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A fluorescent calmodulin that reports the binding of hydrophobic inhibitory ligands.

ABSTRACT: Ca2+ binding to calmodulin in the pCa range 5.5-7.0 exposes hydrophobic sites that bind hydrophobic inhibitory ligands, including calmodulin antagonists, some Ca2+-antagonists and calmodulin-binding proteins. The binding of these hydrophobic ligands to calmodulin can be followed by the approx. 80% fluorescence increase they produce in dansylated (5-dimethylaminonaphthalene-1-sulphonylated) calmodulin (CDRDANS). In the presence of Ca2+, calmodulin binds the calmodulin inhibitor, R24571, with an affinity of approx. 2-3 nM and hydrophobic ligands, including trifluoperazine (TFP), W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide], fendiline, felodipine and prenylamine, with affinities in the micromolar range. This binding is strongly Ca2+-dependent and Mg2+-independent. Calmodulin shows a reasonably high degree of specificity in its binding of these ligands over other ligands tested. CDRDANS, therefore, provides a convenient and simple means of monitoring the interaction of a variety of hydrophobic ligands with the Ca2+-dependent regulatory protein, calmodulin. CDRDANS binds to phospholipid vesicles made of (dimyristoyl)phosphatidylcholine (DMPC) or (dipalmitoyl)phosphatidylcholine (DPPC) and produces fluorescence increases only in the presence of Ca2+ and at temperatures above their gel-to-liquid crystalline phase transition. Although the fluorescence changes in CDRDANS accurately report phase transitions in these liposomes, its binding to these vesicles is weak. Calmodulin probably requires a high-affinity lipid-bound receptor protein for its high-affinity binding to natural membranes.

SUBMITTER: Johnson JD

PROVIDER: S-EPMC1154381 | biostudies-other | 1983 May

REPOSITORIES: biostudies-other

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Project description:BackgroundThe major birch pollen allergen, Bet v 1, is a member of the ubiquitous PR-10 family of plant pathogenesis-related proteins. In recent years, a number of diverse plant proteins with low sequence similarity to Bet v 1 was identified. In addition, determination of the Bet v 1 structure revealed the existence of a large superfamily of structurally related proteins. In this study, we aimed to identify and classify all Bet v 1-related structures from the Protein Data Bank and all Bet v 1-related sequences from the Uniprot database.ResultsStructural comparisons of representative members of already known protein families structurally related to Bet v 1 with all entries of the Protein Data Bank yielded 47 structures with non-identical sequences. They were classified into eleven families, five of which were newly identified and not included in the Structural Classification of Proteins database release 1.71. The taxonomic distribution of these families extracted from the Pfam protein family database showed that members of the polyketide cyclase family and the activator of Hsp90 ATPase homologue 1 family were distributed among all three superkingdoms, while members of some bacterial families were confined to a small number of species. Comparison of ligand binding activities of Bet v 1-like superfamily members revealed that their functions were related to binding and metabolism of large, hydrophobic compounds such as lipids, hormones, and antibiotics. Phylogenetic relationships within the Bet v 1 family, defined as the group of proteins with significant sequence similarity to Bet v 1, were determined by aligning 264 Bet v 1-related sequences. A distance-based phylogenetic tree yielded a classification into 11 subfamilies, nine exclusively containing plant sequences and two subfamilies of bacterial proteins. Plant sequences included the pathogenesis-related proteins 10, the major latex proteins/ripening-related proteins subfamily, and polyketide cyclase-like sequences.ConclusionThe ubiquitous distribution of Bet v 1-related proteins among all superkingdoms suggests that a Bet v 1-like protein was already present in the last universal common ancestor. During evolution, this protein diversified into numerous families with low sequence similarity but with a common fold that succeeded as a versatile scaffold for binding of bulky ligands.

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A fluorescent calmodulin that reports the binding of hydrophobic inhibitory ligands.

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