Protein engineering and characterisation of the human kappa light chain binding interactions of Peptostreptococcal protein L

A program of site directed mutagenesis has been employed to characterise the human kappa light chain (κ-chain) interactions of a single Ig-binding domain of protein L from Peptostreptococcus magnus (PpL). To better understand the intricate nature of the binding interaction of κ-chain at both site 1 and site 2 of PpL, site-specific mutagenesis of residues at each interface of the κ-chain. 1PpL2, κ-chain complex has been carried out. This has allowed the production of the mutants, Y53FL57H PpL and A66W PpL, which have either site 1 or site 2, respectively, eliminated. The interactions of these mutated PpL constructs with κ-chain have been characterised under equilibrium conditions using fluorescence spectroscopy, circular dichroism and isothermal titration calorimetry (ITC) yielding Kd values of 29.3 ± 11.6 nM and 6.05 ± 2.1 μM for site 1 and site 2, respectively. Unique Trp residues have been engineered into PpL in order to probe the environment of these reporter groups at various locations within the domain and to permit the determination of binding constants using fluorimetry. Pre-equilibrium binding studies carried out by stopped-flow fluorescence spectroscopy have shown that Trp residues placed at different positions within the PpL domain all (with the exception of Trp 66 eliminates binding at site 2) indicate binding at two sites. Similar experiments using the site 1 and site 2 ‘knockout’ mutants in combinations with Trp 34 (site 1 reporter) and Trp 63 (site 2 reporter) have enabled a newly proposed binding model between PpL and κ-chain that is compatible with previous data. This model implies that two κ-chains simultaneously and independently have been calculated to be 52.3 ± 16.0 nM and 3.4 ± 16.0 nM and 3.4 ± 0.5 μM for site 1 and site 2, respectively.