[RASMB] vbar

[John Correia]

Chad
 
This requires a discussion of error analysis and the propagation of
error.  If you really are getting a 21/24 reduction in MW that is 12.5%
too low.  This might be due to an error in vbar*rho - I presume you are
also calculating the buffer density, but lets assume its all in vbar -
at a vbar of 0.75 that would correspond to an error in vbar of ~4.2%. 
This seems like a larger error in a calculated vbar but it really
depends upon the solvent and what might be binding to the protein to
alter it.  Alternatively, its a volume change upon assembly & thus
pressure dependent.  ATCase is the classic example of this and in that
case its S value changes by 3% (upon ligand binding) & thus a 1% change
in vbar.  Another example is myosin assembly, but that system exhibits a
remarkable P-dependent disassembly during sedimentation.
 
Of course the whole question depends upon the assumption that its a
tight complex and not exhibiting boundary spreading due to the fact its
an interacting system.  You get a low MW whenever you have a reaction
boundary due to the spreading of the "peak" - think D increasing in an
s/D calculation and thus MW decreases.  Does the data exhibit
concentration dependence?  Spreading can also occur if the system
exhibits kinetic effects.  
 
-------------------------------------------------------------------
 Dr. John J. "Jack" Correia
 Department of Biochemistry
 University of Mississippi Medical Center
 2500 North State Street
 Jackson, MS  39216
 (601) 984-1522                                 
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 email address: jcorreia at biochem.umsmed.edu     
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 dept homepage location:    http://biochemistry.umc.edu/
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>>> Chad Brautigam <Chad.Brautigam at UTSouthwestern.edu> 06/06/06 1:45 PM
>>>

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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