The highly conserved COOH terminus of troponin I forms a Ca2+-modulated allosteric domain in the troponin complex.
The primary structure of the COOH-terminal region of troponin I (TnI) is highly conserved among the cardiac, slow, and fast skeletal muscle TnI isoforms and across species. Although no binding site for the other thin filament proteins is found at the COOH terminus of TnI, truncations of the last 19-23 amino acid residues reduce the activity of TnI in the inhibition of actomyosin ATPase and result in cardiac muscle malfunction. We have developed a specific monoclonal antibody (mAb), TnI-1, against the conserved COOH terminus of TnI. Using this mAb, isolation of the troponin complex by immunoaffinity chromatography from muscle homogenate and immunofluorescence microscopic staining of myofibrils indicate that the COOH terminus of TnI forms an exposed structure in the muscle thin filament. Binding of this mAb to the COOH terminus of cardiac TnI induced extensive conformational changes in the protein, suggesting an allosteric role of this region in the functional integrity of troponin. In the absence of Ca2+, the binding of troponin C and troponin T to TnI had very little effect on the conformation of the COOH terminus of TnI as indicated by the unaffected mAb affinity for the TnI-1 epitope. However, Ca2+ significantly increased the accessibility of the TnI-1 epitope on TnI in the presence of troponin C and troponin T. The results provide evidence that the COOH terminus is an essential structure in TnI and participates in the allosteric switch during Ca2+ activation of contraction.
Jin
J P
JP
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106-4970, USA. jxj12@po.cwru.edu
Yang
F W
FW
Yu
Z B
ZB
Ruse
C I
CI
Bond
M
M
Chen
A
A
D000495:Allosteric Site / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000378:metabolism; D000915:Antibody Affinity; D002118:Calcium / Q000737:chemistry*; D002645:Chickens; D017124:Conserved Sequence / Q000276:immunology; D005260:Female; D006801:Humans; D006825:Hybridomas; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011784:Quail; D051381:Rats; D020317:Salmo salar; D019209:Troponin C / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D020107:Troponin T / Q000378:metabolism; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D002118:Calcium