title: Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa light chains.
abstract: Detailed stopped-flow studies in combination with site-directed mutagenesis, isothermal titration calorimetry data and x-ray crystallographic knowledge have revealed that the biphasic pre-equilibrium fluorescence changes reported for a single Ig-binding domain of protein L from Peptostreptococcus magnus binding to kappa light chain are due to the binding of the kappa light chain at two separate sites on the protein L molecule. Elimination of binding site 2 through the mutation A66W has allowed the K(d) for kappa light chain binding at site 1 to be measured by stopped-flow fluorescence and isothermal titration calorimetry techniques, giving values of 48.0 +/- 8.0 nM and 37.5 +/- 7.3 nM respectively. Conversely, a double mutation Y53F/L57H eliminates binding at site 1 and has allowed the K(d) for binding at site 2 to be determined. Stopped-flow fluorimetry suggests this to be 3.4 +/- 0.8 microM in good agreement with the value of 4.6 +/- 0.8 microM determined by isothermal titration calorimetry. The mutation Y53F reduces the affinity of site 1 to approximately that of site 2.
authors.affiliation: Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, Hants SO16 7PX, United Kingdom.
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Housden
authors.forename: Steven
authors.initials: S
authors.lastname: Harrison
authors.forename: Hazel R
authors.initials: HR
authors.lastname: Housden
authors.forename: Karen-Anne
authors.initials: KA
authors.lastname: Thomas
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Beckingham
authors.forename: Siân E
authors.initials: SE
authors.lastname: Roberts
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Bottomley
authors.forename: Marc
authors.initials: M
authors.lastname: Graille
authors.forename: Enrico
authors.initials: E
authors.lastname: Stura
authors.forename: Michael G
authors.initials: MG
authors.lastname: Gore
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D005453:Fluorescence; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010462:Peptostreptococcus; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; C055047:Ig L-binding protein, Peptostreptococcus; D007145:Immunoglobulin kappa-Chains