TOWARDS CONFORMATIONAL SEQUENCING OF PROTEINS: ASSIGNMENT OF SECONDARY STRUCTURES BY ANTI-PEPTIDE ANTIBODIES
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Issue Date
1987Submitted date
2023-04-26
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Show full item recordAbstract
We performed model studies towards assignment of &-turn and a-helices to protein primary structures with antibodies. Probing of a &-turn was attempted with anti-peptide antibodies directed against the &-turn (DPGQ) of a synthetic &-turn model-peptide (IVIVIDPGQTVTY) adopting the intended conformation &-sheet-&-turn-&-sheet. The specific anti-&-turn model-peptide antibodies have a three orders of magnitude higher affinity for the &-turn containing epitope than the control Gly-peptide (GsDPGQG,, ) of random coil structure. The antibody affinity for the &-turn region (DPGQ) increases from the primary to the hyperimmune response. Although the chosen &-turn sequence is similar to parts of the animal's own proteins, self-tolerance did not raise difficulties in generating antibodies against the R-turn model-peptide. Individual putative &-turn sequences of proteins may be probed by including their sequence between the two &-sheet cartridges of the &-turn model-peptide. Helix assignment was probed with synthetic model peptides of extended conformation including only the superimposed residues of a putative helix (every fourth residue) linked by a spacer amino acid residue (alanine throughout or the corresponding third residue of the sequence to be tested) in order to adjust the translation of the relevant residues of the model-peptide to the helical pitch. The anti-helix modelpeptide antibodies were shown by Western blotting to react in a sequence-specific manner with the corresponding model protein lactose permease of E.coli. "Conformational sequencing" i.e. sequence assignments of secondary structures by anti-peptide antibodies now seems feasable for &-turn regions and helices of proteins of known sequence.Citation
Chemical synthesis in molecular biology, 199 ffAffiliation
Institute for Genetics, University of Cologne Weyertal 121, D-5000 Cologne 41, F.R. GermanyType
Book chapterconference paper
Language
enSeries/Report no.
GBF Monographs, Vol. 8ISSN
0930-4320Collections
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