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<TITLE>Re: [RASMB] problem with fitting</TITLE>
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<BLOCKQUOTE><FONT COLOR="#800000">Hi Everyone<BR>
<BR>
Just one or two quick points on this topic:<BR>
<BR>
(1) I presume, Marina, that by '<B>gly</B>' you do mean <B>gly</B>cerol and not <B>gly</B>cine?<BR>
<BR>
(2) Using the corrected s value calculated by Christopher Chin, and feeding that estimate into the empirical equations of Squire & Himmel (based upon a vast survey of defined 'globular' proteins) and employing your value for vbar yields an estimate of <B>M = 142600 ± 9400</B>. This is plainly a trimer. (bear in mind: your s value is not extrapolated to zero concentration, so it will numerically be a little low, as will therefore the M value)<BR>
<BR>
(3) Having done a 'fit' in Sedfit to give a c(s) profile, why not simply transform it into a c(M) profile? This ought in theory to be a bit more accurate that the Squire-Himmel approach, since it uses an actual estimate* for the frictional ratio of you protein, rather than assuming it to have an 'average' value. <BR>
<BR>
<FONT SIZE="2">*although not, from a single experiment under one set of conditions, a particularly precise value<BR>
</FONT><BR>
(4) the 'disagreement' with a 'model' as defined by XRC surprises me not one bit. The energetics of the protein-protein interaction in the conditions employed for crystal formation are hugely different from those which apply in dilute free solution. An example from my own work with Bill Shaw of some years back: CAT (chloramphenicol acetyl transferase) is a clearly defined trimer in solution, in agreement with XRC. XRC in the presence of manganese ions however clearly shows a hexameric structure - even though AUC analysis continues to show nothing but trimer. It is NOT a question of which method is 'right' - both methods correctly define the oligomeric state of the protein CAT <U>under the conditions under which the method is employed</U>. <BR>
<BR>
(5) finally, Tom - please do not get me started on the issue of 'solvent backflow'. I get tempted into carrying on at some length on that one! I I imagine that Marina's experiment was conducted under conditions of dilution such that for practical purposes we can neglect the issue of s-c dependence.<BR>
<BR>
Regards to all<BR>
<BR>
Arthur<BR>
</FONT><BR>
<BR>
</BLOCKQUOTE><BR>
<BLOCKQUOTE><TT>Hi-<BR>
It should be noted that in addition to the viscosity effects of <BR>
glycerol, there will be solvation effects. Solvation will contribute to <BR>
the Stokes radius of the protein, without necessarily changing the shape <BR>
of the protein. While solvent effects on Rs tend to be small for <BR>
glycerol and other small, inert solutes, they can affect Rs in solutions <BR>
containing small molecules that interact with the protein of interest, <BR>
and they will be substantial solutions containing high concentrations of <BR>
macromolecules. In the latter case, the macromolecule of interest is <BR>
"solvated" with other macromolecules, and it is not unusual to get <BR>
values of f/fo of 4 or greater.<BR>
Also note that the natural frame of reference for the sedimenting <BR>
molecule is the solution, not the cell or the room. This means that <BR>
solvent backflow will contribute to the sedimentation as observed from <BR>
the room frame of reference. Again, solvent backflow is not an issue <BR>
with glycerol, but it is an issue with bulkier solutes (e.g. large <BR>
micelles or vesicles).<BR>
Tom<BR>
<BR>
Chin, Christopher C. wrote:<BR>
><BR>
> Hello Marina,<BR>
><BR>
> As I try to clean up my old e-mail and notice that I miss this <BR>
> discussion about two weeks ago. I remember I did an experiment when <BR>
> Sedfit was release. The reason for doing this experiment is when I <BR>
> analyzed the sample with glycerol presence in the buffer, the S value <BR>
> is always smaller than when I analysis the data using DCDT +, even <BR>
> when I put in the correct density and viscosity value. So I derived an <BR>
> experiment using CRP protein (a dimmer protein with molecular weight <BR>
> about 47KDa) in the presence of different concentration of glycerol. I <BR>
> am sending this in the attachment. If I use the correction constant, <BR>
> then your protein of 6.23S should really be 1.21X 6.23=7.54 S. This <BR>
> will reconfirm it is a trimmer but will not change the trimmer to <BR>
> hexamer as you expected. I hope this information is useful for you.<BR>
><BR>
> All the best,<BR>
><BR>
> Christopher Chin<BR>
><BR>
> -----Original Message-----<BR>
> *From:* rasmb-bounces@rasmb.bbri.org <BR>
> [mailto:rasmb-bounces@rasmb.bbri.org] *On Behalf Of *Thomas Jowitt<BR>
> *Sent:* Monday, March 03, 2008 4:39 AM<BR>
> *To:* thomas.a.jowitt@manchester.ac.uk; Fasolini, Marina [Nervianoms]; <BR>
> rasmb@rasmb.bbri.org<BR>
> *Subject:* RE: [RASMB] problem with fitting<BR>
><BR>
> Hi Marina<BR>
><BR>
> Just a follow up to my previous reply. I notice you are running the <BR>
> sample in 5% glycerol? The viscosity of this alone is 1.127E-2, not <BR>
> what you have mentioned in your mail. If this is the case then this <BR>
> would alter the S20,w of your protein substantially.<BR>
><BR>
> Thanks<BR>
><BR>
> Tom<BR>
><BR>
> ------------------------------------------------------------------------<BR>
><BR>
> *From:* rasmb-bounces@rasmb.bbri.org <BR>
> [mailto:rasmb-bounces@rasmb.bbri.org] *On Behalf Of *Thomas Jowitt<BR>
> *Sent:* 03 March 2008 10:17<BR>
> *To:* Fasolini, Marina [Nervianoms]; rasmb@rasmb.bbri.org<BR>
> *Subject:* RE: [RASMB] problem with fitting<BR>
><BR>
> Dear Marina<BR>
><BR>
> First of all, unless your gel filtration is linked to a light <BR>
> scattering module to estimate the absolute molecular mass, then I <BR>
> wouldnąt trust the estimated molecular weight from a calibrated <BR>
> column. A sedimentation value of 6.23 is most likely to be around <BR>
> 150kDa unless the molecule is substantially elongated, as a hexameric <BR>
> molecule of this size and sedimentation value would have a frictional <BR>
> ratio of around 2.5 (certainly not impossible).<BR>
><BR>
> The two experiments that I would perform to answer these questions <BR>
> are, firstly an equilibrium experiment to establish the correct <BR>
> molecular weight, done with at-least three concentrations to establish <BR>
> if oligomerisation is concentration dependant. Secondly, I would run <BR>
> the crystal structure through a modeling program such as SOMO to <BR>
> generate a bead model, or hydropro to create a shell around the <BR>
> crystal structure. Either of these would give you a decent estimate of <BR>
> the sedimentation value expected for a rigid molecule of that size.<BR>
><BR>
> Thanks<BR>
><BR>
> Tom Jowitt<BR>
><BR>
> ------------------------------------------------------------------------<BR>
><BR>
> *From:* rasmb-bounces@rasmb.bbri.org <BR>
> [mailto:rasmb-bounces@rasmb.bbri.org] *On Behalf Of *Fasolini, Marina <BR>
> [Nervianoms]<BR>
> *Sent:* 03 March 2008 09:10<BR>
> *To:* rasmb@rasmb.bbri.org<BR>
> *Subject:* [RASMB] problem with fitting<BR>
><BR>
> Dear AUC users,<BR>
><BR>
> I have a problem in the fitting of some data and I would like to have <BR>
> your opinion.<BR>
><BR>
> I want to control the sedimentation of a protein in order to define <BR>
> the state of oligomerization. It is published as forming an hexameric <BR>
> homo oligomer. From the crystal structure, it is a wheel like <BR>
> structure of 160 angstrom in diameter, with a central role of 15 <BR>
> angstrom in diameter. The thickness is 20-40 angstrom. How can I <BR>
> expect it to sediment? How can I improve my parameters or model in <BR>
> order to improve the fitting?<BR>
><BR>
> The protein is 50KDa but in gel filtration it cames out as hexamer <BR>
> (300 KDa). In my sedimentation experiment I see one peak of 6.23S <BR>
> which corresponds to 140KDa. Fitting of the sedimentation data was <BR>
> done with SedFit. Do you think I can say that it is a trimer? I would <BR>
> expect it as an hexamer.<BR>
><BR>
> I hope someone can help me.<BR>
><BR>
> the conditions are :<BR>
><BR>
> buffer 20mM Tris pH7, 150mM NaCl, 1mM DTT, 5%gly<BR>
><BR>
> Vbar 0.7397<BR>
><BR>
> Viscosity 1.02530<BR>
><BR>
> Density 0.01183<BR>
><BR>
> Thanks a lot<BR>
><BR>
> Marina<BR>
><BR>
> MARINA FASOLINI<BR>
><BR>
> Structural Chemistry<BR>
><BR>
> Nerviano Medical Sciences <http://www.nervianoms.com/><BR>
><BR>
> Viale Pasteur 10<BR>
><BR>
> 20014 Nerviano - Milano<BR>
><BR>
> marina.fasolini@nervianoms.com <mailto:marina.fasolini@nervianoms.com><BR>
><BR>
> Tel. +390331581462<BR>
><BR>
> Fax. +390331581360<BR>
><BR>
> ------------------------------------------------------------------------<BR>
><BR>
> _______________________________________________<BR>
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> <BR>
<BR>
-- <BR>
Department of Biochemistry and Molecular Biology<BR>
University of New Hampshire<BR>
Durham, NH 03824-3544<BR>
Phone: 603-862-2459<BR>
FAX: 603-862-0031<BR>
E-mail: Tom.Laue@unh.edu<BR>
www.bitc.unh.edu<BR>
www.camis.unh.edu<BR>
<BR>
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