Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues

The interactions between the abundant methionine residues ofthe calcium regulatory protein calmodulin (CaM) and severalof its binding targets were probed using fluorescence spectroscopy.Tryptophan steady-state fluorescence from peptides encompassingthe CaM-binding domains of the target proteins myosin lightchain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE)and caldesmon site A and B (CaD A, CaD B), and the model peptidemelittin showed Ca²⁺-dependent blue-shifts in their maximumemission wavelength when complexed with wild-type CaM. Blue-shiftswere also observed for complexes in which the CaM methionineresidues were replaced by selenomethionine, norleucine and ethionine,and when a quadruple methionine to leucine C-terminal mutantof CaM was studied. Quenching of the tryptophan fluorescenceintensity was observed with selenomethionine, but not with norleucineor ethionine substituted protein. Fluorescence quenching studieswith added potassium iodide (KI) demonstrate that the non-nativeproteins limit the solvent accessibility of the Trp in the MLCKpeptide to levels close to that of the wild-type CaM–MLCKinteraction. Our results show that the methionine residues fromCaM are highly sensitive to the target peptide in question,confirming the importance of their role in binding interactions.In addition, we provide evidence that the nature of bindingin the CaM–CaD B complex is unique compared with the othercomplexes studied, as the Trp residue of this peptide remainspartially solvent exposed upon binding to CaM.