[RASMB] Query concerning membrane protein sedimentation equilibrium experiment

Arthur Rowe arthur.rowe at nottingham.ac.uk
Tue Nov 3 06:00:42 PST 2009


Greetings Karen and all

That's a good point made, Karen, concerning the fact that John's
protein/detergent system might be a distribution rather than anything close
to a single species. Maybe SV data is available to shed light on that issue?

IF (and it's quite a big IF) John's detergent can be matched out in the 1.00
to ~1.1 density range, and the easiest way to do it is probably to use
H2-O18 as the matching agent*. No corrections needed for deuteration of the
protein as when conventional 'heavy water' (D2- O16) is used. And nowadays
the cost of H2-O18 has come down massively, thanks to its widespread
clinical application in use of PET scanners.

Kind regards

Arthur

*see Mullin, et al (2008) The plutipotency rheostat Nanog functions as a
dimer. Biochem J 4 227-231


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Arthur Rowe
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From: Karen Fleming <Karen.Fleming at jhu.edu>
Date: Tue, 3 Nov 2009 08:34:19 -0500
To: Arthur Rowe <arthur.rowe at nottingham.ac.uk>
Cc: rasmb at rasmb.bbri.org
Subject: Re: [RASMB] Query concerning membrane protein    sedimentation
equilibrium experiment


There's a statistical treatment for the issue of more than one protein
molecule per micelle, and this can be described by a Poisson distribution.
See 
The GxxxG-containing transmembrane domain of the CCK4 oncogene does not
encode preferential self-interactions.
<http://www.ncbi.nlm.nih.gov/pubmed/15683231?itool=EntrezSystem2.PEntrez.Pub
med.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=15>
Kobus FJ, Fleming KG.
Biochemistry. 2005 Feb 8;44(5):1464-70.

The other issue is that membrane proteins cannot necessarily be thought of
as binding a "micelle's worth" of detergent molecules. (See Marc Le Maire's
work). This is especially true when their molecular weights get larger.
Therefore, monomers and multimers may bind different numbers of detergent
molecules and therefore the buoyant molecular weight of the
protein-detergent complex may be different for monomers+detergent as
compared to multimers+detergent. We always got around this by density
matching the detergent, but sometimes that's not possible for the system.


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On Nov 3, 2009, at 8:07 AM, Arthur Rowe wrote:

 Hi John (and everyone)

That sounds good to me. Of course you could have more than one protein
monomer contained in each micelle - but that should be immediately obvious
from the M value.

Kind regards

Arthur



--
*******************************************************
Arthur J Rowe
Professor of Biomolecular Technology
NCMH Business Centre
University of Nottingham
School of Biosciences
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Leicestershire LE12 5RD   UK

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From: John_Doran at vrtx.com
Date: Mon, 2 Nov 2009 16:46:47 -0500
To: rasmb at rasmb.bbri.org
Subject: [RASMB] Query concerning membrane protein sedimentation equilibrium
experiment





We have measured the amount of detergent bound to our membrane protein and
hence were able to calculate a Vbar for the protein detergent complex. Is it
sufficient to just substitute this value for Vbar along with the density
value of the detergent buffer solution into the ideal species equation of
heteroanalysis to get an accurate molecular weight value of the
protein-detergent complex, or is it more involved than that? Any thoughts
concerning this would be appreciated. Thank you.

John Doran 
Vertex Pharmaceuticals
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