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Calorimetric Analysis of Mutagenic Effects on Protein-Ligand Interactions

Published

Author(s)

Frederick P. Schwarz

Abstract

Mutagenesis effects can be classified into direct where the mutation occurs at the site of the binding interaction on the protein and indirect where the mutation occurs at a site distal from the binding interaction site. The interaction between the Fv fragment of a lysozyme antibody and lysozyme involving a single mutation at the periphery of the binding site is an example of direct mutagenesis. For direct mutagenesis effects, changes in the binding free energy change and in the binding enthalpy can be directly analyzed in terms of changes in the solvent accessible surface area upon mutagenesis. The linear correlation between changes in the binding free energy and the solvent accessible surface area of the non polar residues proximal to the mutation site shows that this interaction is predominantly hydrophobic. The binding of cAMP to the T127->L mutant of CRP is an example of indirect mutagenesis effects since the mutation occurs in the subunit interface of the CRP dimer and distal from the cAMP binding site in the amino terminal domains of the CRP subunit. Although the mutation is off-site, it alters the cAMP binding thermodynamics from an endothermic cooperative two-site mechanism between the subunits of wild type CRP to an exothermic two-site independent mechanism between the subunits of the mutant. Analysis of how this change occurs is based on a structural model where the mutation alters the structure of the CRP dimer to a structure where cooperativity between the two subunit sites is removed in the mutant. This model is substantiated by comparisons between the x-ray structure of the wild type CRP and a CRP mutant containing the T127->L mutation.
Citation
Methods In Enzymology
Volume
379

Keywords

antibody-antigen interaction, calorimetry, isothermal titration, solvent accessible, surface area, thermodynamics

Citation

Schwarz, F. (2008), Calorimetric Analysis of Mutagenic Effects on Protein-Ligand Interactions, Methods In Enzymology (Accessed May 19, 2024)

Issues

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Created October 16, 2008