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Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa light chains

Record type: Article

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 light chain are due to the binding of the light chain at two separate sites on the protein L molecule. Elimination of binding site 2 through the mutation A66W has allowed the Kd for 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 Kd for binding at site 2 to be determined. Stopped-flow fluorimetry suggests this to be 3.4 ± 0.8 µM in good agreement with the value of 4.6 ± 0.8 µM determined by isothermal titration calorimetry. The mutation Y53F reduces the affinity of site 1 to approximately that of site 2.

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Citation

Housden, N.G., Harrison, S., Housden, H.R., Thomas, K.A., Beckingham, J.A., Roberts, S.E., Bottomley, S.P., Graille, M., Stura, E. and Gore, M.G. (2004) Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa light chains The Journal of Biological Chemistry, 279, (10), pp. 9370-9378. (doi:10.1074/jbc.M312938200).

More information

Submitted date: 26 November 2003
Published date: 1 March 2004
Keywords: PEPTOSTREPTOCOCCUS-MAGNUS, SITE, IGG

Identifiers

Local EPrints ID: 25145
URI: http://eprints.soton.ac.uk/id/eprint/25145
ISSN: 0021-9258
PURE UUID: 41b9eaf1-22d1-48d7-9055-03f6112cabfd

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Date deposited: 06 Apr 2006
Last modified: 17 Jul 2017 16:11

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Contributors

Author: N.G. Housden
Author: S. Harrison
Author: H.R. Housden
Author: K.A. Thomas
Author: J.A. Beckingham
Author: S.E. Roberts
Author: S.P. Bottomley
Author: M. Graille
Author: E. Stura
Author: M.G. Gore

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