Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin.
Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.
Muscle Research Group, Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114, USA.
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Tao
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Gong
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Grabarek
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Gergely
D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D009124:Muscle Proteins / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D019210:Troponin I / Q000737:chemistry*; D020107:Troponin T / Q000737:chemistry*
D009124:Muscle Proteins; D010446:Peptide Fragments; D019210:Troponin I; D020107:Troponin T; D002918:Chymotrypsin; D002118:Calcium