[RASMB] vbar

[John Philo]

RASMB,

With regard to Chad's question about the accuracy of the calculated vbar, I
have seen and published one case where the calculated vbar was clearly way
off, giving an apparent MW 9% lower than the sequence value. The
Edelstein-Schachman D20/H20 method proved the calculated vbar was wrong. See
Philo, J. S., Yang, T. H., and LaBarre, M. (2004). Re-examining the
oligomerization state of macrophage migration inhibitory factor (MIF) in
solution.  Biophys. Chem. 108, 77-87.

John 

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Chad Brautigam
Sent: Tuesday, June 06, 2006 11:46 AM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] vbar


Hello, All,

Sorry if this is a rudimentary question.  I have been using velocity  
sedimentation to examine the oligomeric states of a protein and  
mutants thereof.  Some mutants are trimers, and the molecular weight  
estimates given by sedfit (either a c(M) distribution or a discrete  
species model) are very reasonable.  However, based on a crystal  
structure, we expect the wild-type to be a 24-mer.  Sedfit  
consistently underestimates the molecular weight ( I get something  
more akin to 21-mer).

I assume that there are at least to possibilites here:

1.  The crystal structure is wrong, and the thing really is a 21-mer  
in solution.

2.  The vbar calculated by Sednterp is inaccurate- it is not  
accounting for the fact that some of the volume of the 24-mer is not  
taken up by protein, but by solvent.  The vbar is therefore  
significantly too low, with adverse effects on the MW calculation.

Does anyone know if there is a more accurate way to estimate vbar in  
cases of large macromolecular assemblies?  Can our crystal structure  
help us out in any way?

BTW, yes, I know that sed. equilibrium might be the preferred  
approach in this case, but instrument time is limited at the moment.

Thanks,
Chad


==================================
Chad A. Brautigam, Ph.D.
Research Scientist
The University of Texas
Southwestern Medical Center at Dallas
5323 Harry Hines Blvd.
Dallas, TX 75390
Office:    (214) 645-6384
Fax:        (214) 645-5383




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