[RASMB] vbar

[Ewa Folta-Stogniew]

Peter and Chad,

I maybe too pessimistic, but I do not believe that DLS
has enough precision to distinguish 21 vs. 24
configuration.  The standard fitting protocol for Rh
determination is for Gaussian distribution of sizes
with 15% SD, which would encompass sizes for both
configurations making them virtually identical. 

Static LS for MW determination maybe an option given
the experiment is done with accuracy at least +/- 3%. 


Ewa

--- Peter Schuck <pschuck at helix.nih.gov> wrote:

> Hi Chad,
> if the oligomer is relatively pure, would it be
> possible to do 
> DLS?  Perhaps taken together with s, that would give
> another quick way of 
> confirming M, instead of equilibrium sedimentation. 
> I don't think enclosed 
> solvent adds to the buoyant molar mass, therefore it
> would not contribute 
> an error to the vbar.  However, due to error
> propagation from vbar to the 
> (1-vbar*rho) term, small errors in vbar are
> amplified; my feeling is that 
> this usually could account for a few percent
> uncertainty (i.e. in the 
> ballpark of 1/24).
> Peter
> 
> At 02:45 PM 6/6/2006, you wrote:
> >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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