[RASMB] Amyloid fibrils and Fujita approach

[Stephen Harding]

Dear Sabine
I agree with Ewa.  In this instance SEC-MALS seems to be the best option as it gives an absolute molecular weight distribution without assumption of shape.  The "Extended" Fujita approach with SEDFIT (Fujita's original method was only for random coils) was set up as a companion tool for SEC-MALS for situations where there was a concern about non-inertness of the separation columns or the distribution was outside the separation range - and to provide an independent check on the consistency of the results. 
It was intended for polymers with a quasi-continuous distribution of molecular weight - such as polysaccharides, mucins, large glycoconjugates, synthetic polymers etc (indeed a paper is just out in Carbohydrate Polymers on the characterisation of mucins) & it may well be applicable to amyloid fibrils.

If you wish to apply it you need to give an estimate for the power law coefficient b in s = K.M^b, and an estimate for K.

b: Fortunately the sedimentation coefficient is not as sensitive to conformation as the intrinsic viscosity.  I am not familiar with the conformation of amyloids (Ewa would be best able to comment)  but if the chain flexibility of these structures is not known you could try values between~0.2 (rod) to ~0.4 (coil)?  We did something similar for large glycoconjugate vaccines.

K: For K you will need to do know a value of s for a given M.  I suggest you use the weight average s for the whole distribution from SEDFIT and the weight average M from a sedimentation equilibrium experiment.  You can use current software to analyse the latter, although for polydisperse systems a new version of the old MSTAR programme is to be launched soon on SEDFIT called SEDFIT-MStar - this should be relatively straightforward to use, and itself will give an estimate of the absolute mol. wt. distribution.  A related (and new) algorithm - not part of SEDFIT - called Multisig developed by Arthur Rowe will also give you a good estimate of the mol. wt. distribution.  Both these methods have not as yet been through the peer reviewed process but are about to be submitted for publication.

We can certainly help with the analyses if you need it!
All best wishes
Steve
_________________________________________________________________________
Stephen E. Harding, http://www.nottingham.ac.uk/Biosciences/People/steve.harding

-----Original Message-----
From: rasmb-bounces at list.rasmb.org [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of Ewa Folta-Stogniew
Sent: 05 April 2013 18:36
To: Sabine Kaltofen; rasmb at list.rasmb.org
Subject: Re: [RASMB] Amyloid fibrils and Fujita approach

Sabine,

I had analyzed abeta-peptide assembly distributions by SEC-MALLS.  Since MW determination from LS is independent of shape, SEC-MALLS is thus well suited for this heterogeneous system.

Ewa



At 02:34 PM 4/4/2013, Sabine Kaltofen wrote:
>Content-type: multipart/alternative;
>  boundary="Boundary_(ID_PrmXHidevivbwl7flx4wTQ)"
>Content-language: en-GB
>
>
>
>Dear all,
>
>has anyone of you tried to describe the size/molecular weight of 
>amyloid fibrils using the Fujita approach?
>I am after a rather accurate determination of the MW (distribution) of 
>fibrils.
>Therefore, using a scaling law to relate s with M seems the most 
>rigorous thing to do. I am, however not aware of any literature where 
>this approach has been used for peptide/protein fibrils.
>Am I missing a major point here, why this is not applicable?
>
>All comments, experiences and thoughts are greatly appreciated!
>
>Best wishes
>
>Sabine
>
>
>
>Mind your carbon footprint: please don't print this message.
>
>
>Sabine Kaltofen
>Biochemistry & Structural Biology
>Lund University
>sabine.kaltofen at biochemistry.lu.se
>_______________________________________________
>RASMB mailing list
>RASMB at list.rasmb.org
>http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org

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