Hi This question is outside my area but I was intrigued from a materials characterisation perspective I would suggest SDS-PAGE or NMR and came across this paper that may be of interest:

Lidón-Moya MC, Barrera FN, Bueno M, Pérez-Jiménez R, Sancho J, Mateu MG, Neira JL. Dimeric form of diphtheria toxin: purification and characterization. Protein Sci. 2005 Sep;14(9):2387-404.

Hope this is of use

Examining rotational correlation time (TauC) by NMR will give you the answer for that.

Thank you for your suggestions. However, NMR is not accessible here and as a first step, I have decided to check monomer dimer equilibrium by ITC. The protein is soluble at hight concentration and so I hope this technique may work.

Just recording a 15N-HSQC NMR can be informative in your case, if your protein is small (say under 150 amino acids). If it is a strict monomer you will see around 150 peaks that are very uniform. The peak positions should not change if you change the concentration. If it is a dimer you will probably see lots of overlapping peaks and possible less than 150 peaks. So there is a size constraint on this experiment.

What is the technique that you used to determine that it is a monomer. If it is gel filtration, increase the protein concentration and check whether you can see dimers appearing.

Now that you have the crystal structure, make mutants that disrupt dimer formation and see if they behave any differently from the WT(do you have an activity to measure?).

You can try cross linking experiments followed by tryptic digestion and mass spectrometry. This will validate your dimer interface. Beware of artifacts while cross linking. 

Once you have characterized some dimers, you can try the macromolecular crowding experiments. 

Hope these are useful and not a distraction.

Cheers

Thank you Sujay. NMR could give the answer but I do not have an NMR machine here and so that wont be practical for me. Cross-linking and mutation are good choices. The protein is seen as monomer in lower concentration using gel filtration. For any experiment, my protein dimerise only at crystal condition in which my protein is at concentration of ~30 mg/ml. This could be even higher in the crystal, due to evaporation. As a first step, I am trying dilution equilibrium using ITC and later will use AUC and will update the result here. Thank you.

Analytical ultracentrifugation with a fluorescence detection system is an excellent approach to studying a protein of interest in solutions containing high concentrations of other proteins or cellular components. See: J. S. Kingsbury and T. M. Laue. Fluorescence-detected sedimentation in dilute and highly concentrated solutions. Methods Enzymol. 492:283-304, 2011. Our lab has used it to determine the state of association of enzyme complexes: S. L. Matte, T. M. Laue, and R. H. Cote. Characterization of the conformational changes and protein-protein interactions of the rod photoreceptor phosphodiesterase (PDE6). J.Biol.Chem. 287:20111-20121, 2012.

Hi Stephen, 

I could think of several way but I am not sure what exactly "macromolecular crowded" means. If that is simply high concentrations of your purified protein, you could try one of the following (please excuse if my suggestions are redundant to other previous posts):

- try analytic gel filtration at high concentration

- if available, analytic ultracentrifugation should work

- a very good technique that needs some fine-tuning is chemical/physical cross linking and SDS page analysis. You can use glutaraldehyde (might give to much unspecific super-crosslinks at high concentration) or a zero length cross linker like Ruthenium (see this link for a simple overview and references http://www.koko.gov.my/CocoaBioTech/Membrane%20Transfer4.html). The latter is induced with UV light and the actual cross link can be done very specifically. Ideally, you should see a band at the MW of the dimer as your main product.

More on the crystallography part: is the interface or the interacting residues conserved? If so, there is a high chance that it is a true interface. If not, you might be looking at a crystal packing artifact.  Also the buried surface area can give a hint. Rather small surfaces (below 500-800 A2) might need more critical inspection. 

Good luck!

Christian

Correlation between concentration and ellipticity at 222 nm should be linear for monomeric proteins. I agree with Christian that a fine-tuning experiment of chemical cross linking may give you good results. You may be interested in our article: Colombo et al. BMC Plant Biology 2014, 14:228 http://www.biomedcentral.com/1471-2229/14/228

Thank you Rick. It was inspirational to go with AUC. @ Christian.. The buried surface is >1500 A2 and few amino acids are conserved. @ Eduardo.. My protein form dimer only at crystallization condition which is above 500 uM. Is it possible to monitor the dynode voltage in CD to an acceptable range in such a high protein concentration ?

You Are right. It can not be measured in conventional manner (for example cuvettes of 1 cm light path). For 1 mg/ml protein concentration you should use 0.1 mm short pathlength cells. To reach 5 to 10 mg/ml you should use 0.01 mm pathlength cells

Thank you Eduardo.

you can use analytical gel filtration chromatography to check oligomeric form of your protein at different concentration.

first of all run all the molecular weight marker (commercially available) onto the Sd200 analytical gel filtration column, then plot a standard curve between log of Mol wt and elution volume.

then you can run your protein samples and check the elution volume. calculate molecular weight according to the elution volume plot.

Analytical Ultracentrifugation (AUC) could give the info you need.  Dynamic light scattering (DLS) will give you an indication of reversible dimer formation since the hydrodynamic radius should increase with increasing concentration.

Hi Preyesh, you could use the biophysical technology MicroScale Thermophoresis (MST). This is a free solution technology which works over a broad concentration range, also at very high concentrations. The good thing is also, that it measures in very small volumina (few microliters). Thus your protein consumption wouldn't be too high even if you use very high protein concentrations. For example John Ladbury has used MicroScale Thermophoresis to measure dimerization:"Inhibition of Basal FGF Receptor Signaling by Dimeric Grb2" http://www.cell.com/cell/abstract/S0092-8674(12)00589-2

Hi Preyesh,

You can try light scattering technique. Thanks.

It may be useful to discuss with the reviewer the fact that finding dimers in crystals is completely normal for monomeric proteins. Also, I wonder the physiological significance of the existence of dimers that are detectable only at protein concentrations higher than 10 mg / ml. Under these conditions many monomeric proteins would become dimeric!

Both AUC and DLS are suitable methods to determine if your protein forms dimers or monomers. You can have the buffer conditions same as that for which you get dimer formation. In gel filtration, you may or may  not be able to analyse the protein under the same buffer conditions as those, where your protein forms dimers.  

Native MS, if you have the opportunity to run such experiments, is very sensitive and would most likely pick up the dimers if there are any.

Thank you very much for all your valuable suggestions. I have tested the dimerisation state of of this protein using AUC, with the help of Prof. Jack Kornblatt, Concordia University.