Preorganized cyclic modules facilitate the self-assembly of protein nanostructures

Snoj J, Lapenta F, Jerala R, Chemical Science (2024) DOI

SASDUP2 – DiTET-nc (coiled coil protein origami tetrahedron assembled from one linear and one cyclized chain)

RH1 (Linear coiled-coil protein origami structure consisting of 6 coiled-coil forming segments)
cyRH2 (cyclic bitrigon from 6 coiled coil segments)
MWexperimental 50 kDa
MWexpected 51 kDa
VPorod 161 nm3
log I(s) 4.70×103 4.70×102 4.70×101 4.70×100
RH1 (Linear coiled-coil protein origami structure consisting of 6 coiled-coil forming segments) cyRH2  (cyclic bitrigon from 6 coiled coil segments) small angle scattering data  s, nm-1
ln I(s)
RH1 (Linear coiled-coil protein origami structure consisting of 6 coiled-coil forming segments) cyRH2  (cyclic bitrigon from 6 coiled coil segments) Guinier plot ln 4.71×103 Rg: 3.6 nm 0 (3.6 nm)-2 s2
(sRg)2I(s)/I(0)
RH1 (Linear coiled-coil protein origami structure consisting of 6 coiled-coil forming segments) cyRH2  (cyclic bitrigon from 6 coiled coil segments) Kratky plot 1.104 0 3 sRg
p(r)
RH1 (Linear coiled-coil protein origami structure consisting of 6 coiled-coil forming segments) cyRH2  (cyclic bitrigon from 6 coiled coil segments) pair distance distribution function Rg: 3.7 nm 0 Dmax: 12.5 nm

Data validation


Fits and models


log I(s)
 s, nm-1
RH1 (Linear coiled-coil protein origami structure consisting of 6 coiled-coil forming segments) cyRH2  (cyclic bitrigon from 6 coiled coil segments) SREFLEX model

Synchrotron SAXS data from solutions of DiTET-nc in 50 mM Tris, 150 mM NaCL, 3% glycerol, pH 7.5 were collected on the EMBL P12 beam line at PETRA III (DESY; Hamburg, Germany) using a Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.124 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 90.00 μl sample at 11.6 mg/ml was injected at a 0.75 ml/min flow rate onto a Cytiva Superdex 200 10/300 column at 20°C. 2040 successive 0.995 second frames were collected through the entire SEC elution, where 53 frames were selected through the primary sample elution peak and used for averaging. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

The experimental molecular weight quoted in this entry was evaluated using SEC-MALS combined with the refractive index (RI) measured before the SAXS measurements.

RH1 (Linear coiled-coil protein origami structure consisting of 6 coiled-coil forming segments) (RH1)
Mol. type   Protein
Organism   synthetic construct
Olig. state   Monomer
Mon. MW   25.7 kDa
Sequence   FASTA
 
cyRH2 (cyclic bitrigon from 6 coiled coil segments) (cyRH2)
Mol. type   Protein
Organism   synthetic construct
Olig. state   Monomer
Mon. MW   25.7 kDa
Sequence   FASTA