[RASMB] SV on membrane proteins

Tue May 18 08:22:14 PDT 2010

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
>
>
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>
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>
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>
>
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> 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
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
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>    
>>>> 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
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