In situ orientations of protein domains: troponin C in skeletal muscle fibers.
A recently developed approach for mapping protein-domain orientations in the cellular environment was used to investigate the Ca(2+)-dependent structural changes in the tropomyosin/troponin complex on the actin filament that regulate muscle contraction. Polarized fluorescence from bifunctional rhodamine probes attached along four alpha helices of troponin C (TnC) was measured in permeabilized skeletal muscle fibers. In relaxed muscle, the N-terminal lobe of TnC is less closed than in crystal structures of the Ca(2+)-free domain, and its D helix is approximately perpendicular to the actin filament. In contrast to crystal structures of isolated TnC, the D and E helices are not collinear. On muscle activation, the N lobe orientation becomes more disordered and the average angle between the C helix and the filament changes by 32 degrees +/- 5 degrees. These results illustrate the potential of in situ measurements of helix and domain orientations for elucidating structure-function relations in native macromolecular complexes.
Ferguson
Roisean E
RE
National Institute for Medical Research, Mill Hill, London, NW7 1AA, USA.
Sun
Yin-Biao
YB
Mercier
Pascal
P
Brack
Andrew S
AS
Sykes
Brian D
BD
Corrie
John E T
JE
Trentham
David R
DR
Irving
Malcolm
M
D000199:Actins / Q000737:chemistry*; D000818:Animals; D002118:Calcium / Q000172:deficiency; D020013:Calcium Signaling / Q000502:physiology; D005454:Fluorescence Polarization; D008968:Molecular Conformation; D015335:Molecular Probes; D015394:Molecular Structure; D009119:Muscle Contraction / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000737:chemistry* / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000648:ultrastructure; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011817:Rabbits; D012235:Rhodamines; D015398:Signal Transduction / Q000502:physiology; D019209:Troponin C / Q000737:chemistry*
D000199:Actins; D015335:Molecular Probes; D012235:Rhodamines; D019209:Troponin C; D002118:Calcium