Fluorescence spectroscopic analysis of the proximity changes between the central helix of troponin C and the C-terminus of troponin T from chicken skeletal muscle.
Recent structural studies of the troponin (Tn) core complex have shown that the regulatory head containing the N-lobe of TnC is connected to the IT arm by a flexible linker of TnC. The IT arm is a long coiled-coil formed by alpha-helices of TnI and TnT, plus the C-lobe of TnC. The TnT is thought to play a pivotal role in the linking of Ca(2+) -triggered conformational changes in thin filament regulatory proteins to the activation of cross-bridge cycling. However, a functional domain at the C-terminus of TnT is missing from the Tn core complex. In this study, we intended to determine the proximity relationship between the central helix of TnC and the TnT C-terminus in the binary and the ternary complex with and without Ca2+ by using pyrene excimer fluorescence spectroscopy and fluorescence resonance energy transfer. Chicken fast skeletal TnC contains a Cys102 at the E helix, while TnT has a Cys264 at its C-terminus. These two cysteines were specifically labeled with sulfhydryl-reactive fluorescence probes. The measured distance in the binary complex was about 19 Angstroms and slightly increased when they formed the ternary complex with TnI (20 Angstroms). Upon Ca2+ binding the distance was not affected in the binary complex but increased by approximately 4 Angstroms in the ternary complex. These results suggest that TnI plays an essential role in the Ca(2+) -mediated change in the spatial relationship between the C-lobe of TnC and the C-terminus of TnT.
Liou
Ying-Ming
YM
Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan. ymlion@dragon.nchu.edu.tw
Chao
Huan-Lu
HL
D000818:Animals; D002118:Calcium / Q000502:physiology; D002645:Chickens; D031541:Fluorescence Resonance Energy Transfer; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry; D009282:Naphthalenesulfonates / Q000737:chemistry; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000737:chemistry*; D020107:Troponin T / Q000737:chemistry*
D008301:Maleimides; D009282:Naphthalenesulfonates; D019209:Troponin C; D020107:Troponin T; C016399:N-(4-dimethylamino-3,5-dinitrophenyl)maleimide; C006421:1,5-I-AEDANS; D002118:Calcium