Substrate Induced Movement of the Metal Cofactor between Active and Resting State

Marsden S, Wijma H, Mohr M, Justo I, Hagedoorn P, Laustsen J, Jeffries C Svergun D, Mestrom L, McMillan D, Bento I, Hanefeld U, Angewandte Chemie International Edition (2022) DOI

SASDKU7 – Hydroxypyruvate aldolase (HpcH/HpaI aldolase), S116C mutant in HEPES, NaCl and MgCl2

HpcH/HpaI aldolase (S116C)
MWexperimental 157 kDa
MWexpected 165 kDa
VPorod 223 nm3
log I(s) 1.75×104 1.75×103 1.75×102 1.75×101
HpcH/HpaI aldolase (S116C) small angle scattering data  s, nm-1
ln I(s)
HpcH/HpaI aldolase (S116C) Guinier plot ln 1.75×104 Rg: 3.3 nm 0 (3.3 nm)-2 s2
(sRg)2I(s)/I(0)
HpcH/HpaI aldolase (S116C) Kratky plot 1.104 0 3 sRg
p(r)
HpcH/HpaI aldolase (S116C) pair distance distribution function Rg: 3.3 nm 0 Dmax: 9 nm

Data validation


Fits and models


log I(s)
 s, nm-1
HpcH/HpaI aldolase (S116C) GASBOR model

log I(s)
 s, nm-1
HpcH/HpaI aldolase (S116C) PDB (PROTEIN DATA BANK) model

Synchrotron SAXS data from solutions of S116C mutant hydroxypyruvate aldolase in 20 mM HEPES, 100 mM NaCl, 20 mg/L MgCl2, pH 7.5 were collected using size exclusion chromatography SAXS (SEC-SAXS) on the EMBL P12 beam line at PETRA III (Hamburg, Germany) equipped with a Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.124 nm (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). The SEC parameters were as follows: A 75 μl sample at 10 mg/ml was injected at room temperature onto a GE Healthcare Superdex 200 Increase 10/300 column at a flow rate of 0.6 mL/min. 28 successive 1 second frames were collected through the SEC-elution peak that corresponded to the molecular weight (MW) of the hexamer (as assessed using multi-angle laser light scattering/refractive index measurements; MALLS/RI). The data were normalized to the intensity of the transmitted beam and radially averaged and the scattering of an appropriate solvent-blank was subtracted and the data scaled and averaged to produce the final SAXS profile displayed in this entry. CHROMIXS was used to process the SEC-SAXS data and the MALLS/RI results are made available in the full entry zip archive.

The quoted experimental MW is that determined from MALLS.

HpcH/HpaI aldolase (S116C)
Mol. type   Protein
Organism   Rhizorhabdus wittichii RW1
Olig. state   Hexamer
Mon. MW   27.5 kDa
Sequence   FASTA
 
PDB ID   6R62