Tryptophan fluorescence quenching by methionine and selenomethionine residues of calmodulin: orientation of peptide and protein binding.

Abstract

The two interaction surfaces of the dumbbell-shaped calcium-regulatory protein calmodulin (CaM) are rich in the amino acid Met. In this work we have used fluorescence spectroscopy to study the role of these Met residues in binding the single Trp residue that is found in many CaM-binding domain peptides. This approach is facilitated by the absence of Trp residues in CaM. In addition to the wild-type protein, we studied CaM containing the unnatural amino acid selenomethionine (SeMet), which was biosynthetically substituted for its nine Met residues. Furthermore, a CaM mutant protein in which all four Met residues in the C-terminal domain were mutated to Leu, and the N-terminal domain contained either Met or the unnatural SeMet, was studied. The Trp fluorescence quantum yield of many Trp-containing CaM-binding peptides increases upon binding to calcium-CaM. Moreover, the emission wavelength of the Trp fluorescence is blue-shifted from 353 to 325-333 nm. These parameters indicate movement of Trp from a solvent exposed to a hydrophobic environment. The fluorescence results obtained with these four CaM variants showed that Se is very effective at quenching Trp fluorescence in the calmodulin-bound peptides from myosin light chain kinase (MLCK) and CaM kinase I, while S is somewhat effective (Se > S > C). The quenching effect is markedly distance dependent, as it only influences the Trp residue of the bound peptide (<=7 A) but has little effect on the two Tyr residues in the C-terminal domain of CaM (>=10 A). Since the Trp fluorescence quenching is very dramatic, the protein containing Leu's in the C-terminal domain and SeMet's in the N-terminal domain allowed us to directly determine the orientation of the MLCK and CaM kinase I peptides bound to CaM; in both cases the Trp residue binds to the C-terminal domain of CaM. Our data indicate that SeMet quenching of Trp fluorescence could become a simple and useful tool for studies of protein folding, and protein-protein and protein-peptide interactions.