Conformational stabilization of an engineered binding protein.
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.
Wahlberg
Elisabet
E
School of Biotechnology, Royal Institute of Technology (KTH), S-106 91 Stockholm, Sweden.
Härd
Torleif
T
D000465:Algorithms; D002151:Calorimetry; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D004220:Disulfides; D011506:Proteins; D003545:Cysteine