This Article Full Text Full Text (PDF) Supplement All Versions of this Article: biophysj.107.103879v1 93/3/1008    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Julien, O. Articles by Irving, M. Search for Related Content PubMed PubMed Citation Articles by Julien, O. Articles by Irving, M.

Toward Protein Structure In Situ: Comparison of Two Bifunctional Rhodamine Adducts of Troponin C

Olivier Julien ^*, Yin-Biao Sun , Andrea C. Knowles , Birgit D. Brandmeier , Robert E. Dale , David R. Trentham , John E. T. Corrie , Brian D. Sykes ^* and Malcolm Irving 

^* Canadian Institutes of Health Research Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Canada; Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom; and MRC National Institute for Medical Research, London, United Kingdom

Correspondence: Address reprint requests to Malcolm Irving, Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, United Kingdom. Tel.: 44-207-848-6431; Email: malcolm.irving{at}kcl.ac.uk.

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 Ca²⁺ 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 Ca²⁺-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.