Structure of the inhibitory region of troponin by site directed spin labeling electron paramagnetic resonance

Louise J. Brown, Ken L. Sale, Ron Hills, Clement Rouviere, Likai Song, Xiaojun Zhang, Piotr G. Fajer*

*Corresponding author for this work

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

Site-directed spin labeling EPR (SDSL-EPR) was used to determine the structure of the inhibitory region of TnI in the intact cardiac troponin ternary complex. Maeda and collaborators have modeled the inhibitory region of TnI (skeletal 96-112: the structural motif that communicates the Ca2+ signal to actin) as a kinked α-helix [Vassylyev, D., Takeda, S., Wakatsuki, S., Maeda, K. & Maeda, Y. (1998) Proc. Natl. Acad. Sci. USA 95, 4847-4852), whereas Trewhella and collaborators have proposed the same region to be a flexible β-hairpin [Tung, C. S., Wall, M. E., Gallagher, S. C. & Trewhella, J. (2000) Protein Sci. 9, 1312-1326]. To distinguish between the two models, residues 129-145 of cardiac TnI were mutated sequentially to cysteines and labeled with the extrinsic spin probe, MTSSL. Sequence-dependent solvent accessibility was measured as a change in power saturation of the spin probe in the presence of the relaxation agent. In the ternary complex, the 129-137 region followed a pattern characteristic of a regular 3.6 residues/turn α-helix. The following region, residues 138-145, showed no regular pattern in solvent accessibility. Measurements of 4 intradomain distances within the inhibitory sequence, using dipolar EPR, were consistent with an α-helical structure. The difference in side-chain mobility between the ternary (C·I·T) and binary (C·I) complexes revealed a region of interaction of TnT located at the N-terminal end of the inhibitory sequence, residues 130-135. The above findings for the troponin complex in solution do not support either of the computational models of the binary complex; however, they are in very good agreement with a preliminary report of the x-ray structure of the cardiac ternary complex [Takeda, S. Yamashita, A., Maeda, K. & Maeda, Y. (2002) Biophys. J. 82, 832].

Original languageEnglish
Pages (from-to)12765-12770
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number20
DOIs
Publication statusPublished - 1 Oct 2002
Externally publishedYes

Keywords

  • DEER
  • Dipolar
  • Fourier transform electron paramagnetic resonance
  • Spin labels
  • Troponin I

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