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PDBsum entry 2jxl
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Structural protein
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PDB id
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2jxl
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References listed in PDB file
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Key reference
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Title
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Tryptophan mutants of cardiac troponin c: 3d structure, Troponin i affinity, And in situ activity.
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Authors
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O.Julien,
Y.B.Sun,
X.Wang,
D.A.Lindhout,
A.Thiessen,
M.Irving,
B.D.Sykes.
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Ref.
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Biochemistry, 2008,
47,
597-606.
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PubMed id
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Abstract
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In situ fluorescence/NMR spectroscopic approaches have been used to elucidate
the structure, mobility, and domain orientations of troponin C in striated
muscle. This led us to consider complementary approaches such as solid-state NMR
spectroscopy. The biophysical properties of tryptophan and Trp-analogues, such
as fluorotryptophan or hydroxytryptophan, are often exploited to probe protein
structure and dynamics using solid-state NMR or fluorescence spectroscopy. We
have characterized Phe-to-Trp mutants in the 'structural' C-domain of cardiac
troponin C, designed to immobilize the indole ring in the hydrophobic core of
the domain. The mutations and their fluorinated analogues (F104W, F104(5fW),
F153W, and F153(5fW)) were shown not to perturb the structural properties of the
protein. In this paper, we characterize the mutations F77W and F77W-V82A in the
'regulatory' N-domain of cardiac troponin C. We used NMR to determine the
structure and dynamics of the mutant F77W-V82A-cNTnC, which shows a unique
orientation of the indole ring. We observed a decrease in calcium binding
affinity and a weaker affinity for the switch region of TnI for both mutants. We
present force recovery measurements for all of the N- and C-domain mutants
reconstituted into skeletal muscle fibers. The F77W mutation leads to a
reduction of the in situ force recovery, whereas the C-domain mutants have the
same activity as the wild type. These results suggest that the perturbations of
the N-domain caused by the Trp mutation disturb the interaction between TnC and
TnI, which in turn diminishes the activity in fibers, providing a clear example
of the correlation between in vitro protein structures, their interactions, and
the resulting in situ physiological activity.
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