I agree completely with Anastassis that the equilibrium will be effected by changing the concentration of the sample during gel filtration, however I wanted to point out that the elution volumes of the two species are independent of their populations. I apologize if I was misleading.
Mike
----- Original Message -----
From: "Anastassis Perrakis" <[log in to unmask]>
To: [log in to unmask]
Sent: Wednesday, August 11, 2010 2:10:16 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] monomer-dimer
Dear all,
If I may add that I find the statement
"First, remember that gel filtration elution volumes are independent of conditions like flow rate and protein concentration (unless there are nonspecific interactions at high concentration), but like I described before temp is a factor."
a bit misleading. While concentration will not change where the monomer or the dimer appears in the elution volume, concentration will affect the monomer-dimer equilibrium during your gel-filtration run.
Thus, I would say that concentration is a factor. If your dimer has a kD of ~100uM, and you inject it at a concentration of ~100uM, after getting diluted during gel-filtration (about ten-fold) it will appear >90% as a monomer ... The results of any analytical technique to determine stoichiometry are concentration dependent, and concentration is actually the major variable that needs to be considered to define the oligomerization state (in AUC this can be done nicely). And do not forget that the in-vitro oligomerization state does not necessarily imply the same for in vivo, so please do make mutants to prove it before submitting the paper ...
A.
On Aug 10, 2010, at 1:38, Bostjan Kobe wrote:
Dear Intekhab
Let me just add to this that gel filtration is not an accurate method for
determination of molecular mass, because the migration on the column depends
on the shape of the protein.
The following methods can be used to determine molecular mass irrespective
of shape:
- MALLS (multi-angle laser light scattering or static light sxattering)
- sedimentation equilibrium on analytical ultracentrifuge (AUC)
- native mass spectrometry
For a short recent review on issues associated with determining oligomeric
state from crystal structures, with older references and relevant
bioinformatic tools cited in there, please see
http://www.ncbi.nlm.nih.gov/pubmed/19021571
Bostjan
On 10/08/10 6:26 AM, "Maia Cherney" <[log in to unmask]> wrote:
To determine the oligomeric state of a protein (monomer or dimer in your
case), it's useful to use the PISA server. You upload your pdb file from
the crystal structure.The server calculates the areas of interfaces
(buried area) and deltaG (change in Gibbs energy) upon oligomer
dissociation. (E. Krissinel and K. Henrick (2007). /Inference of
macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*,
774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein
Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife
2005, LNBI 3695, pp. 163--174 <http://dx.doi.org/10.1007/11560500_15>.
E. Krissinel (2009). /Crystal contacts as nature's docking solutions/.
J. Comp. Chem., in press; published on-line 6 May 2009; DOI
10.1002/jcc.21303}
If the interface area (divided by 2 per one protomer) is greater than
1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's
a dimer. However, don't forget that most dimers can dissociate into
monomers upon dilution. There is a dynamic equilibrium between dimers
(oligomers) and monomers that depends on their concentration and the Kdiss.
Separating them in any method will disturb this equilibrium. If the
re-equilibration time is greater than the separation time, you can see
both monomers and dimers. You can even roughly calculate the
dissociation constant:
Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you
an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of
dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes,
protein needs to dissociate easily for the biological function.
Maia
intekhab alam wrote:
Hi everyone
Sorry for some non specific query!!!!!
i am working with a protein that shows a dimer in the crystal
structure but when i tried to figure out that with standard molecular
markers in gel filteration (superdex-200, 24ml column) it turned out
to be a monnomer. Native gel analysis after incubating the protein at
20 degree, 37 degree showed more dimer at 20 degree celcius as
compared to 37. I tried similar strategy in gel filteration by
incubating my protein at various temperature,where a lot of
precipitation was observed at 37 degree celcius and after removing the
precipitates i run the gel filteration that has 0.5 ml higher elution
volume as compared to samples incubated at 20 degree celcius and 4
degree celcius.( Is this significant)
Furthermore i have done some experiments in cold room (4 degree) where
the elution volume is stuck at a point irrespective of the conditions
(as Flow rate, concentration of protein etc) and that is higher than
that of the room temperature by 1 ml.
Standard moleculr weight markers also show higher elution volume in
cold room in comparison to the room temperature by 1 ml.
I will be highly obliged if someone suggest some literature or any
otherway to do gel filtrtaion so that i can clearly resolve this
issue. Also let me know if there is some literature
available on effect of temperature on the elution volume of proteins.
Thanks in advance
--
INTEKHAB ALAM
LABORATORY OF STRUCTURAL BIOINFORMATICS
KOREA UNIVERSITY, SEOUL
---
Bostjan Kobe
ARC Federation Fellow
Professor of Structural Biology
School of Chemistry and Molecular Biosciences
and Institute for Molecular Bioscience (Division of Chemistry and Structural
Biology) and Centre for Infectious Disease Research
Cooper Road
University of Queensland
Brisbane, Queensland 4072
Australia
Phone: +61 7 3365 2132
Fax: +61 7 3365 4699
E-mail: [log in to unmask]
URL: http://profiles.bacs.uq.edu.au/Bostjan.Kobe.html
Office: Building 76 Room 329
Notice: If you receive this e-mail by mistake, please notify me, and do not
make any use of its contents. I do not waive any privilege, confidentiality
or copyright associated with it. Unless stated otherwise, this e-mail
represents only the views of the Sender and not the views of The University
of Queensland.
P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
--
Michael C. Thompson
Graduate Student
Biochemistry & Molecular Biology Division
Department of Chemistry & Biochemistry
University of California, Los Angeles
[log in to unmask]
|