Low-resolution structures of modular nanotransporters shed light on their functional activity

Yury Ryzhykau.

SASDJY7 – A modular diphtheria toxin/flavohemoprotein nanotransporter (MNT) fused to a melanocyte stimulating hormone ligand module (MNT-MSH)

Modular nanotransporter with a melanocyte stimulating hormone ligand module
MWexperimental 86 kDa
MWexpected 70 kDa
VPorod 127 nm3
log I(s) 2.30×10-2 2.30×10-3 2.30×10-4 2.30×10-5
Modular nanotransporter with a melanocyte stimulating hormone ligand module small angle scattering data  s, nm-1
ln I(s)
Modular nanotransporter with a melanocyte stimulating hormone ligand module Guinier plot ln 2.30×10-2 Rg: 3.7 nm 0 (3.7 nm)-2 s2
(sRg)2I(s)/I(0)
Modular nanotransporter with a melanocyte stimulating hormone ligand module Kratky plot 1.104 0 3 sRg
p(r)
Modular nanotransporter with a melanocyte stimulating hormone ligand module pair distance distribution function Rg: 3.8 nm 0 Dmax: 13.3 nm

Data validation


Fits and models


log I(s)
 s, nm-1
A modular diphtheria toxin/flavohemoprotein nanotransporter (MNT) fused to a melanocyte stimulating hormone ligand module (MNT-MSH) Rg histogram Rg, nm
Modular nanotransporter with a melanocyte stimulating hormone ligand module EOM/RANCH model
Modular nanotransporter with a melanocyte stimulating hormone ligand module EOM/RANCH model
Modular nanotransporter with a melanocyte stimulating hormone ligand module EOM/RANCH model
Modular nanotransporter with a melanocyte stimulating hormone ligand module EOM/RANCH model

log I(s)
 s, nm-1
Modular nanotransporter with a melanocyte stimulating hormone ligand module GASBOR model

Synchrotron SAXS data from solutions of MNT-MSH in 150 mM NaCl, 10 mM phosphate buffer saline, pH 8 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.9 m and at a wavelength of λ = 0.09918 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 500.00 μl sample at 4.2 mg/ml was injected at a 0.75 ml/min flow rate onto a GE Superdex 200 Increase 10/300 column at 20°C. 10 successive 1 second frames were collected through the SEC elution peak of the sample. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

The first part of the amino acid sequence of MNT-MSH encompasses sequences homologous to Diphtheria toxin (https://www.uniprot.org/uniprot/P00587) followed by Flavohemoprotein (https://www.uniprot.org/uniprot/P24232). The C-terminal region has a sequence homology to the Large T antigen from Simian virus 40 (SV40) (https://www.uniprot.org/uniprot/P03070).

Modular nanotransporter with a melanocyte stimulating hormone ligand module (MNT-MSH)
Mol. type   Protein
Organism   synthetic construct
Olig. state   Monomer
Mon. MW   70.4 kDa
Sequence   FASTA