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Updated: Jul 6, 2026

Engineering Cell-permeable Protein

Engineering Cell-permeable Protein

Published on: December 28, 2009

Conformational stabilization of an engineered binding protein.

Elisabet Wahlberg¹, Torleif Härd

¹School of Biotechnology, Royal Institute of Technology (KTH), S-106 91 Stockholm, Sweden.

Journal of the American Chemical Society

|June 8, 2006

Summary

Disulfide engineering significantly enhanced binding protein affinity by stabilizing specific conformations. This stabilization improved binding enthalpy and enthalpy-entropy compensation, leading to an order of magnitude increase in binding affinity.

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Area of Science:

Protein engineering
Biophysical chemistry
Molecular biology

Background:

The Z(SPA)(-)(1) affibody exhibits moderate binding affinity (K(d) = 1.6 microM) to its Z domain partner.
Previous studies suggested conformational heterogeneity, not interface limitations, underlies this moderate affinity.

Purpose of the Study:

To investigate the thermodynamic basis for improving binding protein affinity through disulfide engineering.
To enhance the binding affinity of the Z(SPA)(-)(1) affibody to its Z domain partner.

Main Methods:

Disulfide engineering was employed to create stabilized Z(SPA)(-)(1) double cysteine mutants.
Binding affinity was measured using dissociation constants (K(d)).
Thermodynamic analysis of binding was performed, examining conformational entropy, desolvation entropy, and binding enthalpy.

Main Results:

Five stabilized Z(SPA)(-)(1) mutants showed an order of magnitude improvement in affinity, reaching K(d) = 130 nM.
Thermodynamic analysis revealed a balance between conformational and desolvation entropy changes.
Higher binding affinity correlated with more favorable binding enthalpy and enthalpy-entropy compensation.

Conclusions:

Protein-protein binding affinity can be significantly improved by stabilizing specific protein conformations via disulfide engineering.
Stabilizing conformations allows for better exploration of favorable enthalpic effects during binding.
Disulfide engineering offers a viable strategy to enhance affibody affinity for therapeutic applications.