Journal article
Conformation of the N-Terminal Segment of a Monocysteine Mutant of Troponin I from Cardiac Muscle
Biochemistry (Easton), Vol.36(22), pp.6745-6753
06/03/1997
Handle:
https://hdl.handle.net/2376/116094
PMID: 9184156
Abstract
A monocysteine mutant of cardiac muscle troponin I, cTnI(S5C/C81I/C98S), was generated from a mouse cTnI cDNA clone and expressed in a bacterial system. Cys-5 was modified with the fluorescent sulfhydryl reagent IAANS to probe the conformation of the N-terminal extension of the mutant and the mutant complexed with cardiac muscle troponin C. Our emphasis was on the effect of phosphorylation of Ser-23 and Ser-24 by protein kinase A on the conformation of the N-terminal segment. Phosphorylation resulted in an 8-nm red-shift of the emission spectrum of the attached IAANS probe and a reduction of its quantum yield by a factor of 4−5. The intensity decay of nonphosphorylated IAANS-labeled mutant was complex and had to be described by a sum of three exponential terms, with lifetimes in the range 0.1−5 ns. A fourth component in the range 7−9 ns was required to describe the intensity decay of the phosphorylated mutant. Phosphorylation also reduced the weighted mean lifetime, consistent with the changes observed in the steady-state fluorescence parameters and a 33% decrease in the global rotational correlation time calculated from anisotropy decay data. This change in correlation time suggested a decrease in the axial ratio of the protein. The fluorescence changes of the labeled mutant induced by phosphorylation were carried over to its complex with troponin C. The Stern−Volmer plots of acrylamide quenching of the steady-state fluorescence were essentially linear for nonphosphorylated mutant but displayed pronounced concave downward curvatures for the phosphorylated protein under all conditions studied. The present results are interpreted in terms of a more compact hydrodynamic shape of the phosphorylated cTnI mutant and are consistent with a folded conformation of the N-terminal extension induced by phosphorylation of the two serines. These conformational changes may play a role in the modulation of cardiac muscle contractility by troponin I phosphorylation.
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Details
- Title
- Conformation of the N-Terminal Segment of a Monocysteine Mutant of Troponin I from Cardiac Muscle
- Creators
- Wen-Ji DongMurali ChandraJun XingR. John SolaroHerbert C Cheung
- Publication Details
- Biochemistry (Easton), Vol.36(22), pp.6745-6753
- Academic Unit
- Integrative Physiology and Neuroscience, Department of; Chemical Engineering and Bioengineering, School of
- Publisher
- American Chemical Society
- Identifiers
- 99900547811101842
- Language
- English
- Resource Type
- Journal article