Dataset Information

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

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 Ca(2+) signal to actin) as a kinked alpha-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 beta-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 alpha-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 alpha-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].

SUBMITTER: Brown LJ

PROVIDER: S-EPMC130534 | biostudies-literature | 2002 Oct

REPOSITORIES: biostudies-literature

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

Brown Louise J LJ Sale Ken L KL Hills Ron R Rouviere Clement C Song Likai L Zhang Xiaojun X Fajer Piotr G PG

Proceedings of the National Academy of Sciences of the United States of America 20020918 20

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 Ca(2+) signal to actin) as a kinked alpha-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 t ...[more]

PMID: 12239350

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Project description:KCNE1 is a single transmembrane protein that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in the genes encoding either protein can result in diseases such as congenital deafness, long QT syndrome, ventricular tachyarrhythmia, syncope, and sudden cardiac death. Despite the biological significance of KCNE1, the structure and dynamic properties of its physiologically relevant native membrane-bound state are not fully understood. In this study, the structural dynamics and topology of KCNE1 in bilayered lipid vesicles was investigated using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A 53-residue nitroxide EPR scan of the KCNE1 protein sequence including all 27 residues of the transmembrane domain (45-71) and 26 residues of the N- and C-termini of KCNE1 in lipid bilayered vesicles was analyzed in terms of nitroxide side-chain motion. Continuous wave-EPR spectral line shape analysis indicated the nitroxide spin label side-chains located in the KCNE1 TMD are less mobile when compared to the extracellular region of KCNE1. The EPR data also revealed that the C-terminus of KCNE1 is more mobile when compared to the N-terminus. EPR power saturation experiments were performed on 41 sites including 18 residues previously proposed to reside in the transmembrane domain (TMD) and 23 residues of the N- and C-termini to determine the topology of KCNE1 with respect to the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) lipid bilayers. The results indicated that the transmembrane domain is indeed buried within the membrane, spanning the width of the lipid bilayer. Power saturation data also revealed that the extracellular region of KCNE1 is solvent-exposed with some of the portions partially or weakly interacting with the membrane surface. These results are consistent with the previously published solution NMR structure of KCNE1 in micelles.

Dataset Information

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

Publications

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

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