Interactions between a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus and a human κ 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 K-d of the complex under a range of conditions by using both pre-equilibrium and equilibrium methods. The K-d 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-how fluorescence shows that complex formation takes place by two discrete, sequential processes. A fast bimolecular reaction, with a rate constant of 8.3 x 10(5) M(-1.)s(-1) (at pH 8.0 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 pH 8.0 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.