[RASMB] SV on membrane proteins

Tue May 18 09:20:51 PDT 2010

Hi, Christine,

Yes, that was a typo.  Thanks for the correction.

We have been using the vbarD from le Maire (2000), rounding to 1.13.

Yes, our s and Mb are lower than one would expect for this protein if  
there were no detergent around.

Doing a very careful c(s), we find that there is a dominant, sharp  
peak, and well as a few minority peaks (~2-3% of sedimenting material)  
well separated from the main peak.  I am therefore modeling all of  
that.  The Mp that we get, after transforming with the (correct)  
formula, gives a number that agrees pretty well with SEC-MALS  
measurements on the same protein/detergent system.  We have performed  
the SV experiment at several different concentrations, and all seem to  
give the same s, so we think that this is not a reaction boundary.

Thanks for your input!

Chad

On May 18, 2010, at 10:22 AM, Christine EBEL wrote:

> Dear Chad,
>
> Perhaps it was a typographic error, but Chad wrote:
> " Mp = Mb (1-phiprime*rho),
> where (neglecting bound lipid)
> (1 - phiprime*rho) = (1 - vbarp*rho) + deltasubD(1-vbarD*rho)"
>
> The first formula is inexact: The appropriate one is:
> Mb = Mp(1-phiprime*rho)
>
> ref  for vbar LDAO=1.128-1.134(le maire) or 1.0597 (calculated,  
> Maslennikov 2007)
>  ref: le Maire et al.  Biochimica et Biophysica Acta 1508 (2000)  
> 86-111
> ref: Maslennikov et al BMC Structural Biology 2007, 7:74
> So that   (1 - phiprime*rho) < (1 - vbarp*rho)
> And Mb and s are expected to be lower than for a soluble protein of  
> same molar mass and frictional ratio.
> The determination of Mb from  one non -interacting particle analysis  
> may be imprecise related to protein heterogeneity equilibrium of  
> association-dissociation or unconsidered LDAO flotation (but you  
> know...).
>
> All the best
> Chritsine
>
>
>
>
>
> Le 17/05/2010 16:28, Mark Agacan a écrit :
>>
>> Hello,
>>
>> I'm also running SV on a membrane protein in 150 mM NaCl, 50 mM NaCl,
>> and 0.1 % Foscholine-12.
>>
>> I have no data for the detergent density or viscosity and so I  
>> tried to
>> omit it completely from the input parameters and see what sedfit c(s)
>> would output.
>>
>> The mass values were way off from what was expected... as expected.
>>
>> Can anyone suggest a way I can deal with density, viscosity, vbar
>> (without actually measuring the density) for this buffer / detergent
>> combination?
>>
>> Cheers
>>
>> Mark
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>> Dr Mark Agacan, Scientific Officer for the Division of Biological
>> Chemistry and Drug Discovery,
>> Wellcome Trust Biocentre, College of Life Sciences, University of
>> Dundee, Dundee, DD1 5EH
>> Tel: +44 1382 386095    Fax: +44 1382 345764    Mobile: 07525 451 117
>> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>>
>>
>> ************************************************************
>> Please consider the environment. Do you really need to print this
>> email?
>>
>>
>>
>>>>> Chad Brautigam <chad.brautigam at utsouthwestern.edu> 5/13/2010 15:41
>>>>>
>>>>>
>> Greetings, All, from the newbiest of membrane-protein newbies,
>>
>> First, let me thank all of you experts who answered (both on- and  
>> off-
>>
>> board) my last question on the topic of membrane proteins.  'Preciate
>>
>> ya, as we say here in Texas.
>>
>> I recently did some SV experiments on a membrane protein solubilized
>> in the accursed floating detergent LDAO (DDAO to some of you).  I was
>>
>> able to fit the data nicely in SEDFIT using a discrete species model.
>>
>> By using the trick of setting the vbar to 0, I fitted for s and for
>> the buoyant molar mass (Mb).  Hooray.
>>
>> In a separate experiment, I used a combination of interference optics
>>
>> and absorbance optics to measure the amount of detergent bound to the
>>
>> protein.  This was very consistent from sample to sample (I tried 2
>> different protein concentrations).  This method is à la le Maire et  
>> al
>>
>> (2008) Nat. Protocols, vol. 3, 1782.
>>
>> OK, so I have Mb and I know the amount of detergent bound to the
>> protein on a g/g basis (this quantity is deltasubD).
>>
>> We have the justly famous formula:
>> Mp = Mb (1-phiprime*rho),
>> where (neglecting bound lipid)
>> (1 - phiprime*rho) = (1 - vbarp*rho) + deltasubD(1-vbarD*rho)
>>
>> So, since I have Mb and deltasubD, and I know rho and the vbars,  
>> can I
>>
>> just plug the Mb that I get from SV into these equations and get a
>> good estimate for Mp, or is there some SV subtlety here that my  
>> newbie
>>
>> brain hasn't apprehended?  I know this can be done for SE, but I've
>> never seen anyone attempt it for SV.
>>
>> Any and all responses will be 'preciated.
>>
>> Chad
>>
>> ======================================
>> Chad A. Brautigam, Ph.D.
>> Assistant Professor
>> Department of Biochemistry
>> The University of Texas
>> Southwestern Medical Center at Dallas
>> Department of Biochemistry
>> 5323 Harry Hines Blvd.
>> Rm. ND10.214A
>> Dallas, TX 75390-8816
>> Office:  214-645-6384
>> Fax:  214-645-6353
>> Email:  chad.brautigam at utsouthwestern.edu
>>
>>
>>
>>
>>
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>
>
> -- 
> Christine EBEL
> Institut de Biologie Structurale CEA-CNRS-UJF
> 41 rue Jules Horowitz, F-38027 Grenoble France
> Tel (33) (0) 4 38 78 95 70; Fax (33) (0) 4 38 78 54 94
> christine.ebel at ibs.fr
> http://www.ibs.fr/laboratories/molecular-biophysics-lab-lbm/ssimpas-435/

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