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<TITLE>Re: [RASMB] frctional ratio</TITLE>
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<BLOCKQUOTE>Ewa -<BR>
<BR>
I think the bottom line is that, without further experimentation, one can only deduce that expectations of elongated conformation (based upon sequence, perhaps ?) seem very plausible.<BR>
<BR>
I entirely agree with John Champagne's point that working at 690 nm, even a rod-shaped particle would behave more or less as an isotropic scatterer. An expected dissymmetry ratio ball-parking at around1.02 or so does not sound very hopeful !<BR>
<BR>
All the best for any further work on your system<BR>
<BR>
Arthur<BR>
<TT><BR>
Arthur and others,<BR>
<BR>
thank you very much for responses.<BR>
<BR>
I will look up the reference.<BR>
<BR>
The protein is not glycosylated and it is expected to<BR>
be elongated, but I was wondering how much more can I<BR>
infer, so Arthur's comments were helpful since I have<BR>
plenty of it left so in a spare time.....<BR>
<BR>
Yes, I had used DAWN for static (and dynamic from one<BR>
angle simultaneously) and cannot get Rg since the<BR>
protein is too small for when using 690 nm.<BR>
<BR>
Thank once more, Ewa<BR>
<BR>
<BR>
<BR>
<BR>
--- Arthur Rowe <arthur.rowe@nottingham.ac.uk> wrote:<BR>
> Dear Ewa<BR>
> <BR>
> A spherical, unhydrated protein of mass 41.7 kD<BR>
> would have an expected Rh =<BR>
> 2.3 nm (if a vbar = 0.73 ml/g is assumed). That<BR>
> means a frictional ratio =<BR>
> 4.2/2.3 = 1.83. <BR>
> <BR>
> As you have correctly inferred, that is clearly not<BR>
> a simple globular<BR>
> protein, for which a frictional ratio in the region<BR>
> of 1.1-1.2 is expected.<BR>
> There are two possibilities:<BR>
> <BR>
> (1) you have one of these 'natively unfolded'<BR>
> proteins, whose normal state<BR>
> is a loosely structured swollen one, or<BR>
> <BR>
> (2) you have an elongated protein, ball-park axial<BR>
> ratio of around 10 giving<BR>
> a Perrin function (that part of the frictional ratio<BR>
> which can be attributed<BR>
> to shape rather than hydration/swelling) = around<BR>
> 1.5 (derived from<BR>
> 1.83/1.2)<BR>
> <BR>
> If you have done static light LS, you ought to be<BR>
> able to extract an Rg<BR>
> value (you can download a pdf about Rg values etc<BR>
> from<BR>
> rabbit.cbe.uiowa.edu/Regression.pdf) from the slope<BR>
> of the Zimm plot.<BR>
> Unfortunately, the difference between the two models<BR>
> is not great, and SLS<BR>
> does not tend to give values of the precision you<BR>
> can get with SANS or SAXS.<BR>
> <BR>
> If - and its a big IF - you have enough material and<BR>
> the kit to hand to do<BR>
> an intrinsic viscosity and an s upon c plot, you are<BR>
> home and dry, as the<BR>
> Ratio Function ks/[eta] has a value of 1.6 for the<BR>
> swollen spherical model,<BR>
> and half that value for an elongated model.<BR>
> <BR>
> But, that is quite a bit more work. To be honest, on<BR>
> the basis of the data<BR>
> you have to date, you might as well toss a coin.<BR>
> Only hint I can give is<BR>
> that natively unfolded proteins of low-ish molecular<BR>
> mass are pretty unusual<BR>
> things. See Uversky, V.N. (2002) Eur J Biochem 269<BR>
> 2-12. So - if I was<BR>
> putting money it, I would go for an elongated shape<BR>
> !<BR>
> <BR>
> All best wishes for your work<BR>
> <BR>
> Arthur Rowe<BR>
> <BR>
> <BR>
> --<BR>
><BR>
*******************************************************<BR>
> Arthur J Rowe<BR>
> Professor of Biomolecular Technology<BR>
> NCMH Business Centre<BR>
> University of Nottingham<BR>
> School of Biosciences<BR>
> Sutton Bonington<BR>
> Leicestershire LE12 5RD UK<BR>
> <BR>
> Tel: +44 (0)115 951 6156<BR>
> +44 (0)116 271 4502<BR>
> Fax: +44 (0)115 951 6157<BR>
> email: arthur.rowe@nottingham.ac.uk<BR>
> arthur.rowe@connectfree.co.uk (home)<BR>
> Web: www.nottingham.ac.uk/ncmh/business<BR>
><BR>
*******************************************************<BR>
> <BR>
> <BR>
> <BR>
> <BR>
> <BR>
> <BR>
> I had just analyzed a protein which has MW of 41.4<BR>
> kDa from static LS<BR>
> analysis (monomer 41.7kDa) and Rh=4.2nm from dynamic<BR>
> LS.<BR>
> <BR>
> What can I infer about the shape (beyond that it is<BR>
> not globular)?<BR>
> Pointing to references appreciated.<BR>
> <BR>
> Thank you, Ewa<BR>
> <BR>
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