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<TITLE>Re: [RASMB] Query concerning membrane protein sedimentation equilibrium experiment</TITLE>
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Greetings Karen and all<BR>
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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?<BR>
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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.<BR>
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Kind regards<BR>
<BR>
Arthur<BR>
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*see Mullin, <U>et al</U> (2008) The plutipotency rheostat Nanog functions as a dimer. Biochem J <B>4</B> 227-231<BR>
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<BLOCKQUOTE><BR>
<B>From: </B>Karen Fleming <Karen.Fleming@jhu.edu><BR>
<B>Date: </B>Tue, 3 Nov 2009 08:34:19 -0500<BR>
<B>To: </B>Arthur Rowe <arthur.rowe@nottingham.ac.uk><BR>
<B>Cc: </B>rasmb@rasmb.bbri.org<BR>
<B>Subject: </B>Re: [RASMB] Query concerning membrane protein sedimentation equilibrium experiment<BR>
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</BLOCKQUOTE><BR>
<BLOCKQUOTE>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 <BR>
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.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=15> <BR>
Kobus FJ, Fleming KG.<BR>
Biochemistry. 2005 Feb 8;44(5):1464-70.<BR>
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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.<BR>
<BR>
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On Nov 3, 2009, at 8:07 AM, Arthur Rowe wrote:<BR>
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<BLOCKQUOTE> Hi John (and everyone)<BR>
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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.<BR>
<BR>
Kind regards<BR>
<BR>
Arthur<BR>
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Arthur J Rowe<BR>
Professor of Biomolecular Technology<BR>
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<BLOCKQUOTE><B>From: </B>John_Doran@vrtx.com<BR>
<B>Date: </B>Mon, 2 Nov 2009 16:46:47 -0500<BR>
<B>To: </B>rasmb@rasmb.bbri.org<BR>
<B>Subject: </B>[RASMB] Query concerning membrane protein sedimentation equilibrium experiment<BR>
<BR>
<BR>
</BLOCKQUOTE><BR>
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<BLOCKQUOTE><BR>
<FONT SIZE="2">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.</FONT> <BR>
<BR>
<FONT SIZE="2">John Doran</FONT> <BR>
<FONT SIZE="2">Vertex Pharmaceuticals</FONT> <BR>
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