THE STABILITY AND UNFOLDING OF AN IGG BINDING-PROTEIN BASED UPON THE B-DOMAIN OF PROTEIN-A FROM STAPHYLOCOCCUS-AUREUS PROBED BY TRYPTOPHAN SUBSTITUTION AND FLUORESCENCE SPECTROSCOPY

The stability and unfolding of an immunoglobulin (Ig) G binding protei n based upon the B domain of protein A (SpA(B)) from Staphylococcus au reus were studied by substituting tryptophan residues at strategic loc ations within each of the three alpha-helical regions alpha 1-alpha 3) of the domain. The role of the C-terminal helix, alpha 3, was investi gated by generating two protein constructs, one corresponding to the c omplete SpA(B), the other lacking a part of alpha 3; the Trp substitut ions were made in both one and two-domain versions of each of these co nstructs. The fluorescence properties of each of the single-tryptophan mutants were studied in the native state and as a function of guanidi ne-HCl-mediated unfolding, and their IgG binding activities were deter mined by a competitive enzyme-linked immunosorbent assay. The free ene rgies of folding and of binding to IgG for each mutant were compared w ith those for the native domains. The effect of each substitution upon the overall structure and upon the IgG binding interface was modelled by molecular graphics and energy minimization. These studies indicate that (i) alpha 3 contributes to the overall stability of the domain a nd to the formation of the IgG binding site in alpha 1 and alpha 2, an d (ii) alpha 1 unfolds first, followed by alpha 2 and alpha 3 together .