Toward protein structure in situ: comparison of two bifunctional rhodamine adducts of troponin C.
As part of a program to develop methods for determining protein structure in situ, sTnC was labeled with a bifunctional rhodamine (BR or BSR), cross-linking residues 56 and 63 of its C-helix. NMR spectroscopy of the N-terminal domain of BSR-labeled sTnC in complex with Ca(2+) and the troponin I switch peptide (residues 115-131) showed that BSR labeling does not significantly affect the secondary structure of the protein or its dynamics in solution. BR-labeling was previously shown to have no effect on the solution structure of this complex. Isometric force generation in isolated demembranated fibers from rabbit psoas muscle into which BR- or BSR-labeled sTnC had been exchanged showed reduced Ca(2+)-sensitivity, and this effect was larger with the BSR label. The orientation of rhodamine dipoles with respect to the fiber axis was determined by polarized fluorescence. The mean orientations of the BR and BSR dipoles were almost identical in relaxed muscle, suggesting that both probes accurately report the orientation of the C-helix to which they are attached. The BSR dipole had smaller orientational dispersion, consistent with less flexible linkers between the rhodamine dipole and cysteine-reactive groups.
Julien
Olivier
O
Canadian Institutes of Health Research Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Canada.
Sun
Yin-Biao
YB
Knowles
Andrea C
AC
Brandmeier
Birgit D
BD
Dale
Robert E
RE
Trentham
David R
DR
Corrie
John E T
JE
Sykes
Brian D
BD
Irving
Malcolm
M
D000818:Animals; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D009682:Magnetic Resonance Spectroscopy; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000737:chemistry / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D012235:Rhodamines / Q000737:chemistry*; D012997:Solvents; D019209:Troponin C / Q000737:chemistry*
D002247:Carbon Isotopes; D012235:Rhodamines; D012997:Solvents; D019209:Troponin C