Caldesmon-binding sites on tropomyosin.
The interaction of chicken gizzard caldesmon with fragments of tropomyosin, generated by chemical, enzymatic, and mutational means, was studied to determine the caldesmon-binding site(s) on tropomyosin. Binding was examined by fluorescence spectroscopy and affinity chromatography. Removal of residues 1-141 and 228-284, respectively, from the NH2 and COOH ends of tropomyosin did not affect its binding to caldesmon significantly, indicating that the major, caldesmon-binding region lies between residues 142-227. The Escherichia coli produced chicken gizzard beta-tropomyosin mutant, CSM-beta (1/8/12-227), bound caldesmon about 2-fold stronger than a similar mutant of residues 8-200. This further focused the primary caldesmon-binding site to residues 201-227. Cleavage of tropomyosin at CYS-190 weakened markedly the binding of the two resulting fragments, residues 1-189 and 190-284, to caldesmon suggesting the requirement for the integrity of the caldesmon-binding region between residues 142227 of tropomyosin for strong interaction with caldesmon. Based on data from this study and others, we have proposed models for the interaction of tropomyosin with caldesmon in vitro, as well as the possible arrangement of the smooth muscle thin filament proteins in vivo.
Watson
M H
MH
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Kuhn
A E
AE
Novy
R E
RE
Lin
J J
JJ
Mak
A S
AS
D000818:Animals; D001665:Binding Sites; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002645:Chickens; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005895:Gizzard, Avian; D007700:Kinetics; D008958:Models, Molecular; D009130:Muscle, Smooth / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D015183:Restriction Mapping; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism*
D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D014335:Tropomyosin