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 protein based upon the B domain of protein A (SpAB) from Staphylococcus aureus were studied by substituting tryptophan residues at strategic locations 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 investigated by generating two protein constructs, one corresponding to the complete SpAB, the other lacking a part of alpha 3; the Trp substitutions were made in both one- and two-domain versions of each of these constructs. The fluorescence properties of each of the single-tryptophan mutants were studied in the native state and as a function of guanidine-HCl-mediated unfolding, and their IgG binding activities were determined by a competitive enzyme-linked immunosorbent assay. The free energies of folding and of binding to IgG for each mutant were compared with 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 and to the formation of the IgG binding site in alpha 1 and alpha 2, and (ii) alpha 1 unfolds first, followed by alpha 2 and alpha 3 together.
Bottomley
S P
SP
Department of Biochemistry, University of Southampton, UK.
Popplewell
A G
AG
Scawen
M
M
Wan
T
T
Sutton
B J
BJ
Gore
M G
MG
D001483:Base Sequence; D001665:Binding Sites; D007136:Immunoglobulins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013205:Staphylococcal Protein A / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*
D007136:Immunoglobulins; D013205:Staphylococcal Protein A; D014364:Tryptophan