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 A^2 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
_______________________________________________
phenixbb mailing list
phenixbb@phenix-online.org
http://phenix-online.org/mailman/listinfo/phenixbb
phenixbb mailing list
phenixbb@phenix-online.org
http://phenix-online.org/mailman/listinfo/phenixbb