[RASMB] vbar

[Peter Schuck]

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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