Electrostatic contributions to the kinetics and thermodynamics of protein assembly.
The role of electrostatic interactions in the assembly of a native protein structure was studied using fragment complementation. Contributions of salt, pH, or surface charges to the kinetics and equilibrium of calbindin D(9k) reconstitution was measured in the presence of Ca(2+) using surface plasmon resonance and isothermal titration calorimetry. Whereas surface charge substitutions primarily affect the dissociation rate constant, the association rates are correlated with subdomain net charge in a way expected for Coulomb interactions. The affinity is reduced in all mutants, with the largest effect (260-fold) observed for the double mutant K25E+K29E. At low net charge, detailed charge distribution is important, and charges remote from the partner EF-hand have less influence than close ones. The effects of salt and pH on the reconstitution are smaller than mutational effects. The interaction between the wild-type EF-hands occurs with high affinity (K(A) = 1.3 x 10(10) M(-1); K(D) = 80 pM). The enthalpy of association is overall favorable and there appears to be a very large favorable entropic contribution from the desolvation of hydrophobic surfaces that become buried in the complex. Electrostatic interactions contribute significantly to the affinity between the subdomains, but other factors, such as hydrophobic interactions, dominate.
Dell'Orco
Daniele
D
Department of Biophysical Chemistry, Lund University, S-221 00 Lund, Sweden.
Xue
Wei-Feng
WF
Thulin
Eva
E
Linse
Sara
S
D064026:Calbindins; D002118:Calcium / Q000737:chemistry*; D003460:Crystallization / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046912:Multiprotein Complexes / Q000032:analysis* / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D064030:S100 Calcium Binding Protein G / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D064026:Calbindins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D002118:Calcium