Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: About system absence in P4222?
From: Ronald E Stenkamp stenkamp {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2008-12-11

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Subject: Re: About system absence in P4222?
From: "Borhani, David" David {- dot -} Borhani {- at -} DESHAWRESEARCH {- dot -} COM
Date: 2008-12-11

Subject: Re: O/T: can a protein which dimerizes in solution crystallize as a monomer?
From: "Miller, Mitchell D {- dot -} " mmiller {- at -} SLAC {- dot -} STANFORD {- dot -} EDU
Date: 2008-12-11

Hi Bernie,
We had a case recently which was a dimer in the crystal (with 2
Ca binding sites in the symmetric dimer interface) but anSEC gave
monomer under standard conditions ( 20mM Tris, 200mM NaCl,
0.5mM TCEP at pH7.5, Temperature at 8C ).

The crystals had 0.2 M Ca Acetate. We had a little protein left over
and tried running anSEC+SLS after adding Ca2+ to the protein sample and
using a mobile phase of 20 mM Tris pH 7.5, 200 mM NaCl, 0.2 M CaCl2.
It then ran as a dimer. See comments in remark 300 for pdb id 3DB7
http://www.pdb.org/pdb/explore/explore.do?structureId=3DB7 and
the related TOPSAN page for this protein --
http://www.topsan.org/explore?pdbId=3db7

This supports Pat Loll & Ethan Merritt's comments about the conditions
(crystal and anSEC) influencing on the oligomerizaiton state. Neither
of these conditions are what we expect the protein sees in the periplasm
and we did not any protein left to investigate the concentration of Ca
needed to shift the distribution from monomer to dimer, so it is hard to
say for sure how it functions physiologically.

Regards,
Mitch

-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ethan A Merritt
Sent: Thursday, December 11, 2008 8:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?

On Thursday 11 December 2008, Santarsiero, Bernard D. wrote:
> In parallel with the discussion around this off-CCP4-topic, are they any
> good examples of the opposite case, where the protein is a monomer in
> solution (as evident from light scattering, MW determination through
> centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer?

I don't think such a question is entirely well-defined, for two reasons.

1) The monomer/dimer equilibrium in solution may well depend on the specific
conditions (pH, concentration, presence of ligands, temperature, etc).
Unless these conditions are replicated in your crystallization medium,
it is uncertain to what extent the solution measurement is relevant.

2) How extensive an interface is required in order for it to be considered
a dimer/multimer interaction? In the limiting case of very small
interfaces, the entire crystal might be consider a single oligomer,
with each lattice-packing contact constituting a monomer:monomer
interaction. That's not a very useful place to set the threshold,
but where do you set it - 100 A^2 ? 500 A^2 ? 1000 A^2?
Some definition other than surface area?

That said, I have some interest in the question as a practical matter.
We have a new structure that is "obviously", but totally unexpectedly,
a tetramer in the crystal. In this case the monomer:monomer interaction
surface is >1500 A^2. But exactly what criteria would I use to
argue that this is a real tetramer? What criteria would I use to
argue that it is a crystal artifact? Yes, of course ideally one would
go back to the lab and survey for solution measurements that are
consistent with tetramerization, but that is not always practical,
and may lead right back to your original question.

--
Ethan A Merritt
Biomolecular Structure Center
University of Washington, Seattle 98195-7742

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: About system absence in P4222?
From: Ronald E Stenkamp stenkamp {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2008-12-11

Next message:
Subject: Re: About system absence in P4222?
From: "Borhani, David" David {- dot -} Borhani {- at -} DESHAWRESEARCH {- dot -} COM
Date: 2008-12-11