From: Jo Butler <pjgb@mrc-lmb.cam.ac.uk>
  To  : hlashuel <hlashuel@picower.edu>
  Date: Thu, 22 Mar 2001 10:12:43 -0000

Re: A trimer with a central channel

Dear Hilal,

How did you determine the density increment (or, more probably, partial 
specific volume) which you used to interpret the sedimentation data?
Your description of the structure of the crystal trimer certainly sounds 
like a protein where one could get anomolous solvation, with either binding 
or exclusion of salt ions, and this would be likely to lead to an abnormal 
density increment.  You do not mention the solution conditions, but if you 
have got partial salt exclusion in the channel this would have a 
particularly big effect if you have a reasonably high salt concentration 
(including buffer ions in the total salt).
The density increment (often expressed as [1 - v-bar*rho]) comes into all 
methods of determining molecular mass from sedimentation data and so any 
error will affect both your equilibrium and velocity measurements.

I gave a paper addressing these issues at a Meeting of the UK AUC Users 
Group a couple of years ago (Butler, P.J.G. (1998). Use of density 
increment in assessing protein aggregation under unusual conditions. 
Biochem. Soc. Trans. 26, 749-753).

Jo

--On Sunday, March 21, 2004 3:57 pm -0800 hlashuel <hlashuel@picower.edu> 
wrote:

> I am currently working with a protein that is 12.4 KDa in MW and
> crystallizes as a symmetric trimer. Association of the three monomers
> results in the formation of a solvent accessible channel that runs
> through the molecule giving it an unusual donut shape. The solution
> characterization of this protein has been controversial. gel-filtration,
> NMR and sedimentation equilibrium (rat protein) of this protein have
> reported a dimer in solution. However, cross linking studies have
> suggested a mixture of monomer, dimer and trimer. Sedimentation velocity
> studies carried out in our laboratory show that this molecule sediments
> as a single species with an s value of 2.94 S and a molecular weight of
> 24-25 KDa. However, sedimentation equilibrium yielded a MW of 27 KDa. I
> would like to know what everyone thinks about the central channel of this
> protein, and how it might effect the hydrodynamic properties of this
> protein. Can a trimer (37 KDa behaves like a dimer (24.7 KDa) in
> solution. This proteins elutes as three peaks in gel filtration exp, but
> the data could not be fitted to a monomer, dimer and trimer model.
> Sedimentation equilibrium carried out for each fraction revealed a MW of
> 26-27 KDa for each peak. NO monomer could be detected.
>
>
>
> your comments will be highly appreciated.
>
>
> Hilal



P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296