Development and characterization of fluorescently-labeled myosin light chain kinase calmodulin-binding domain peptides.
Calmodulin-dependent protein kinases such as myosin light chain kinase (MLCK), calmodulin kinase II, and phosphorylase kinase contain specific sequences responsible for binding calmodulin. These regions are known as calmodulin-binding domains and in many cases are contained within sequences that are short enough to be synthesized by solid-phase techniques. The ability to chemically-synthesize target enzyme calmodulin-binding domains has permitted the use of a variety of biophysical techniques to study the interactions between calmodulin and calmodulin-binding domain peptides. The work reviewed here describes the development and characterization of peptides based on the sequence of the calmodulin-binding domain of skeletal muscle myosin light chain kinase which were labeled with the fluorescent reagent, acrylodan. Data are presented demonstrating the use of fluorescently-labeled peptides to study various aspects of calmodulin-peptide interactions including binding affinity, stoichiometry, specificity, changes in peptide conformation, and thermal stability of the peptide-calmodulin complex. These data indicate the peptides exhibit many of the salient features seen with calmodulin-target enzyme interactions. The fluorescently-labeled peptides should thus serve as useful models for studying calmodulin-target enzyme interactions at the molecular level.
Blumenthal
D K
DK
Department of Pharmacology & Toxicology, University of Utah, Salt Lake City 84112.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D009219:Myosin-Light-Chain Kinase