Identification of troponin C antagonists from a phage-displayed random peptide library.
Affinity purification of a phage-displayed library, expressing random peptide 12-mers at the N terminus of protein III, has identified 10 distinct novel sequences which bind troponin C specifically. The troponin C-selected peptides yield a consensus binding sequence of (V/L)(D/E)XLKXXLXXLA. Sequence comparison revealed as much as a 62.5% similarity between phiT5, the peptide sequence of the phage clone with the highest level of binding to troponin C, and the N-terminal region of troponin I isoforms. Biotinylated peptides corresponding to library-derived sequences and similar sequences from various isoforms of troponin I were synthesized shown to bind troponin C specifically. Alkaline phosphatase fusion proteins of two of the phage clone sequences bound troponin C specifically, and were specifically competed by both library-derived and native troponin I peptides. Measurement of equilibrium dissociation constants of the peptides by surface plasmon resonance yielded dissociation constants for troponin C as low as 0.43 microM for pT5; in contrast, dissociation constants for calmodulin were greater than 6 microM for all peptides studied. Nondenaturing polyacrylamide gel electrophoresis demonstrated that pT5 formed a stable complex with troponin C in the presence of calcium. We also found that the pT5 peptide inhibited the maximal calcium-activated tension of rabbit psoas muscle fibers.
Pierce
H H
HH
Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Schachat
F
F
Brandt
P W
PW
Lombardo
C R
CR
Kay
B K
BK
D000595:Amino Acid Sequence; D000818:Animals; D017104:Bacteriophage M13; D001665:Binding Sites; D015374:Biosensing Techniques; D016384:Consensus Sequence; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D016658:Psoas Muscles / Q000187:drug effects; D011817:Rabbits; D019209:Troponin C / Q000037:antagonists & inhibitors* / Q000378:metabolism
D019151:Peptide Library; D010455:Peptides; D019209:Troponin C