PDBsum entry 2jxl

Structural protein

PDB id: 2jxl

Contents

Protein chain

89 a.a. *

Metals

_CA

* Residue conservation analysis

PDB id:

2jxl

Name:

Structural protein

Title:

Solution structure of cardiac n-domain troponin c mutant f77w-v82a

Structure:

Troponin c, slow skeletal and cardiac muscles. Chain: a. Fragment: regulatory domain. Synonym: tn-c. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: tnnc1, tnnc. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

30 models

Authors:

O.Julien,Y.Sun,X.Wang,D.A.Lindhout,A.Thiessen,M.Irving,B.D.Sykes

Key ref:

O.Julien et al. (2008). Tryptophan mutants of cardiac troponin C: 3D structure, troponin I affinity, and in situ activity. Biochemistry, 47, 597-606. PubMed id: 18092822

Date:

20-Nov-07 Release date: 04-Dec-07

Protein chain

P63316  (TNNC1_HUMAN) -  Troponin C, slow skeletal and cardiac muscles from Homo sapiens

Seq: 161 a.a.

Struc: 89 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Biochemistry 47:597-606 (2008)

PubMed id: 18092822

Tryptophan mutants of cardiac troponin C: 3D structure, troponin I affinity, and in situ activity.

O.Julien, Y.B.Sun, X.Wang, D.A.Lindhout, A.Thiessen, M.Irving, B.D.Sykes.

ABSTRACT

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.