The mechanism behind acetyl-CoA-mediated activation in Mycobacterium tuberculosis isocitrate lyase 2 and the contribution of its unique protein features to allostery and selectivity

Ashish Sethi.

SASDVN7 – Mycobacterium tuberculosis Isocitrate Lyase 2 mutant (Q635C, V708C, V734C)

Isocitrate lyase 2 (Q635C, V708C, V734C)
MWexperimental 330 kDa
MWexpected 87 kDa
VPorod 526 nm3
log I(s) 2.59×10-1 2.59×10-2 2.59×10-3 2.59×10-4
Isocitrate lyase 2 (Q635C, V708C, V734C) small angle scattering data  s, nm-1
ln I(s)
Isocitrate lyase 2 (Q635C, V708C, V734C) Guinier plot ln 2.59×10-1 Rg: 5.2 nm 0 (5.2 nm)-2 s2
(sRg)2I(s)/I(0)
Isocitrate lyase 2 (Q635C, V708C, V734C) Kratky plot 1.104 0 √3 sRg
p(r)
Isocitrate lyase 2 (Q635C, V708C, V734C) pair distance distribution function Rg: 5.4 nm 0 Dmax: 20.6 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Isocitrate lyase 2 (Q635C, V708C, V734C) DAMMIN model
Synchrotron SAXS data from solutions of Mycobacterium tuberculosis Isocitrate Lyase 2 mutant (Q635C, V708C, V734C) in 50 mM HEPES, 150 mM NaCl, pH 7.5 were collected on the SAXS/WAXS beam line at the Australian Synchrotron (Melbourne, Australia) using a Pilatus3 S 2M detector at a sample-detector distance of 2.5 m and at a wavelength of λ = 0.10332 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) co-flow SAXS was employed. The SEC parameters were as follows: A 50.00 μl sample at 5 mg/ml was injected at a 0.40 ml/min flow rate onto a GE Superdex 200 Increase 5/150 column at 20°C. 300 successive 1 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.

Isocitrate lyase 2 (Q635C, V708C, V734C) (ICL2 mutant)
Mol. type   Protein
Organism   Mycobacterium tuberculosis (strain ATCC 35801 / TMC 107 / Erdman)
Olig. state   Monomer
Mon. MW   87.5 kDa
 
UniProt   H8F3R6 (1-766)
Sequence   FASTA