From: Vermaas, Joshua (
Date: Wed Nov 08 2017 - 14:19:21 CST

Hi Lizelle,

Maybe? One of the big limitations for ILS is that the assumption is that
the particle will only weakly react with the protein, and that the
protein structure (or in this case your sugars) wouldn't be perturbed by
the species being introduced. This is probably not the case for
radicals, where the extra charge can lead to some pretty radical
rearrangements. That being said, if your glycans raise the barrier along
the pathway going from the protein surface to the active site for O2
(probably by sweeping away the molecules and guarding the entrance to
the tunnel), you could make the case that something similar would happen
for superoxide as well. Quantitatively, your numbers would be all wrong
(O2!=superoxide), but qualitatively, you could point to this type of
mechanism as an explanation of the phenomena you see through experiment,
and why glycosylation matters in this case.

-Josh Vermaas

On 11/08/2017 12:53 PM, Lizelle Lubbe wrote:
> Hi VMD users,
> I have performed explicit solvent MD using AMBER on a glycosylated zinc metalloprotease and just came across the implicit ligand sampling method of mapping gas migration pathways in VMD.
> The MD's aim was to investigate the role of glycans in protecting the active site from diffuse radical oxidation observed experimentally. I simulated two highly homologous proteins (90% active site identity) that differ greatly in glycan location and abundance. Experimentally, I see time-dependent oxidative inactivation of the enzyme in the presence of molecular oxygen, H2O2 and ascorbate. I still need to determine if the species responsible for inactivation is superoxide or hydroxide. The enzyme with the least glycans is the most susceptible to diffuse radical oxidation. The solvent accessibility will be mapped to these proteins and the glycan influence assessed in combination with mass spectrometry (similar to hydroxyl radical protein footprinting mass spectrometry).
> I found the gas migration pathway mapping analyses very interesting and thought of applying it to my work.
> I understand that it is an analysis for gas molecules but could the O2 gas pathway perhaps be used as an indication for the superoxide entry to the active site prior to enzyme inactivation?
> The pathway won't be taken as the final result since further experimental work will involve mass spectrometric identification of the oxidation sites. If the sites superimpose onto the oxygen migration pathway it would serve as validation (to my understanding) and yield a more thorough mechanism than simply relating the solvent accessibility to oxidation sites as in radical footprinting mass spectrometry.
> Could someone please comment on whether this type of analysis could be considered scientifically sound?
> Kind regards
> Lizelle Lubbe
> PhD (Medical biochemistry) candidate
> Department of Integrative Biomedical Sciences
> University of Cape Town
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