Fluorescence energy transfer analysis of calmodulin-peptide complexes.
The interactions between calmodulin and the tryptophan residues of synthetic peptides corresponding to the calmodulin binding domains of skeletal muscle myosin light-chain kinase and the plasma membrane calcium pump were examined. The single tryptophan residue contained in each peptide became relatively immobilized and inaccessible to iodide ion upon binding to calmodulin, indicating that the indole side chain was inserted into a hydrophobic cleft in the surface of calmodulin. Fluorescence energy transfer from peptidyl tryptophan residues to an AEDANS moiety attached to cysteine-26 of spinach calmodulin was measured. Included in these analyses was a tryptophan-containing peptide analog of the calmodulin binding domain of neuromodulin. These data indicated that the indole ring of each peptide inserted 32-35 A away from cysteine-26 and may therefore interact with the carboxyl-terminal lobe of CaM in its "bent" conformation [Persechini & Kretsinger (1988a) J. Cardiovasc. Pharmacol. 12 (Suppl 5), S1-S12; Ikura et al. (1992) Science 256, 632-638; Vorherr et al. (1992) Eur. J. Biochem. 204, 931-937]. The interchange of tryptophan-3 and phenylalanine-21 of the calcium pump peptide increased the efficiency of energy transfer to the AEDANS-moiety approximately 12-fold, reducing the calculated distance to 20 A. These data suggest that phenylalanine-21 of the calcium pump peptide interacts with the hydrophobic cleft in the amino-terminal lobe of CaM.
Chapman
E R
ER
Department of Pharmacology SJ-30, University of Washington, Seattle 98195.
Alexander
K
K
Vorherr
T
T
Carafoli
E
E
Storm
D R
DR
D000595:Amino Acid Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003460:Crystallization; D004735:Energy Transfer; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry* / Q000378:metabolism; D009282:Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014965:X-Rays
C032162:AEDANS-calmodulin; D002147:Calmodulin; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; C006421:1,5-I-AEDANS; D014364:Tryptophan; D009219:Myosin-Light-Chain Kinase