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 Tr...

Full description

Saved in:
Bibliographic Details
Published in:Molecular diversity 1996-08, Vol.1 (4), p.259-265
Main Authors: Pierce, H H, Adey, N, Kay, B K
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Bacteriophage M13 - genetics
Base Sequence
Calcium-Calmodulin-Dependent Protein Kinases - antagonists & inhibitors
Calmodulin - antagonists & inhibitors
Cattle
Cloning, Molecular
Directed Molecular Evolution - methods
DNA, Complementary - genetics
Enzyme Inhibitors - chemistry
Enzyme Inhibitors - pharmacology
In Vitro Techniques
Ligands
Mice
Peptide Library
Peptides, Cyclic - chemistry
Peptides, Cyclic - genetics
Peptides, Cyclic - pharmacology
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:1381-1991
1573-501X
DOI:10.1007/bf01715530