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