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" <a.perra...@nki.nl>
To: CCP4BB@JISCMAIL.AC.UK
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" <ch...@ualberta.ca> 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: b.k...@uq.edu.au 
URL: http://profiles.bacs.uq.edu.au/Bostjan.Kobe.html 
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Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member 
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Netherlands Cancer Institute, 
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-- 
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