Equilibrium and pre-equilibrium fluorescence spectroscopic studies of the binding of a single-immunoglobulin-binding domain derived from protein G to the Fc fragment from human IgG1.
A single-immunoglobulin-binding protein based upon the C2 domain of Protein G from Streptococcus has been shown to bind tightly to the Fc fragment of IgG1. The binding interaction results in a decrease in the fluorescence intensity from the sole Trp residue (Trp-48) in this domain. This spectral change has been used to monitor the binding interactions between the two proteins using equilibrium and pre-equilibrium fluorescence spectroscopy. Comparison of the data from the two techniques suggests that a conformational change occurs after the initial formation of the complex. Mutagenesis studies have shown that the Trp residue is important for binding and that replacement by a Phe residue is important for binding and that replacement by a Phe residue leads to a 300-fold decrease in the affinity for Fc gamma 1. Determination of the rate constants kon and koff at different values of pH between 4.0 and 9.0 suggest that variations in Kd are mediated predominantly by changes in kon. Competition experiments between SpG1 and a single-IgG-binding domain from Protein A from Staphylococcus aureus have been used to determine the affinity of the latter for Fc gamma 1.
Walker
K N
KN
Department of Biochemistry, University of Southampton, U.K.
Bottomley
S P
SP
Popplewell
A G
AG
Sutton
B J
BJ
Gore
M G
MG
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017452:Receptors, IgG / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013205:Staphylococcal Protein A / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; C034284:IgG Fc-binding protein, Streptococcus; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D017452:Receptors, IgG; D013205:Staphylococcal Protein A; D014364:Tryptophan