Fluorescence Spectroscopic Studies of Tyrosine Environment and Ligand Binding of Plant Calmodulin

摘要

Recent studies in our laboratories have focused on using tyrosine (Tyr) fluorescence of calmodulin (CaM) and tryptophan (Trp) fluorescence of CaM-bound peptides as intrinsic probes of structure and interactions of this Ca{sup}(2+) regulatory protein. Plant CaM contains a single Tyr (Tyr-138) and vertebrate CaM contains two (Tyr-99 and Tyr-138). Neither protein contains Trp. The fluorescence properties of Tyr-138 of wheat-germ CaM is sensitive to conformational changes induced by perturbations such as Ca{sup}(2+) ligation or depletion, and pH changes. Effects of these perturbations on quantum yield, lifetime and dynamic quenching of Tyr-138 fluorescence are reported. We have also studied binding of amphiphilic peptides to wheat-germ CaM. A comparison of wheat CaM induced changes in the fluorescence properties of a single Trp of these peptides with those induced by bovine testes CaM indicate general similarities of the peptide binding surfaces of plant and mammalian CaMs. Frequency domain measurements of decay of intensity and anisotropy have suggested some orientational freedom and local motion of the Trp residue of CaM-bound peptide, independent of the overall protein motion, even when the Trp is expected to be buried in the doubly apolar protein-peptide interface.