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Biophysical characterization of Z_SPA-1—A phage-display selected binder to protein A

¹ KTH Biotechnology, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden
² Department of Medical Biochemistry, Göteborg University, SE-405 30 Göteborg, Sweden

(RECEIVED March 10, 2004; FINAL REVISION April 30, 2004; ACCEPTED May 8, 2004)

Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z_SPA-1 affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z_SPA-1 complex and noted that Z_SPA-1 in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z_SPA-1 and four Z_SPA-1 mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z_SPA-1 was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z_SPA-1 is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z_SPA-1 with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.

Keywords: protein engineering; affibody; protein stability; osmolyte; NMR spectroscopy

Abbreviations: ¹⁵N-HSQC, ¹H-¹⁵N heteronuclear single quantum correlation • ANS, 8-anilino-1-naphtalenesulfonic acid • CD, circular dichroism • D50%, denaturant concentration at which 50% of the protein is unfolded • GuHCl, guanidine hydrochloride • ITC, isothermal titration calorimetry • NMR, nuclear magnetic resonance spectroscopy • NOE, nuclear Over-hauser effect • SEC, size exclusion chromatography • SPA, staphylococcal protein A • TMAO, trimethylamine N-oxide.

Reprint requests to: Christofer Lendel, KTH Biotechnology, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden; e-mail: lendel{at}kth.se; fax: 46-8-5537-8358.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04728604.

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