[Myotox] toxin/lipid interactions

[Borries Demeler]

Hello everybody,

it has been a while since we have reported back on our NMR findings,
so it is about time to pick up this interesting project again and get
everyone up-to-date on the latest status.

If you recall, Amy found that by using significantly lower SDS
concentrations than CMC that myotoxin-II formed a very stable and
homogeneous oligomer with about 6 or 7 myotoxin-II molecules in it. When
SDS concentration was increased, it was possible to disrupt this oligomer
and generate increasing amounts of heterogeneous oligomeric forms, with
the predominant form being dimer. This observation matched nicely with
the SDS PAGE results reported earlier by Susumu Uchiyama's group.

But of course, more interesting to all of us should be what the
structure of the oligomeric species was, and if it potentially formed 
a membrane penetrating pore, or has some other functional relevance.

If you recall, and in summary, Paul and his group has investigated the
interaction of SDS with myotoxin-II, and after multiple iterations of
different ratios of SDS to myotoxin-II, concluded the following:

   "There seems to be an unusually strong interaction with SDS. The
   alpha methylene exhibits high stress similar to that seen in an epoxy
   ring. Ar first sight it appears to be an AB quartet. Simulations show
   that the 2JHH of the methylene is very small indicating strain."

Paul also mentioned that the NMR studies are hindered by the large
background signals and really would require labeling to allow more
sophisticated multi-dimensional analysis.

I also pursued the potential to get more info with cryo-EM by sending
the samples to the University of Manitoba for study by Pauline Padilla
and Joerg Stetefeld, but this angle unfortunately did not go anywhere,
since negative staining screens did not produce the desired results, 
suggesting cryoEM is out of the question.

Separately, I contacted Bruce Bowler at the University of Montana, who
studies protein-membrane interactions, and he offered an array of other
lipids he had used for crystallizing cytochrome-C interacting with
membranes and still has in the fridge. So we could try to use some of
these lipids to replicate Amy's results with other lipid systems.

Recently, I heard a very interesting talk by Sebastien Poget on the
interactions of various animal toxins with lipids, so I shared our
findings with him. He wrote the following:

   "Thanks for your e-mails, it looks like you have a very interesting
   toxin to study. Is there any precedent for a snake phospholipase
   forming membrane channels? It would make sense physiologically,
   as a quick way to disrupt the membrane and place the phospholipase
   in the right environment to hydrolyze the lipids. One way that
   you could quickly look into this would be to add the phospholipase
   to artificial bilayers - one could maybe use ether-linked lipids
   to prevent hydrolysis and see if any pores are formed just by the
   presence of the toxin alone. We have a planar bilayer system in my lab,
   and I would be happy to give this a try if it would be of interest to
   you. As you mentioned, testing oligomerization in the presence of other
   detergents may also be interesting, including maybe lysolipids. Maybe
   we can set up a zoom call to discuss some of these questions in more
   detail if you are interested?  Best, Sebastien"

I am passing his questions on to this group to get your comments.
Sebastien is now included on our email list, so please feel free to
comment by simply replying to this message. I personally would be very
curious to see if such a membrane pore theory could be tested with
Sebastien's methods.

I would like to follow Sebastien's suggestion and propose a zoom call to
discuss next steps. Here is a Doodle poll for a zoom meeting, please 
respond by the end of tomorrow.

https://doodle.com/poll/xbx5rhhf9g7mkxb3?utm_source=poll&utm_medium=link

If none of the proposed times work for most of us, we could try again 
next week with another doodle poll.

Thanks! -Borries