Interactions between a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus and a human kappa light chain.
The placement of a tryptophan residue into a single Ig-binding-domain of protein L from Peptostreptococcus magnus has been used to examine the binding interactions between the binding domain and kappa light chains (kappa-chains). The fluorescence intensity of the mutant domain increases on the formation of a complex with kappa-chains. This has been used to determine the Kd of the complex under a range of conditions by using both pre-equilibrium and equilibrium methods. The Kd values determined for the complex with kappa-chains at a number of different pH values are very close to those obtained with the wild-type domain, indicating that the mutation has not substantially affected its binding properties. Examination of the reaction between the mutant domain and kappa-chains by stopped-flow fluorescence shows that complex formation takes place by two discrete, sequential processes. A fast bimolecular reaction, with a rate constant of 8.3x10(5) M-1. s-1 (at pH8.0 and 25 degrees C), is followed by a slow unimolecular process with a rate (1.45 s-1) that is independent of the concentration of the reactants. This suggests that a conformational change occurs after the initial encounter complex is formed. The dissociation of the complex at equilibrium occurs in a single process of rate 0.095 s-1 at pH8.0 and 25 degrees C. Stopped-flow CD studies show that a slow decrease in ellipticity at 275 nm occurs with a rate of 1.3 s-1 when wild-type protein binds to kappa-chains, suggesting that the conformational transition might involve a change in environment around one or more tyrosine residues.
Beckingham
J A
JA
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, Hants. SO16 7PX, UK.
Bottomley
S P
SP
Hinton
R
R
Sutton
B J
BJ
Gore
M G
MG
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010462:Peptostreptococcus; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D001426:Bacterial Proteins; C055047:Ig L-binding protein, Peptostreptococcus; D007074:Immunoglobulin G; D007145:Immunoglobulin kappa-Chains; D010446:Peptide Fragments; D014443:Tyrosine; D014364:Tryptophan