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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

Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa light chains
Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa light chains
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.
PEPTOSTREPTOCOCCUS-MAGNUS, SITE, IGG
0021-9258
9370-9378
Housden, N.G.
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Harrison, S.
0e3bb8ab-7b95-4686-9b74-f889065ad068
Housden, H.R.
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Thomas, K.A.
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Beckingham, J.A.
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Roberts, S.E.
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Bottomley, S.P.
9e5f98e5-d93d-40b1-89ea-db79b8990182
Graille, M.
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Stura, E.
a37324b9-b03c-42ca-b642-6ba684b864ee
Gore, M.G.
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Housden, N.G.
5fc2b5f1-a7da-4f20-b794-d7d8ef597d5d
Harrison, S.
0e3bb8ab-7b95-4686-9b74-f889065ad068
Housden, H.R.
1bc65e47-6f9a-4c68-9bc2-0424f12ad681
Thomas, K.A.
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Beckingham, J.A.
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Roberts, S.E.
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Bottomley, S.P.
9e5f98e5-d93d-40b1-89ea-db79b8990182
Graille, M.
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Stura, E.
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Gore, M.G.
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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), 9370-9378. (doi:10.1074/jbc.M312938200).

Record type: Article

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 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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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: 15 Mar 2024 07:00

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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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