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Title: Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR

Abstract

We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca{sup 2+}-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca{sup 2+} state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time {tau} = 1.9 ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca{sup 2+} state showed that Cys133 existed on a stable segment ({tau} = 4.8 ns) held by TnC. Spectra of reconstituted thin filaments (-Ca{sup 2+} state) revealed that slow mobility ({tau} = 45 ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca{sup 2+} concentrations.

Authors:
 [1];  [1];  [1];  [2]
  1. Department of Biological Sciences, Graduate School of Science, Osaka University and CREST/JST, Toyonaka, Osaka 560-0043 (Japan)
  2. Department of Biological Sciences, Graduate School of Science, Osaka University and CREST/JST, Toyonaka, Osaka 560-0043 (Japan). E-mail: arata@bio.sci.osaka-u.ac.jp
Publication Date:
OSTI Identifier:
20798791
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 340; Journal Issue: 2; Other Information: DOI: 10.1016/j.bbrc.2005.12.030; PII: S0006-291X(05)02746-4; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ACTIN; CALCIUM; CALCIUM IONS; ELECTRON SPIN RESONANCE; FILAMENTS; LABELLING; MUSCLES; POLYPEPTIDES; RELAXATION; SENSITIVITY; TROPOMYOSIN

Citation Formats

Aihara, Tomoki, Ueki, Shoji, Nakamura, Motoyoshi, and Arata, Toshiaki. Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR. United States: N. p., 2006. Web. doi:10.1016/j.bbrc.2005.12.030.
Aihara, Tomoki, Ueki, Shoji, Nakamura, Motoyoshi, & Arata, Toshiaki. Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR. United States. doi:10.1016/j.bbrc.2005.12.030.
Aihara, Tomoki, Ueki, Shoji, Nakamura, Motoyoshi, and Arata, Toshiaki. Fri . "Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR". United States. doi:10.1016/j.bbrc.2005.12.030.
@article{osti_20798791,
title = {Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR},
author = {Aihara, Tomoki and Ueki, Shoji and Nakamura, Motoyoshi and Arata, Toshiaki},
abstractNote = {We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca{sup 2+}-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca{sup 2+} state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time {tau} = 1.9 ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca{sup 2+} state showed that Cys133 existed on a stable segment ({tau} = 4.8 ns) held by TnC. Spectra of reconstituted thin filaments (-Ca{sup 2+} state) revealed that slow mobility ({tau} = 45 ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca{sup 2+} concentrations.},
doi = {10.1016/j.bbrc.2005.12.030},
journal = {Biochemical and Biophysical Research Communications},
number = 2,
volume = 340,
place = {United States},
year = {Fri Feb 10 00:00:00 EST 2006},
month = {Fri Feb 10 00:00:00 EST 2006}
}
  • Highlights: •Dipolar EPR detects the distance between the spin-labeled kinesin α-1 and α-2 helices. •The distance has at least two populations: 1.5 nm (in crystal form: 20%) and >2.5 nm. •The short distance conformer was populated 40% in the apo state with microtubules. •ATP analog or ADP binding caused the 1.5 nm distance to be less populated (∼20%). •The α-1 helix moves closer to the neck-linker (away from α-2) to facilitate docking. -- Abstract: In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal regionmore » of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening. The inter-helix distance distribution showed that 20% of the spins have a peak characteristic of 1.4–1.7 nm separation, which is similar to what is predicted from the X-ray crystal structure, albeit 80% were beyond the sensitivity limit (>2.5 nm) of the method. Upon MT binding, the fraction of kinesin exhibiting an inter-helix distance of 1.4–1.7 nm in the presence of AMPPNP (a non-hydrolysable ATP analog) and ADP was 20% and 25%, respectively. In the absence of nucleotide, this fraction increased to 40–50%. These nucleotide-induced changes in the fraction of kinesin undergoing displacement of the α-1 helix were found to be related to the fraction in which the NL undocked from the motor core. It is therefore suggested that a shift in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.« less
  • The duration of activation in cardiac muscle is a function of the load. On the basis of studies of Ca{sup 2+} transients in muscles subjected to quick release, it has been suggested that force or shortening-mediated changes in Ca{sup 2+}-troponin C affinity may provide a mechanism for a contraction-activation feedback. This study was designed to test the hypothesis that the formation of force-generating complexes between actin and myosin enhances the affinity of cardiac troponin C for Ca{sup 2+}. This was done by first establishing the normal relationship between Ca{sup 2+} binding and force development in chemically skinned bovine ventricular musclemore » bundles and then comparing the Ca{sup 2+}-saturation curves obtained with relaxed and contracting muscle bundles. A double isotope technique was used to measure Ca{sup 2+} binding during ATP-induced force generation and during relaxation maintained by the phosphate analogue vanadate. The results showed that the generation of force was associated with an enhanced binding of Ca{sup 2+} to the Ca{sup 2+}-specific regulatory site of cardiac troponin C. These data provide direct evidence that feedback between force and activation in the heart may be mediated by the Ca{sup 2+}-regulatory site of troponin C.« less
  • The EPR spectra of the spin labels attached to loops L11 and L12 of kinesin were resolved into slow (rotational correlation time, {tau} = 10-45 ns) and fast ({tau} = 2 ns) components. The fraction of the slow component increased considerably when kinesin was complexed with a microtubule (MT). On MT binding and in the presence of nucleotides ADP and AMPPNP, the spin labels on L11, particularly at A252C and L249C, significantly decreased the fraction of the slow component. Moreover, dipolar EPR detected a wide distribution in distance range, 1-2 nm between the two spin labels attached to T242C/A252C ormore » A247C/A252C; this distribution was slightly narrower in the presence of MTs than in their absence. These results suggested that the L11 residues undergo conformational transition on the binding of nucleotides and MT, while these residues remained to fluctuate over a nanometer range.« less
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