Ca 2+ ‐induced structural changes and intramolecular interactions in N‐terminal region of diacylglycerol kinase alpha

Takahashi D, Yonezawa K, Okizaki Y, Caaveiro J, Ueda T, Shimada A, Sakane F, Shimizu N, Protein Science 31(7) (2022) DOI

SASDN55 – A recoverin homology domain with EF hand domain of diacylglycerol kinase alpha under the condition with CaCl2

Diacylglycerol kinase alpha
MWI(0) 29 kDa
MWexpected 22 kDa
VPorod 44 nm3
log I(s) 1.91×10-2 1.91×10-3 1.91×10-4 1.91×10-5
Diacylglycerol kinase alpha small angle scattering data  s, nm-1
ln I(s)
Diacylglycerol kinase alpha Guinier plot ln 1.92×10-2 Rg: 1.9 nm 0 (1.9 nm)-2 s2
(sRg)2I(s)/I(0)
Diacylglycerol kinase alpha Kratky plot 1.104 0 √3 sRg
p(r)
Diacylglycerol kinase alpha pair distance distribution function Rg: 2.0 nm 0 Dmax: 6.2 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Diacylglycerol kinase alpha DAMMIF model
Synchrotron SAXS data from solutions of a recoverin homology domain with EF hand domain of diacylglycerol kinase alpha in 20 mM TrisHCl, 200 mM NaCl, 5% Glycerol, 5mM DTT, 3mM CaCl2 pH 7.4 were collected on the beam line BL-10C at the Phton Factory (PF; Tsukuba, Ibaraki, Japan) using a Pilatus3 2M detector at a sample-detector distance of 1.08 m and at a wavelength of λ = 0.1 nm (I(s) vs s, where s = 4πsinθ/λ and 2θ is the scattering angle). 360 successive 20 second frames were collected at 20°C using size-exclusion chromatography SAXS. The sample (200 μL at 11.56 mg/ml) was loaded to Superdex 75 increase 10/300 (Cytiva). The flow rate was set at 0.05 mL/min. The data were normalized to the intensity of the incident beam and circularly averaged; the scattering of the solvent-blank was subtracted. The scattering profiles above the half intensity of the peak (frames 193-215) are averaged by Serial Analyzer. In order to estimate the molecular mass from I(0), PSV (0.737 cm3/g) and the scattering mass contrast (2.695×10^10 cm-2) of this protein were calculated by using the Contrast module of MULCh (http://smb-research.smb.usyd.edu.au/NCVWeb/input.jsp).

Diacylglycerol kinase alpha (RVH-EF)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   22.4 kDa
 
UniProt   P23743 (1-197)
Sequence   FASTA