Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements.

Fuertes G Banterle N, Ruff KM, Chowdhury A, Mercadante D, Koehler C, Kachala M, Estrada Girona G, Milles S, Mishra A, Onck PR, Gräter F, Esteban-Martín S, Pappu RV, Svergun DI, Lemke EA, Proc Natl Acad Sci U S A 114(31):E6342-E6351 (2017) Europe PMC

SASDEA3 – Labeled thioredoxin (TRX-Alexa488/Alexa594) with denaturant

Thioredoxin 1
Alexa Fluor™ 594 C5 Maleimide
Alexa Fluor™ 488 C5 Hydroxylamine
MWexperimental 10 kDa
MWexpected 13 kDa
VPorod 31 nm3
log I(s) 3.15×102 3.15×101 3.15×100 3.15×10-1
Thioredoxin 1 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine small angle scattering data  s, nm-1
ln I(s)
Thioredoxin 1 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine Guinier plot ln 3.15×102 Rg: 3.2 nm 0 (3.2 nm)-2 s2
(sRg)2I(s)/I(0)
Thioredoxin 1 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine Kratky plot 1.104 0 3 sRg
p(r)
Thioredoxin 1 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine pair distance distribution function Rg: 3.5 nm 0 Dmax: 13.9 nm

Data validation


There are no models related to this curve.

Synchrotron SAXS data from solutions of Labeled thioredoxin (TRX-Alexa488/Alexa594) with denaturant in PBS, 10 mM DTT, 6 M urea, 0.3 M KCl, pH 7.4 were collected on the EMBL P12 beam line at the PETRA III storage ring (DESY; Hamburg, Germany) using a Pilatus 2M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.1 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 10.00 mg/ml was measured at 23°C. 20 successive 0.050 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

The protein contains a penultimate non-canonical amino acid p-acetylphenylalanine (207 Da) that is represented as U (selenocysteine, 168 Da) in the amino acid sequence for the entry. Therefore, the calculated MW from sequence (MW(expected)) must be adjusted accordingly (ca. 40 Da).

Tags: idp
Thioredoxin 1 (TRX-488/594)
Mol. type   Protein
Organism   Escherichia coli
Olig. state   Monomer
Mon. MW   11.8 kDa
 
UniProt   P0AA25 (2-106)
Sequence   FASTA
 
Alexa Fluor™ 594 C5 Maleimide (Alexa594)
Mol. type   Other
Olig. state   Monomer
Mon. MW   0.9 kDa
Alexa Fluor™ 488 C5 Hydroxylamine (Alexa488)
Mol. type   Other
Olig. state   Monomer
Mon. MW   0.7 kDa