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
