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Hi Tim, <br>
I have just looked at the your paper, and also at Malvern web-site.
How can one get access to this polymer? <br>
regards, Dmitry<br>
<br>
<br>
Timothy Dafforn wrote:
<blockquote
cite="mid:FC95ADD26BA5804F92CDA82BCB7BEE78BDB9ADED@LESMBX1.adf.bham.ac.uk"
type="cite">
<pre wrap="">Hi Tom,
This is what I thought...
We published the method recently in JACS..
Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer.
Knowles TJ, Finka R, Smith C, Lin YP, Dafforn T, Overduin M.
J Am Chem Soc. 2009 Jun 10;131(22):7484-5.
Happy to discuss its use..
Cheers
Tim
-----Original Message-----
From: Tom Laue [<a class="moz-txt-link-freetext" href="mailto:Tom.Laue@unh.edu">mailto:Tom.Laue@unh.edu</a>]
Sent: 26 January 2010 15:52
To: Timothy Dafforn
Cc: Tina Daviter; <a class="moz-txt-link-abbreviated" href="mailto:rasmb@server1.bbri.org">rasmb@server1.bbri.org</a>
Subject: Re: [RASMB] density matching ASB-14
Hi-
To a good first approximation, yes the masses will add. The only caveat
is that you are assuming the partial specific volumes of the components
(polyer, lipid and protein) are unchanged in the complex from what they
are uncomplexed. This assumption usually is valid to a percent or so, so
it is not a big problem.
What is the polymer? (Can you share more info?). It is wonderful to
finally have ways to study membrane proteins as soluble entities.
Best wishes,
Tom
Timothy Dafforn wrote:
</pre>
<blockquote type="cite">
<pre wrap="">Hi All,
First post here..
Just responding to Toms comments on Lipodiscs..
We have an analogous system that replaces the protein that stabilizes the disc with a simple polymer.
We have run a load of SV of these with various membrane proteins in them and empirically it seems that if we do a simple sum along the lines of:
mass of protein in disc = mass of disc with protein - mass of disc
Then we get masses that match with what we expect for the protein.
My question is, is this a reasonable approach, or am I missing some technical issue.
Cheers
Tim
(A CD expert not an AUC expert!)
-----Original Message-----
From: <a class="moz-txt-link-abbreviated" href="mailto:rasmb-bounces@rasmb.bbri.org">rasmb-bounces@rasmb.bbri.org</a> [<a class="moz-txt-link-freetext" href="mailto:rasmb-bounces@rasmb.bbri.org">mailto:rasmb-bounces@rasmb.bbri.org</a>] On Behalf Of Tom Laue
Sent: 09 January 2010 16:13
To: Tina Daviter
Cc: <a class="moz-txt-link-abbreviated" href="mailto:rasmb@server1.bbri.org">rasmb@server1.bbri.org</a>
Subject: Re: [RASMB] density matching ASB-14
Hi-
The benefits to doing density matching are marginal for sedimentation
velocity, whereas they can be considerable for sedimentation
equilibrium. Density matching the detergent for equilibrium allows the
buoyant mass of the protein to be determined without regard to the
extent of detergent binding. For velocity sedimentation, however,
density matching does not remove the contribution of the bound detergent
to the frictional coefficient.
By varying the solvent density it is possible to determine the amount of
bound detergent and the protein molar mass, but it will not account for
the effect on f. Be aware, too, that solvent density matching using
other solvent components (e.g. salts, sugars) may result in a
significant shift in the protein's vbar if there is preferential
solvation, leading to inaccuracies in interpretation. Depending on what
you use to match density, preferential solvation may result in
substantial (5 - 10% error) in vbar. Since there is a 3-fold lever arm
that results in a 3% error in M for every 1% error in vbar, there may be
a 15-30% error in M if the preferential solvation is not accounted for.
Judging from the detergent composition, I would anticipate the vbar for
ASB-14 (my guess is ~0.75 ml/g) to be too low for solvent density
matching using D2O, and that it will appear as 'extra protein' due to
the nearness of its vbar to that of protein.
We have done some experiments with nano-discs (essentially HDL belt
protein + lipid). These are wonderful biophysical tools. The nanodiscs
we used sedimented as a single boundary (~3.5 s) yielding the correct
molecular weight. They were rock stable at 4 C for 5 months (try that
with liposomes!), did not stick to windows or centerpieces and showed no
signs of aggregation upon resuspension after sedimentation. There are
few proteins that are as well behaved. Furthermore, we were able to
determine by sedimentation velocity that a protein integrated into the
nanodiscs as a stable monomer or dimer or tetramer, with no evidence for
trimer or interactions between nanodiscs. The results suggested that the
protein may undergo a 1<>2<>4 reversible association, at least during
the time when the nanodiscs were being synthesized. The nanodiscs could
be used with interference, absorbance and fluorescence optics. Care had
to be taken with absorbance data to use the correct extinction
coefficient for the different protein/lipid complexes. Though we saw no
evidence for it, one always needs to pay attention to the possibility of
variation in the quantum yield with different assembly stoichiometries
with the fluorescence optics. The 100-fold lower concentrations needed
with the fluorescence optics were useful for saving the scarce protein.
Best wishes,
Tom Laue
Tina Daviter wrote:
</pre>
<blockquote type="cite">
<pre wrap="">Dear All,
I will have to run an SV experiment in the presence of the detergent
ASB-14 at 0.5%. It is a solid and I wondered if anyone has any
information on the density of it in solution so I can do density
matching? Or is it not that easy in this case?
The aim of the experiment is to get the oligomeric state of a membrane
protein.
If the density is unavailable, any suggestion on how to most easily
approach the problem would be highly welcome. I fear to determine the
density by running ASB-14 in H20/D20 mixes might fail as it does not
seem to absorb much at any wavelength (is interference sensitive
enough?).
Also, I have not entirely understood how to account for the influence
detergents have on the viscosity. I should be able to organise access
to an Anton-Paar densitometer, but I don't think there is a
viscosimeter that I could use.
Is SE inavoidable?
For more information on the detergent, if that helps, here is the full
name and link to Sigma:
Amidosulfobetaine-14,3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate.
<a class="moz-txt-link-freetext" href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?D7=0&N5=SEARCH_CONCAT_PNO">http://www.sigmaaldrich.com/catalog/ProductDetail.do?D7=0&N5=SEARCH_CONCAT_PNO</a>|BRAND_KEY&N4=A1346|SIGMA&N25=0&QS=ON&F=SPEC
Literature recommendations (preferably overviews and experimental
methods) are also welcome.
Thank you very much in advance.
Regards
Tina
</pre>
</blockquote>
<pre wrap="">
</pre>
</blockquote>
<pre wrap=""><!---->
</pre>
</blockquote>
<br>
<pre class="moz-signature" cols="72">--
Dr. Dmitry Veprintsev
MRC Laboratory of Molecular Biology
Hills Road, Cambridge CB2 0QH, UK
<a class="moz-txt-link-freetext" href="mailto:dbv@mrc-lmb.cam.ac.uk">mailto:dbv@mrc-lmb.cam.ac.uk</a>
Ph +44 (0)1223 402027
Fax +44 (0)1223 402140</pre>
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