Calcium-induced flexibility changes in the troponin C-troponin I complex.
The contraction of vertebrate striated muscle is modulated by Ca(2+) binding to the regulatory protein troponin C (TnC). Ca(2+) binding causes conformational changes in TnC which alter its interaction with the inhibitory protein troponin I (TnI), initiating the regulatory process. We have used the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific sites in the TnC-TnI complex in the presence and absence of Ca(2+) or Mg(2+). Using sequences based on rabbit skeletal muscle proteins, we prepared functional, binary complexes of wild-type TnC and a TnI mutant which contains no Cys residues and a single Trp residue at position 106 within the TnI inhibitory region. We used TnI Trp-106 as the FRET donor, and we introduced energy acceptor groups into TnC by labeling at Met-25 with dansyl aziridine or at Cys-98 with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our distance distribution measurements indicate that the TnC-TnI complex is relatively rigid in the absence of Ca(2+), but becomes much more flexible when Ca(2+) binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, helping to release the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distributions between TnC and TnI in their binary complex.
Zhao
X
X
Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA.
Kobayashi
T
T
Gryczynski
Z
Z
Gryczynski
I
I
Lakowicz
J
J
Wade
R
R
Collins
J H
JH
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D017931:DNA Primers; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000737:chemistry / Q000378:metabolism*; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D019209:Troponin C; D019210:Troponin I; D002118:Calcium