Date of Approval 2017/4/7
Approver Kaoru Shibata
Date of Report 2017/4/7

Experimental Report
Project No.

Name of Instrument scientist

2015A0141

Kaoru Shibata
Title of experiment

Name of Instrument/ BL No.

Dynamics of the cardiomyopathy-causing mutant of troponin

BL02
Date of Experimentstudied by neutron scattering
Name of principal investigator

16/05/19 16/05/23

Satoru Fujiwara
Affiliation
National Institutes for Quantum and Radiological Science andTechnology

Please report your samples, experimental method and results, discussion and conclusions. Please add figures andtables for better explanation.
1.
Name of sample(s) and chemical formula, or compositions including physical form.
The following five D2O solution samples were used in this experiment.
(1) Wild-type human cardiac Troponin (MW: 52 kDa) in the –Ca2+ state (21.2 mg/ml)(2) Wild-type human cardiac Troponin in the +Ca2+ state (22.1 mg/ml)(3) K247R mutant of human cardiac Troponin in the –Ca2+ state (21.8 mg/ml)(4) K247R mutant of human cardiac Troponin in the +Ca2+ state (22.7 mg/ml)(5) D2O Buffer (50 mM HEPES (pD 8.0), 0.5 M NaCl, 5 mM MgCl2, 5 mM EGTA)
Note: For the samples in the +Ca2+ state (2 and 4), 5 mM CaCl2 were added to the buffer instead of 5mM EGTA.

2.
Experimental method and results.

If you failed to conduct experiment as planned, please describe reasons.

Troponin (Tn) is a protein that regulates cardiac muscle contraction depending on the intracellular Ca2+concentration. It consists of three subunits (TnC, TnI, and TnT). Upon Ca2+-binding to TnC, this signal
is transmitted to TnI, TnT, and other regulatory proteins, leading to tension development. This
regulatory function of Tn is essential for cardiac muscle contraction. Various mutations within Tn
molecules, however, have been identified as a cause of inherited cardiomyopathy. Understanding how
these mutations affect the physical properties of Tn is indispensable for elucidation of the molecularmechanism of the pathogenesis of inherited cardiomyopathy. In this experiment, we focused on K247R
mutation of TnT, which is one of the mutants that cause inherited hypertrophic cardiomyopathy, andinvestigated how this mutation affects the internal dynamics of Tn at picosecond timescale byquasielastic neutron scattering (QENS) using the near-backscattering spectrometer BL02 DNA.

Experimental method and results (continued)

2.

We used troponin core domain (Tn-CD), which consists of TnC, TnI, and TnT2 (part of TnT). All thesubunits of human cardiac Tn-CD were bacterially expressed, purified, and then reconstituted into theTn-CD. The Tn-CD containing either wild-type TnT2 (wtTn-CD) or K247R mutant of TnT2 (mtTn-CD)
were dissolved in D2O buffer containing 50 mM HEPES (pD 8.0), 0.5 M NaCl, 5 mM MgCl2. For thesamples in the -Ca2+ state, 5 mM EGTA was added to the buffer, while for the samples in the +Ca2+state, 5 mM CaCl2 were added. Each of these samples (1.65 ml) was put into a cylindrical aluminumcell and then sealed with indium wire. QENS spectra were recorded for 18 hours per sample at 300 K
with the energy resolution of ~12 eV. MLF was operating at ~200 kW.
The QENS spectra of proteins were extracted by subtracting the spectra of the D2O buffer from those of
the samples with an appropriate scaling factor calculated based on the scattering cross-section. The
net spectra of proteins were analyzed by fitting the spectra using a sum of Lorentz functions describing
macromolecular global diffusion and local diffusive motions with a Dirac delta function describing thecontribution of “immobile” atoms. An example of the QENS spectra is shown in Fig. 1 with acorresponding fit (wtTn-CD in the +Ca2+ state; Q=1.650 Å-1).

Fig. 1. Example of the QENS spectra.

Fig. 2. Summary of the dynamics parameters.

In the current analysis, the apparent translational diffusion coefficients (Dapp) of the Tn-CD molecules,
the residence time ( ) and geometry of atomic local motions were evaluated (Concerning geometry ofmotions, average values of the amplitudes of atomic motions are presented here). Regarding the globalmotions, the Dapp values were found to be the same within errors for all the samples. Analysis of these
Dapp values using molecular models of the Tn-CD suggests that the Dapp values include not only theglobal diffusion but also the segmental motions of flexible regions in the Tn-CD, the latter of which arefound not to be affected by the mutation. As for the local motions, it was found that the2+different between the wtTn-CD and the mtTn-CD in the –Ca2+

Ca -binding to the wtTn-CD reduces both thevalues arestate (Fig. 2). In addition, whereas

values and the amplitudes, Ca2+-binding to mtTn-CD

increases the amplitudes without any change in thevalues. This experiment thus shows that themutation changes the internal dynamics of the Tn-CD in both states. More detailed analysis and thepreparation of a paper are currently underway.