determining oligomeric structure of a protein

Russ_Lehrman ou_lehrman at wmich.edu
Fri Mar 4 21:22:27 EST 1994


In article <2l675s$mna at charm.magnus.acs.ohio-state.edu>, rsaldanh at magnus.acs.ohio-state.edu (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.

It isn't clear from this evidence that the protein is only a dimer.  For
example, your crosslinking agents are on the short side, it may be that the
subunits have functional groups that are placed appropiately apart for
reaction.  
, b

> 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 
> machine.

Try dynamic light scatter, if its available.  For analytical
ultracentrifugation, you may want to talk to Dr. Emory Braswell at University
of Conneticut.   Good luck/.

> 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.



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