[RASMB] density matching ASB-14

Tue Jan 26 08:42:49 PST 2010

Hi Tim (and all)

If you can match out the detergent density, preferably with 18O water  
rather than Deuterium Oxide* (now that the former has come down hugely  
in price thanks to its hospital use in PET-scanners), then the  
precision with which you can get the MW of the protein far exceeds what  
you need to say 'monomer or dimer?'

See reference below for an example of this

Kind regards

Arthur

*to be avoided if possible, it can destabilise some proteins, and  
always changes their mass by an amount not accurately known

Mullin et al (2008) Biochem J 411 227-231


On Jan 26, 2010, at 16:34, Timothy Dafforn wrote:

> Whoops forgot to also mention..
> We have also done a fair amount of AUC SV of membrane proteins in  
> detergent..
> Most of the time this is just to check the samples contain single  
> species (for the Xtalographers!)
> However they always ask if I can give them an approximate mass to  
> ensure that, for example, if they expect a dimer it is in fact a  
> dimer..
> I have to say I am always cagey, but just how inaccurate are the  
> masses likely to be?
> +/- 100%? Or +/- 10%...
> Tim
>
> -----Original Message-----
> From: Tom Laue [mailto:Tom.Laue at unh.edu]
> Sent: 26 January 2010 15:52
> To: Timothy Dafforn
> Cc: Tina Daviter; rasmb at server1.bbri.org
> 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:
>> 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: rasmb-bounces at rasmb.bbri.org  
>> [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Tom Laue
>> Sent: 09 January 2010 16:13
>> To: Tina Daviter
>> Cc: rasmb at server1.bbri.org
>> 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:
>>
>>> 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
>
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