title: Fluorescence of a tryptophan bearing peptide from smooth muscle myosin light chain kinase upon binding to two closely related calmodulins.
abstract: We have investigated the fluorescence of a calmodulin binding peptide (AS19) based on the sequence of the calmodulin binding domain of smooth muscle myosin light chain kinase and bearing a tryptophan residue at position 5 upon binding to two closely related calmodulins. The emission maximum of peptide AS19 bound to the engineered SYNCAM calmodulin was 318 nm and a vibrational structure was clearly apparent. The emission maximum of peptide AS19 bound to chicken gizzard calmodulin (ChG CaM) was 327 nm and its spectrum was featureless. Red edge excitation effect supports the assumption that the polarity of Trp-5 environment is larger in the complex with ChG CaM than with SYNCAM, in agreement with fluorescence spectra. Time-resolved fluorescence and anisotropy measurements showed that, in both complexes, the tryptophan emitting state was 1La. The X-ray structure of the calmodulin-peptide complex has been resolved (W. E. Meador, A. R. Means, and F. A. Quiocho, 1992, Science 257, 1251-1255). The Trp binding site has been characterized. It differs by a single-point mutation between the two calmodulins: Met-144 of ChG CaM has been replaced by Val in SYNCAM. This suggests that the spectral relaxation of Trp-5 in the complex with ChG CaM as compared to SYNCAM is due to the polarizability of the sulfur atom containing Met side chain that is higher than that of Val. This provides an ideal system to investigate the origin of the Stokes shift of the indole moiety in proteins.
authors.affiliation: CNRS UA 491, Laboratoire de Biophysique, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, Illkirch, France.
authors.forename: M
authors.initials: M
authors.lastname: Chabbert
authors.forename: E
authors.initials: E
authors.lastname: Piémont
authors.forename: F G
authors.initials: FG
authors.lastname: Prendergast
authors.forename: H
authors.initials: H
authors.lastname: Lami
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D013725:Terbium; D014364:Tryptophan; D009219:Myosin-Light-Chain Kinase