Biophysical characterization of Z(SPA-1)--a phage-display selected binder to protein A.

• 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.

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Annan industriell bioteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Other Industrial Biotechnology (hsv//eng)

Nyckelord

Bacterial Proteins

chemistry

metabolism

Biophysics

Nuclear Magnetic Resonance

Biomolecular

Peptide Library

Peptides

chemistry

genetics

metabolism

Protein Binding

Protein Denaturation

Protein Folding

Protein Structure

Tertiary

Staphylococcal Protein A

chemistry

metabolism

Staphylococcus aureus

chemistry

Thermodynamics

protein engineering

Structural biochemistry

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