Journal article
The Cardiac Ca2+-Sensitive Regulatory Switch, a System in Dynamic Equilibrium
Biophysical journal, Vol.95(10), pp.4772-4789
11/15/2008
Handle:
https://hdl.handle.net/2376/100364
PMID: 18676638
Abstract
The Ca
2+
-sensitive regulatory switch of cardiac muscle is a paradigmatic example of protein assemblies that communicate ligand binding through allosteric change. The switch is a dimeric complex of troponin C (TnC), an allosteric sensor for Ca
2+
, and troponin I (TnI), an allosteric reporter. Time-resolved equilibrium Förster resonance energy transfer (FRET) measurements suggest that the switch activates in two steps: a TnI-independent Ca
2+
-priming step followed by TnI-dependent opening. To resolve the mechanistic role of TnI in activation we performed stopped-flow FRET measurements of activation after rapid addition of a lacking component (Ca
2+
or TnI) and deactivation after rapid chelation of Ca
2+
. Time-resolved measurements, stopped-flow measurements, and Ca
2+
-titration measurements were globally analyzed in terms of a new quantitative dynamic model of TnC-TnI allostery. The analysis provided a mesoscopic parameterization of distance changes, free energy changes, and transition rates among the accessible coarse-grained states of the system. The results reveal that 1), the Ca
2+
-induced priming step, which precedes opening, is the rate-limiting step in activation; 2), closing is the rate-limiting step in de-activation; 3), TnI induces opening; 4), there is an incompletely deactivated population when regulatory Ca
2+
is not bound, which generates an accessory pathway of activation; and 5), there is incomplete activation by Ca
2+
—when regulatory Ca
2+
is bound, a 3:2 mixture of dynamically interconverting open (active) and primed-closed (partially active) conformers is observed (15°C). Temperature-dependent stopped-flow FRET experiments provide a near complete thermokinetic parameterization of opening: the enthalpy change (Δ
H
= −33.4 kJ/mol), entropy change (Δ
S
= −0.110 kJ/mol/K), heat capacity change (Δ
C
p
= −7.6 kJ/mol/K), the enthalpy of activation (
δ
‡
= 10.6 kJ/mol) and the effective barrier crossing attempt frequency (
ν
adj
= 1.8 × 10
4
s
−1
).
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Details
- Title
- The Cardiac Ca2+-Sensitive Regulatory Switch, a System in Dynamic Equilibrium
- Creators
- John M Robinson - Department of Biochemistry and Molecular GeneticsHerbert C Cheung - Department of Biochemistry and Molecular GeneticsWenji Dong - Department of Biochemistry and Molecular Genetics
- Publication Details
- Biophysical journal, Vol.95(10), pp.4772-4789
- Academic Unit
- Chemical Engineering and Bioengineering, School of
- Publisher
- The Biophysical Society
- Identifiers
- 99900546570501842
- Language
- English
- Resource Type
- Journal article