Hi Karolina,
I would suggest you do SAXS. You can compare the scattering profile you get from your protein in solution to the calculated profiles of the three possible dimers (or other oligomers) you have in the crystal structure. This should give you a definitive answer. See for example this paper where SAXS was used to resolve exactly the same kind of problem: doi: 10.1016/j.molcel.2008.02.020 Regards, Julia On Thu, 25 Jul 2013 17:33:57 -0500, Karolina Michalska wrote: Hi all, I'm working with a protein that appears to be a dimer in solution, on SEC in runs as 24 kDa, while the actual mass of a dimer is 30. And I am trying to figure out which dimer is the biological one (it is a regulatory protein but details are uknown). The crystal structure gives me a few options (mol A and B in ASU plus P6122 symmetry), but none of them is really convincing: for deltaGdiss PISA goes from -0.4 to 1.5 kcal/mol. Theoretically, I have two compact dimers (1 and 2) and one elongated (3). In 1, I have two hydrophobic helices interacting and four hydrogen bonds, 1550 A2 buried area (out of 14200 total). This interface applies only to molecule A and its crystallographic mate, equivalent molecules B are too far from each other. Moreover, even in the dimer made of mol A there are channel at the interface. Interface 2 is purely hydrophobic, but at least it's consistent, i.e there are comparable interactions for A-A and B-B pairs, 1850 A2 buried area Interface 3 involves non-crystallographic copies, buried area is 1040 A2. The interacting elements are proline-rich, and there are four main-chain - main chain hydrogen bonds plus two main-chain - side chain ones. Formally, these fragments are not classified as beta-strands, but the association does look like an intermolecular beta-sheet. This dimer is not consistent with the SEC data though. I'm assuming that with an elongated shape it would run as a bigger particle than it actually is, not as a smaller one. So I think I can discard first option but I am still debating on 2 and 3. I'll appreciate your comments on this. Karolina
