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Protein Science (2004), 13:640-651. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Using lanthanide ions to align troponin complexes in solution: Order of lanthanide occupancy in cardiac troponin C

Grant L. Gay, Darrin A. Lindhout and Brian D. Sykes

Canadian Institutes of Health Research (CIHR) Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton AB T6G 2H7, Alberta, Canada

(RECEIVED September 11, 2003; FINAL REVISION November 12, 2003; ACCEPTED November 23, 2003)



Abstract

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional {1H, 15N} HSQC NMR spectroscopy has been used to examine the binding order of Ce3+, Tb3+, and Yb3+ to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce3+ to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca2+ ions in the C-domain binding sites. By using the species cTnC•Yb3+•2 Ca2+ it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound 15N-labeled cTnI129–148 by using two-dimensional {1H, 15N} inphase antiphase (IPAP) NMR spectroscopy.

Keywords: Troponin C; lanthanides; NMR; ion binding order; residual dipolar couplings

Abbreviations: TnC, troponin C • cTnC, cardiac muscle TnC • sTnC, skeletal muscle TnC • TnI, troponin I • cTnI, cardiac muscle TnI • cTnI129–148, cTnI peptide (residues 129–148) • HSQC, heteronuclear single quantum coherence • IPAP, inphase antiphase HSQC

Reprint requests to: Brian D. Sykes, 4-19 Medical Sciences Building, Department of Biochemistry, University of Alberta, Edmonton AB T6G 2H7, Canada; e-mail: brian.sykes{at}ualberta.ca; fax: (780) 492-0886.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03412704.


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