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TOWARDS CONFORMATIONAL SEQUENCING OF PROTEINS: ASSIGNMENT OF SECONDARY STRUCTURES BY ANTI-PEPTIDE ANTIBODIES
Beyreuther, Konrad Schulze-Gahmen, Ursula Bieseler, Barbara Prinz, Heinrich
Beyreuther, Konrad
Schulze-Gahmen, Ursula
Bieseler, Barbara
Prinz, Heinrich
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Issue Date
1987
Submitted date
2023-04-26
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Abstract
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 ff
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Book chapter
conference paper
conference paper
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en
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GBF Monographs, Vol. 8
ISSN
0930-4320
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Attribution-NonCommercial-ShareAlike 4.0 International