Spectroscopic characterization of the interaction between calmodulin-dependent protein kinase I and calmodulin.
Calmodulin-dependent protein kinase I (CaM kinase I) is a member of the expanding class of protein kinases that are regulated by calmodulin (CaM). Its putative CaM-binding region is believed to occur within a 22-residue sequence (amino acids 299-320). This sequence was chemically synthesized and utilized for CaM interaction studies. Gel band shift assays and densitometry experiments with intact CaM kinase I and the CaM-binding domain peptide (CaMKIp) reveal that they bind in an analogous manner, giving rise to 1:1 complexes. Fluorescence analysis using dansyl-CaM showed that conformational changes in CaM on binding CaM kinase I or CaMKIp were nearly identical, suggesting that the peptide mimicked the CaM-binding ability of the intact protein. In the presence of CaM, the peptide displays an enhancement of its unique Trp fluorescence as well as a marked blue shift of the emission maximum, reflecting a transfer to a more rigid, less polar environment. Quenching studies, using acrylamide, confirmed that the Trp in the peptide on binding CaM is no longer freely exposed to solvent as is the case for the free peptide. Studies with a series of Met mutants of CaM showed that the Trp-containing N-terminal end of CaMKIp was bound to the C-terminal lobe of CaM. Near-UV CD spectra also indicate that the Trp of the peptide and Phe residues of the protein are involved in the binding. These results show that the CaM-binding domain of CaM kinase I binds to CaM in a manner analogous to that of myosin light chain kinase.
Gomes
A V
AV
Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, T2N 1N4, Canada.
Barnes
J A
JA
Vogel
H J
HJ
D020106:Acrylamide / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry*; D002147:Calmodulin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
C035684:5-dimethylaminonaphthalene-1-sulfonyl-calmodulin; D002147:Calmodulin; D010446:Peptide Fragments; D020106:Acrylamide; D014364:Tryptophan; D008715:Methionine; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases