[RASMB] density matching ASB-14

[Tom Laue]

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:
> 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. 
>
> http://www.sigmaaldrich.com/catalog/ProductDetail.do?D7=0&N5=SEARCH_CONCAT_PNO|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
>
>

-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
www.bitc.unh.edu
www.camis.unh.edu