Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa light chains
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. Journal of Biological Chemistry, 279, (10), 9370-9378. (doi:10.1074/jbc.M312938200).
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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.
|Subjects:||Q Science > QD Chemistry
Q Science > QH Natural history > QH301 Biology
|Divisions:||University Structure - Pre August 2011 > School of Biological Sciences
|Date Deposited:||07 Aug 2008|
|Last Modified:||12 May 2013 01:18|
|Contributors:||Housden, N.G. (Author)
Harrison, S. (Author)
Housden, H.R. (Author)
Thomas, K.A. (Author)
Beckingham, J.A. (Author)
Roberts, S.E. (Author)
Bottomley, S.P. (Author)
Graille, M. (Author)
Stura, E. (Author)
Gore, M.G. (Author)
|Date:||1 March 2004|
|RDF:||RDF+N-Triples, RDF+N3, RDF+XML, Browse.|
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