determining oligomeric structure of a protein

David I Resnick dresnick at
Sat Mar 5 16:23:21 EST 1994

In article <2l675s$mna at> rsaldanh at (Roland J Saldanha) writes:

   I am working on the mitochondrial tyrosyl synthetase that functions as both a 
   synthetase and as a facilitator of group I intron splicing.  In characterizing 
   the protein we find that it elutes from gel filtration columns with an apparent
   molecular weight of a tetramer (monomer MW=72,000).  However the protein 
   crosslinks as a dimer with dimethyl suberimidate and glutaraldehyde.  This 
   would suggest a highly elongated structure that has an aberrant mobility 
   (reflecting the elongated size) in gel filtration.
   I would welcome suggestions for other methods for demonstrating the oligomeric 
   structure of the protein.  I am aware that sedimentation analysis is the best 
   method of approaching this problem but do not have access to an analytical 
   ultracentrifuge and would welcome collaborative offers from anyone who has a 

Gel filtration gives you elution based on Stokes radius, not really on
molecular weight -- unless of course your standards are all the same
shape and are the same shape as your protein.  You can use the
Stokes radius you get from gel filtration and a sedimentation velocity
that you can get from a prep ultracentrifuge (use a glycerol or
sucrose gradient to prevent convection) to calculate a molecular
weight.  You also need a partial specific volume for this calculation
- if you know the amino acid comp you can calculate this, if not you
can determine that as well.  This approach is a bit crude, but it
works.  If the issue is tetramer vs dimer I'd think it would be

Sedimentation equilibrium in an analytical ultracentrifuge is
better, assuming you have modest amounts of pure material.  As I
understand it, there are also methods based on light scattering for
such determinations.  I'd also go for some different crosslinkers -
try long spacer arms, different active groups, and maybe even
unspecific photoactivatable ones (see the Pierce catalog).  

   We would also like to try native page but the protein is very basic
   (computer calculated pI of 10.3).  I would thus welcome suggestions for
   native gels of basic proteins and suitable standards for basic proteins.

As far as native electrophoresis, you could try it with agarose.  You
need a fairly decent seiving though, as the charge-to-mass of your
oligomers would of course be the same as your monomers.  The only
problem with this approach is interpreting the result.  Say you do a
concentration curve with your protein (labelled with 125I or
something) and you see a concentration dependant retardation of mobility.
How do you know what has happened?  You may get the affinity of the
interaction, but it isn't clear that you'd expect to see any
intermediates if your final oligomerization state is a tetramer since
association could be highly cooperative.

Good luck!

       David Resnick                           dresnick at      

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