al, 2001;Chalikian, 2003;Gekko et al, 2003;Gekko et al, 2004;Bano and Marek, 2006 Taulier and Chalikian, 2002;Mori et al, 2006). …”

“…According to the definition of compressibility coefficient (equation 7), the partial specific adiabatic compressibility coefficient, o S β , is expressed as: Thus, equation 15 gives (Gekko and Noguchi, 1974;Gekko and Noguchi, 1979;Gekko and Hasegawa, 1986;Gekko and Hasegawa, 1989;Gekko and Yamagami, 1991;Paci and Marchi, 1996;Kamiyama and Gekko, 2000;Gekko et al, 2003;Gekko et al, 2004) …”

“…calculated the unit compressibility contributions of charged, cha γ , polar, pol γ , and non-polar, non γ , protein surface area from 18 °C to 55 °C in aqueous solutions. The negative values of unit compressibility contributions shown in Table 2.1 indicate that the interaction between the solvent (23) molecules and proteins contributes negatively to the protein compressibility properties (Gekko and Noguchi, 1979;Gekko and Hasegawa, 1986;Gekko and Hasegawa, 1989;Gekko and Yamagami, 1991;Kharakoz, 1991;Kharakoz and Sarvazyan, 1993;Paci and Marchi, 1996;Kharakoz, 1997;Heremans and Smeller, 1998;Gekko, 2002;Taulier and Chalikian, 2002;Valdez et al, 2001;Chalikian, 2003;Gekko et al, 2003;Marchi, 2003;Gekko et al, 2004;Bano and Marek, 2006;Dadarlat and Post, 2006;Mori et al, 2006). pol γ -66 ± 3 -62 ± 3 -60 ± 3 -58 ± 3 -57 ± 3 non γ -13 ± 2 -9 ± 2 -6 ± 2 -5 ± 2 -4 ± 2 compared the unit compressibility contribution of charged, cha γ , polar, pol γ , and non-polar, non γ , protein surfaces with those on low-molecular-mass compounds to illustrate the hydration properties of proteins.…”