I am using AUC to determine if my protein is an oligomer (more specifically, a dimer). It crystallizes as a dimer, and homologs from other species also crystallize as dimers. However, my size exclusion chromatography and hydrogen deuterium exchange data suggest it is a monomer. Dimerization can sometimes be an artifact of crystallization. Therefore, I want to use AUC to distinguish between these two possibilities.
I am performing velocity sedimentation-AUC, and I analyze this data with either SEDFIT or SEDVIEW. The sedimentation coefficient is calculated to be similar among different runs, ~4.3-4.9. I know the theoretical mass of my protein based on its amino acid sequence as well as denatured intact protein mass spectrometry. However, I do not know the protein's frictional coefficient. Using SEDFIT, a person can manipulate the various parameters at will (sedimentation coefficient, frictional coefficient, viscosity, etc). Under a particular set of fixed initial conditions, I can conclude either that the protein is a monomer or a dimer. Of course, this is not what I desire.
So my question is this: How can I unambiguously determine whether my protein is a monomer or a dimer? Can this be done solely from velocity sedimentation AUC? Or will it be necessary to perform equilibrium AUC as well? Our AUC facility does not presently have an equilibrium AUC cell set, so it would be optimal if I could use velocity AUC for this question. However, I leave it to you (the experts, lol) to tell me if I need to perform the equilibrium experiment. If so, I will have to wait until we get the equipment.
Dr. Guera
Would you like to use electrophoresis (absence and presence of SDS) ? My teacher elucidated whether his target enzyme was monomer or dimer using electrophoresis(please read attachment file).
I don't know the answer to your question, but I would like to suggest as an alternative experimental approach the use of chemical crosslinking and SDS-PAGE to distinguish monomer from dimer. See this paper for an example:
Both techniques may be telling you the truth- it is possible that your protein undergoes a mass-action monomer-dimer equilibrium. AUC will answer your question unambiguously. Here is what you need to do:
1- Run 3 samples that cover as wide a concentration range as possible.
2- Determine the average sedimentation coefficient for each sample (any of the packages will do this)
3- If s-average increases with concentration, you have thermodynamic proof of a mass-action equilibrium that has significant populations of both monomer and dimer in the concentration range you covered. You may see that the g(s) or c(s) distributions are skewed, with the amount of skewing being concentration dependent. If the skewing becomes more apparent on the low-s side at lower concentrations, it means that your loading (initial) concentration was much above the Kd. If the skewing becomes more apparent towards the high-s side as the concentration is increased, it means your initial concentration is much below the Kd.
You will not necessarily observe two peaks (c(s) may show two peaks, but not necessarily).
4- If s is constant or decreasing, it means that you have a single component. If you can tell us the expected monomer mass, you can interpret the observed s as being one or the other oligomeric form.
I don't know the answer either to your question, but I do know we used equilibrium AUC and we were able to unambiguously determine we had a dimer.
- Badges
- Science topic