Triazinylaniline derivatives as fluorescence probes. Part 3. Effects of calcium and other metal ions on the steady-state and time-resolved fluorescence of bovine brain calmodulin labelled at lysine-75

Abstract

The fluorescence characteristics of bovine brain calmodulin labelled at lysine-75 by the reactive triazinylaniline (TA) dye p-Et₂NC₆H₄C₃N₃(Cl)₂ reveal structural changes critically dependent on calcium ion binding. Binding of two calcium ions to the C-terminal lobe exposes a hydrophobic region to the TA fluorophore, enhancing the fluorescence yield four-fold. Addition of two further calcium ions, to the N-terminal lobe, decreases the TA probe fluorescence three-fold by a displacement of the hydrophobic probe imposed by mutual interaction of two protein lobes/regions. The protein matrix induces circular dichorism in the attached TA dye in a calcium-dependent manner. Auxiliary binding of divalent metal ions at mmol dm^–3 concentrations greatly enhances probe fluorescence. Despite the above changes the probe rotational relaxation times (8.0 ns) and limiting anisotropies (0.340) are insensitive to the calcium status of the triazinylaniline-labelled calmodulin (TACaM). Close co-rotation of the probe with a single lobe of the calmodulin carrier is likely, but the possibility of the TA probe optical transition axis lying parallel to a major axis of protein deformations is not excluded. Complex formation with melittin, and the potential utility of TACaM in the study of calmodulin-peptide interaction kinetics, are described.