Identification of cyclized calmodulin antagonists from a phage display random peptide library.
To isolate peptide ligands that bound calmodulin (CaM) specifically, we screened an M13 phage library displaying cyclized octamer random peptides with immobilized bovine CaM. Isolates were recovered, sequenced, and deduced to express nine independent peptides, five of which contained the sequence Trp-Gly-Lys (WGK). Four of the nine peptide sequences were synthesized in cyclized, biotinylated form. All of the peptides required Ca2+ to bind CaM. The cyclized, disulfide-bonded form of one such peptide, SCLRWGKWSNCGS, bound CaM better than its reduced form or an analogue in which the cysteine residues were replaced by serine. The cyclized peptide also exhibited the ability to inhibit CaM-dependent kinase activity. Systematic alanine substitution of residues in this peptide sequence implicate the tryptophan residue as being critical for binding, with other residues contributing to binding to varying degrees. Cloning of ligand targets (COLT) confirmed the specificity of one of the cyclized peptides, yielding full-length and C-terminal CaM clones, in addition to a full-length clone of troponin C, a CaM-related protein. This study has demonstrated that conformationally constrained peptides isolated from a phage library acted as specific, Ca(2+)-dependent CaM ligands.
Pierce
H H
HH
Department of Biology, University of North Carolina at Chapel Hill 27599, USA.
Adey
N
N
Kay
B K
BK
D000595:Amino Acid Sequence; D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
D002147:Calmodulin; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D008024:Ligands; D019151:Peptide Library; D010456:Peptides, Cyclic; D017871:Calcium-Calmodulin-Dependent Protein Kinases