Abstract
The placement of a tryptophan residue into a single Ig-binding-domain of protein L from Peptostreptococcus magnus has been used to examine the binding interactions between the binding domain and kappa light chains (kappa-chains). The fluorescence intensity of the mutant domain increases on the formation of a complex with kappa-chains. This has been used to determine the Kd of the complex under a range of conditions by using both pre-equilibrium and equilibrium methods. The Kd values determined for the complex with kappa-chains at a number of different pH values are very close to those obtained with the wild-type domain, indicating that the mutation has not substantially affected its binding properties. Examination of the reaction between the mutant domain and kappa-chains by stopped-flow fluorescence shows that complex formation takes place by two discrete, sequential processes. A fast bimolecular reaction, with a rate constant of 8.3x10(5) M-1. s-1 (at pH8.0 and 25 degrees C), is followed by a slow unimolecular process with a rate (1.45 s-1) that is independent of the concentration of the reactants. This suggests that a conformational change occurs after the initial encounter complex is formed. The dissociation of the complex at equilibrium occurs in a single process of rate 0.095 s-1 at pH8.0 and 25 degrees C. Stopped-flow CD studies show that a slow decrease in ellipticity at 275 nm occurs with a rate of 1.3 s-1 when wild-type protein binds to kappa-chains, suggesting that the conformational transition might involve a change in environment around one or more tyrosine residues.
Full Text
The Full Text of this article is available as a PDF (171.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ashby B., Wootton J. C., Fincham J. R. Slow conformational changes of a Neurospora glutamate dehydrogenase studied by protein fluorescence. Biochem J. 1974 Nov;143(2):317–329. doi: 10.1042/bj1430317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Björck L., Kronvall G. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J Immunol. 1984 Aug;133(2):969–974. [PubMed] [Google Scholar]
- Bottomley S. P., Beckingham J. A., Murphy J. P., Atkinson M., Atkinson T., Hinton R. J., Gore M. G. Cloning, expression and purification of Ppl-1, a kappa-chain binding protein, based upon protein L from Peptostreptococcus magnus. Bioseparation. 1995;5(6):359–367. [PubMed] [Google Scholar]
- Bottomley S. P., Popplewell A. G., Scawen M., Wan T., Sutton B. J., Gore M. G. The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy. Protein Eng. 1994 Dec;7(12):1463–1470. doi: 10.1093/protein/7.12.1463. [DOI] [PubMed] [Google Scholar]
- Chaiyen P., Brissette P., Ballou D. P., Massey V. Thermodynamics and reduction kinetics properties of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase. Biochemistry. 1997 Mar 4;36(9):2612–2621. doi: 10.1021/bi962325r. [DOI] [PubMed] [Google Scholar]
- Derrick J. P., Wigley D. B. Crystal structure of a streptococcal protein G domain bound to an Fab fragment. Nature. 1992 Oct 22;359(6397):752–754. doi: 10.1038/359752a0. [DOI] [PubMed] [Google Scholar]
- Dorrington K. J., Smith B. R. Conformational changes accompanying the dissociation and association of immunoglobulin-G subunits. Biochim Biophys Acta. 1972 Mar 15;263(1):70–81. doi: 10.1016/0005-2795(72)90160-2. [DOI] [PubMed] [Google Scholar]
- Edmundson A. B., Ely K. R., Abola E. E. Conformational flexibility in immunoglobulins. Contemp Top Mol Immunol. 1978;7:95–118. doi: 10.1007/978-1-4757-0779-3_3. [DOI] [PubMed] [Google Scholar]
- Enokizono J., Wikström M., Sjöbring U., Björck L., Forsén S., Arata Y., Kato K., Shimada I. NMR analysis of the interaction between protein L and Ig light chains. J Mol Biol. 1997 Jul 4;270(1):8–13. doi: 10.1006/jmbi.1997.1090. [DOI] [PubMed] [Google Scholar]
- Ghose A. C. Conformational studies on the constant region halves of heavy and light chains of human immunoglobulin G. Biochim Biophys Acta. 1972 Sep 29;278(2):337–343. doi: 10.1016/0005-2795(72)90238-3. [DOI] [PubMed] [Google Scholar]
- Gore M. G., Greasley P., McAllister G., Ragan C. I. Mammalian inositol monophosphatase: the identification of residues important for the binding of Mg2+ and Li+ ions using fluorescence spectroscopy and site-directed mutagenesis. Biochem J. 1993 Dec 15;296(Pt 3):811–815. doi: 10.1042/bj2960811. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goward C. R., Scawen M. D., Murphy J. P., Atkinson T. Molecular evolution of bacterial cell-surface proteins. Trends Biochem Sci. 1993 Apr;18(4):136–140. doi: 10.1016/0968-0004(93)90021-e. [DOI] [PubMed] [Google Scholar]
- Ikeda K., Hamaguchi K., Migita S. Circular dichroism of Bence-Jones proteins and immunoglobulins G. J Biochem. 1968 May;63(5):654–660. doi: 10.1093/oxfordjournals.jbchem.a128825. [DOI] [PubMed] [Google Scholar]
- Kastern W., Sjöbring U., Björck L. Structure of peptostreptococcal protein L and identification of a repeated immunoglobulin light chain-binding domain. J Biol Chem. 1992 Jun 25;267(18):12820–12825. [PubMed] [Google Scholar]
- Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Langone J. J. Protein A of Staphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumonococci. Adv Immunol. 1982;32:157–252. [PubMed] [Google Scholar]
- Patella V., Casolaro V., Björck L., Marone G. Protein L. A bacterial Ig-binding protein that activates human basophils and mast cells. J Immunol. 1990 Nov 1;145(9):3054–3061. [PubMed] [Google Scholar]
- Sasso E. H., Silverman G. J., Mannik M. Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup. J Immunol. 1991 Sep 15;147(6):1877–1883. [PubMed] [Google Scholar]
- Sjöquist J., Movitz J., Johansson I. B., Hjelm H. Localization of protein A in the bacteria. Eur J Biochem. 1972 Oct 17;30(1):190–194. doi: 10.1111/j.1432-1033.1972.tb02086.x. [DOI] [PubMed] [Google Scholar]
- Thumser A. E., Wilton D. C. Characterization of binding and structural properties of rat liver fatty-acid-binding protein using tryptophan mutants. Biochem J. 1994 Jun 15;300(Pt 3):827–833. doi: 10.1042/bj3000827. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wikström M., Drakenberg T., Forsén S., Sjöbring U., Björck L. Three-dimensional solution structure of an immunoglobulin light chain-binding domain of protein L. Comparison with the IgG-binding domains of protein G. Biochemistry. 1994 Nov 29;33(47):14011–14017. doi: 10.1021/bi00251a008. [DOI] [PubMed] [Google Scholar]
- Wikström M., Forsén S., Drakenberg T. Backbone dynamics of a domain of protein L which binds to immunoglobulin light chains. Eur J Biochem. 1996 Feb 1;235(3):543–548. doi: 10.1111/j.1432-1033.1996.00543.x. [DOI] [PubMed] [Google Scholar]
- Wikström M., Sjöbring U., Drakenberg T., Forsén S., Björck L. Mapping of the immunoglobulin light chain-binding site of protein L. J Mol Biol. 1995 Jul 7;250(2):128–133. doi: 10.1006/jmbi.1995.0364. [DOI] [PubMed] [Google Scholar]
- Wikström M., Sjöbring U., Kastern W., Björck L., Drakenberg T., Forsén S. Proton nuclear magnetic resonance sequential assignments and secondary structure of an immunoglobulin light chain-binding domain of protein L. Biochemistry. 1993 Apr 6;32(13):3381–3386. doi: 10.1021/bi00064a023. [DOI] [PubMed] [Google Scholar]