A natural antibody missing a cysteine in VH: consequences for thermodynamic stability and folding.
While the disulfide bridge is highly conserved within the immunoglobulin fold, a few antibody variable domains lack one of the essential cysteine residues. In the levan binding antibody ABPC48 one of the essential cysteine residues (Cys H92) of the heavy chain variable domain is replaced by tyrosine. We expressed scFv fragments with the ABPC48 sequence and a mutant in which the VH disulfide bond has been restored in Escherichia coli, purified both proteins by antigen affinity chromatography and characterized them by equilibrium denaturation. While the ABPC48 protein was found to be significantly less stable than an average scFv molecule, the restored disulfide increased its stability above that of other, unrelated scFv fragments, explaining why it tolerates the disulfide loss. Surprisingly, we observed that under some refolding conditions, the unpaired cysteine residue of functional scFv of ABPC48 is derivatized by glutathione. It is easily accessible to other reagents and thus appears to be solvent-exposed, in contrast to the deeply buried disulfide of ordinary variable domains. This implies a very unusual conformation of stand b containing the unpaired Cys H22, which might be stabilized by interactions with the tyrosine residue in position H92.
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Biochemisches Institut Universität Zürich, Switzerland.
Honegger
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Plückthun
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D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005803:Genes, Immunoglobulin; D005813:Genes, Synthetic; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009194:Myeloma Proteins / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics
D004220:Disulfides; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D009194:Myeloma Proteins; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; C040835:myeloma protein A48, mouse; D003545:Cysteine