Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Kusharyoto, W.
Right arrow Articles by Schmid, R. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kusharyoto, W.
Right arrow Articles by Schmid, R. D.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Protein Engineering, Vol. 15, No. 3, 233-241, March 2002
© 2002 Oxford University Press

Mapping of a hapten-binding site: molecular modeling and site-directed mutagenesis study of an anti-atrazine antibody

Wien Kusharyoto, Jürgen Pleiss, Till T. Bachmann and Rolf D. Schmid,1

Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany

A three-dimensional model of the variable domain of the atrazine-specific Fab fragment K411B was constructed by molecular modeling using known structures of highly homologous immunoglobulins as templates. Molecular dynamic simulations and cross-reactivity data were used to predict residues responsible for the binding of the hapten 4-chloro-6-(isopropylamino)-1,3,5-triazine-2-(6-aminohexanecarboxylic acid) (iPr/Cl/C6) instead of atrazine. Specific binding pockets could be defined for the chlorine, the isopropylamino group and the C6-spacer of the hapten. The influence of various amino acids on hapten binding was investigated by site-directed mutagenesis, and the effect of these mutations was analyzed by capture ELISA using the hapten iPr/Cl/C6 and 4-amino-6-chloro-1,3,5-triazine-2-(6-aminohexanecarboxylic acid) (H/Cl/C6). GlyH100a seems to be important in determining the conformation of the heavy-chain complementarity determining region H3; replacing it with any other residue prevented the binding of the hapten. Altering residues responsible for the binding of the chlorine atom (TrpH33, GluH50 and TyrL96) decreased the affinity significantly. Hapten-spacer recognition can be attributed to the interaction with PheL32; replacing PheL32 by leucine reduced the affinity towards iPr/Cl/C6. A triple mutant Fab fragment (GlnL89Glu, ValH37Ile and GluL3Val) showed an affinity 5-fold greater towards iPr/Cl/C6 compared to the wild-type K411B, as a result of better recognition of the isopropylamino group of iPr/Cl/C6.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Protein Eng Des SelHome page
T.K. Nevanen, M.-L. Hellman, N. Munck, G. Wohlfahrt, A. Koivula, and H. Soderlund
Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody
Protein Eng. Des. Sel., December 1, 2003; 16(12): 1089 - 1097.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.