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Conformational stabilization of an engineered binding protein.

Journal article
Authors Elisabet Wahlberg
Torleif Härd
Published in Journal of the American Chemical Society
Volume 128
Issue 23
Pages 7651-60
ISSN 0002-7863
Publication year 2006
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 7651-60
Language en
Keywords Algorithms, Calorimetry, Circular Dichroism, Cysteine, chemistry, genetics, metabolism, Disulfides, chemistry, Mutation, Protein Binding, Protein Conformation, Protein Denaturation, Protein Engineering, Protein Folding, Proteins, chemistry, genetics, metabolism, Thermodynamics
Subject categories Medical and Health Sciences


We analyzed the thermodynamic basis for improvement of a binding protein by disulfide engineering. The Z(SPA)(-)(1) affibody binds to its Z domain binding partner with a dissociation constant K(d) = 1.6 microM, and previous analyses suggested that the moderate affinity is due to the conformational heterogeneity of free Z(SPA)(-)(1) rather than to a suboptimal binding interface. Studies of five stabilized Z(SPA)(-)(1) double cystein mutants show that it is possible to improve the affinity by an order of magnitude to K(d) = 130 nM, which is close to the range (20 to 70 nM) observed with natural Z domain binders, without altering the protein-protein interface obtained by phage display. Analysis of the binding thermodynamics reveals a balance between conformational entropy and desolvation entropy: the expected and favorable reduction of conformational entropy in the best-binding Z(SPA)(-)(1) mutant is completely compensated by an unfavorable loss of desolvation entropy. This is consistent with a restriction of possible conformations in the disulfide-containing mutant and a reduction of average water-exposed nonpolar surface area in the free state, resulting in a smaller conformational entropy penalty, but also a smaller change in surface area, for binding of mutant compared to wild-type Z(SPA)(-)(1). Instead, higher Z domain binding affinity in a group of eight Z(SPA)(-)(1) variants correlates with more favorable binding enthalpy and enthalpy-entropy compensation. These results suggest that protein-protein binding affinity can be improved by stabilizing conformations in which enthalpic effects can be fully explored.

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