Journal article
Disparate Fluorescence Properties of 2-[4′-(Iodoacetamido)anilino]-Naphthalene-6-Sulfonic Acid Attached to Cys-84 and Cys-35 of Troponin C in Cardiac Muscle Troponin
Biophysical journal, Vol.72(2 Pt 1), pp.850-857
02/1997
Handle:
https://hdl.handle.net/2376/104602
PMCID: PMC1185608
PMID: 9017210
Abstract
Two monocysteine mutants of cardiac muscle troponin C, cTnC(C35S) and cTnC(C84S), were genetically generated and labeled with the fluorescent probe 2-[4′-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS) at Cys-84 and Cys-35, respectively. Cys-84 is located on helix D in the regulatory N-domain, and Cys-35 is at the
-y
position of the inactive 12-residue loop of site I. These labeled mutants were studied by a variety of steady-state and time-resolved fluorescence methods. In the absence of divalent cation, the fluorescence of the attached IAANS indicated an exposed environment at Cys-35 and a relatively less-exposed environment at Cys-84. The binding of Ca
2+
to the single regulatory site elicited a large enhancement of the emission of IAANS attached to Cys-84, but only marginal fluorescence changes of the probe at Cys-35. Upon reconstitution of the labeled cTnC mutants with troponin I and troponin T to form the three-subunit troponin, the fluorescence of IAANS-Cys-84 in apo-troponin was spectrally similar to that observed with the Ca
2+
-loaded uncomplexed cTnC mutant. Only very moderate changes in the fluorescence of IAANS-Cys-84 were observed when the regulatory site in reconstituted troponin was saturated. The exposed Cys-35 environment of the uncomplexed cTnC mutant became considerably less exposed and less polar when the mutant was incorporated into apo-troponin. In contrast to the Cys-84 site, saturation of the regulatory site II by Ca
2+
in reconstituted troponin resulted in a reversal of the environment of the Cys-35 site toward a more exposed and more polar environment. These results indicated involvement of the inactive loop I in the Ca
2+
trigger mechanism in cardiac muscle. The fluorescence of IAANS at both Cys-84 and Cys-35 was sensitive to phosphorylation of cTnI in reconstituted troponin, and the sensitivity was observed with both apo-troponin and Ca
2+
-loaded troponin.
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Details
- Title
- Disparate Fluorescence Properties of 2-[4′-(Iodoacetamido)anilino]-Naphthalene-6-Sulfonic Acid Attached to Cys-84 and Cys-35 of Troponin C in Cardiac Muscle Troponin
- Creators
- Wen-Ji Dong - Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294-2041, USAChien-Kao Wang - Department of Physiology and Biophysics, University of Washington, Seattle, Washington 89195 USAAlbert M Gordon - Department of Physiology and Biophysics, University of Washington, Seattle, Washington 89195 USAHerbert C Cheung - Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294-2041, USA
- Publication Details
- Biophysical journal, Vol.72(2 Pt 1), pp.850-857
- Academic Unit
- Chemical Engineering and Bioengineering, School of
- Identifiers
- 99900546886401842
- Language
- English
- Resource Type
- Journal article