Localization of Cys133 of rabbit skeletal troponin-I with respect to troponin-C by resonance energy transfer.
We have used the technique of resonance energy transfer in conjunction with distance geometry analysis to localize Cys133 of troponin-I (TnI) with respect to troponin-C (TnC) in the ternary troponin complex and the binary TnC.TnI complex in the presence and absence of Ca2+. Cys133 of TnI was chosen because our previous work has shown that the region of TnI containing this residue undergoes Ca2+-dependent movements between actin and TnC, and may play an important role in the regulatory function of troponin. For this purpose, a TnI mutant with a single Cys at position 133, and TnC mutants, each with a single Cys at positions 5, 12, 21, 41, 49, 89, 98, 133, and 158, were constructed by site-directed mutagenesis. The distances between TnI Cys133 and each of the nine residues in TnC were then measured. Using a least-squares minimization procedure, we determined the position of TnI Cys133 in the coordinate system of the crystal structure of TnC. Our results show that in the presence of Ca2+, TnI Cys133 is located near residue 12 beneath the N-terminal lobe of TnC, and moves away by 12.6 A upon the removal of Ca2+. TnI Cys133 and the region of TnC that undergoes major change in conformation in response to Ca2+ are located roughly on opposite sides of TnC's central helix. This suggests that the region in TnI that undergoes Ca2+-dependent interaction with TnC is distinct from that interacting with actin.
Luo
Y
Y
Muscle Research Group, Boston Biomedical Research Institute, Massachusetts 02114, USA.
Wu
J L
JL
Gergely
J
J
Tao
T
T
D000251:Adenosine Triphosphatases / Q000032:analysis*; D000818:Animals; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000032:analysis*; D005454:Fluorescence Polarization / Q000379:methods; D007700:Kinetics; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000201:enzymology; D009210:Myofibrils / Q000737:chemistry* / Q000201:enzymology; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry* / Q000378:metabolism
D019209:Troponin C; D019210:Troponin I; D000251:Adenosine Triphosphatases; D003545:Cysteine