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Göteborgs universitets publikationer

Conformational stabilization of an engineered binding protein.

Författare och institution:
Elisabet Wahlberg (-); Torleif Härd (Institutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi)
Publicerad i:
Journal of the American Chemical Society, 128 ( 23 ) s. 7651-60
Artikel, refereegranskad vetenskaplig
Sammanfattning (abstract):
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.
Ämne (baseras på Högskoleverkets indelning av forskningsämnen):
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
Postens nummer:
Posten skapad:
2007-10-31 12:00
Posten ändrad:
2010-01-26 12:43

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