GST dimer/monomer question

[Paul N Hengen]

Pete (pkursula at cc.oulu.fi) wrote:

> Is it possible to break a GST dimer into a monomer, and if so, under 
> which conditions would this happen?

Yes. There was a discussion here some time ago which was reviewed
in my monthly column on the topic of GST fusions:

@article{Hengen1996Octtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Purification of {GST} fusion proteins",
journal = "Trends in Biochemical Sciences",
volume = "21",
number = "10",
pages = "400-401",
month = "oct",
year = "1996"}

Since the editors had removed a section regarding the resolution of
dimers from that article (they didn't think it was important enough
to mention), a follow-up section on this will be in the January 1997
issue, which I have not yet seen in print...

@article{Hengen1997Jantibs,
author = "P. N. Hengen",
title = "Methods and reagents - False positives from the yeast
two--hybrid system",
journal = "Trends in Biochemical Sciences",
volume = "22",
number = "1",
pages = "33-34",
month = "jan", 
year = "1997"}

In the paper discussed, the authors were able to resolve GST-CD2 dimers
by denaturing them with 3M guanidine HCl and allowing them to renature.
You should have a look at the original paper for more information on how
they did it:

@article{Murray1995,
author = "A. J. Murray
     and S. J. Lewis
     and A. N. Barclay
     and R. L. Brady",
title = "One sequence, two folds: a metastable structure of {CD2}",
journal = "Proc. Natl. Acad. Sci. U.S.A.",
volume = "92",
pages = "7337-7341",
year = "1995"}

Abstract:

When expressed as part of a glutathione S-transferase fusion protein the
NH2-terminal domain of the lymphocyte cell adhesion molecule CD2 is shown to
adopt two different folds. The immunoglobulin superfamily structure of the
major (85%) monomeric component has previously been determined by both x-ray
crystallography and NMR spectroscopy. We now describe the structure of a
second, dimeric, form present in about 15% of recombinant CD2 molecules. After
denaturation and refolding in the absence of the fusion partner, dimeric CD2 is
converted to monomer, illustrating that the dimeric form represents a
metastable folded state. The crystal structure of this dimeric form, refined to
2.0-A resolution, reveals two domains with overall similarity to the IgSF fold
found in the monomer. However, in the dimer each domain is formed by the
intercalation of two polypeptide chains. Hence each domain represents a
distinct folding unit that can assemble in two different ways. In the dimer the
two domains fold around a hydrophilic interface believed to mimic the cell
adhesion interaction at the cell surface, and the formation of dimer can be
regulated by mutating single residues at this interface. This unusual misfolded
form of the protein, which appears to result from inter- rather than
intramolecular interactions being favored by an intermediate structure formed
during the folding process, illustrates that evolution of protein oligomers is
possible from the sequence for a single protein domain.

--
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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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