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1.
J Sep Sci ; 32(10): 1686-95, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19472280

RESUMO

The binding site of a monoclonal anti-L-amino acid antibody (anti-L-AA) was modeled using the program SWISS-MODEL. Docking experiments with the enantiomers of phenylalanine revealed that the antibody interacts with L-phenylalanine via hydrogen bonds and hydrophobic contacts, whereas the D-enantiomer is rejected due to steric hindrance. Comparison of the sequences of this antibody and an anti-D-amino acid antibody (anti-D-AA) indicates that both immunoglobulins derived from the same germline progenitor. Substitution of four amino acids residues, three in the framework and one in the complementarity determining regions (CDRs), allowed in silico conversion of the anti-L-AA into an antibody that stereoselectively binds D-phenylalanine.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Simulação por Computador , Modelos Moleculares , Fenilalanina/química , Fenilalanina/imunologia , Sequência de Aminoácidos , Reações Antígeno-Anticorpo , Sítios de Ligação , Dados de Sequência Molecular , Alinhamento de Sequência , Estereoisomerismo
2.
Chirality ; 20(3-4): 559-70, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18172831

RESUMO

The structure of the binding site of the stereoselective anti-D-amino acid antibody 67.36 was modeled utilizing web antibody modeling (WAM) and SWISS-MODEL. Although docking experiments performed with an aromatic amino acid as model ligand were unsuccessful with the WAM structure, ligand binding was achieved with the SWISS-MODEL structure. Incorporation of side-chain flexibility within the binding site resulted in a protein structure that stereoselectively binds to the D-enantiomer of the model ligand. In addition to four hydrogen bonds that are formed between amino acid residues in the binding site and the ligand, a number of hydrophobic interactions are involved in the formation of the antibody-ligand complex. The aromatic side chain of the ligand interacts with a tryptophan and a tyrosine residue in the binding site through pi-pi stacking. Fluorescence spectroscopic investigations also suggest the presence of tryptophan residues in the binding site, as ligand binding causes an enhancement of the antibody's intrinsic fluorescence at an emission wavelength of 350 nm. Based on the modeled antibody structure, the L-enantiomer of the model ligand cannot access the binding site due to steric hindrance. Additional docking experiments performed with D-phenylalanine and D-norvaline showed that these ligands are bound to the antibody in a way analogous to the D-enantiomer of the model ligand.


Assuntos
Aminoácidos/química , Aminoácidos/imunologia , Anticorpos Monoclonais/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Complexo Antígeno-Anticorpo/química , Reações Antígeno-Anticorpo , Sítios de Ligação de Anticorpos , Simulação por Computador , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estereoisomerismo
3.
Chirality ; 17 Suppl: S9-18, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15612044

RESUMO

This article describes the production of stereoselective antibodies using both classical immunological and modern molecular biological techniques. Stereoselective antibodies against alpha-hydroxy acids were raised in rabbits and mice and compared with previously produced anti-alpha-amino acid antibodies. It was found that both types of antibodies combine stereoselectivity with class-specificity. Sequence analyses revealed that antibodies with opposing stereoselectivities can be formed during the affinity maturation process from a common progenitor or independently using nonhomologous binding sites. For the first time, phage display was employed to obtain stereoselective antibody fragments. The versatility of stereoselective antibodies as chiral selectors was demonstrated by applying them in several immunosensors and in chiral chromatography. A simple, membrane-based optical sensor allowed detection of enantiomeric impurities at the 1/2,000 level (99.9% ee). Silica-based antibody chiral stationary phases could be used for enantiomer separation of aliphatic amino acids in standard-sized columns, while miniaturized columns allowed interfacing with an MS-detector.


Assuntos
Formação de Anticorpos , Especificidade de Anticorpos , Hidroxiácidos/química , Hidroxiácidos/imunologia , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/imunologia , Animais , Cromatografia de Afinidade/métodos , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Coelhos , Homologia de Sequência de Aminoácidos , Estereoisomerismo
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