Hello Intekhab, Your results do not seem surprising at all. It is not uncommon for molecular interactions such as dimerization to be more stable at lower temperatures, and this is exactly why you are seeing the shift to higher elution volumes at lower tempratures. At lower temperatures, both the monomer and dimer are likely to be more compact in solution due to less thermal fluctuations in the overall structures. Remember that protein structures are always in motion, and lowering the temperature restricts these motions, and therefor lowers the effective radius of the molecule in solution as it moves through the column. And of course smaller molecules elute at higher volumes, so this probably explains what you see in the cold room.
As for some of the other concerns you have with your gel filtration experiments, I can offer the following suggestions. 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. That being said, often analytical gel filtration experiments are more informative at moderate concentrations instead of high concentrations, because this will favor the formation of relevant oligomers, instead of oligomers and aggregates that form only at high concentrations and aren't really "biological." When you do your incubation experiments, try using lower protein concentrations or shorter incubation times. This might prevent the formation of precipitates and will give you more biologically relevant information - after all, most proteins are not available in the cell at very high concentrations, so if your dimer is biological, the kd is likely pretty low. Also you could try another experiment like a pulldown with tagged/untagged constructs, or SPR. These experiments (SPR particularly) would also tell you if the kd is reasonable for a biologically relevant interaction. One more thought is to be sure your protein is not degrading at high temperatures, which may be the reason your 37 degree incubation results in increased elution volume. Mass spec could help you here. Finally, to determine the biological relevance of your dimer you should do an analysis of the "dimer" interface seen in the crystal structure. I believe that for the "average" biological oligomer, the oligomerization interface buries approx 1200-2000A2 of surface area, whereas the average crystal contact buries approx 400-800A2. Some older work related to these analyses has been published by Joel Janin and Janet Thornton. Also, some webservers like PISA attempt to predict the relevant oligomerization states of proteins in the PDB based on interfaces seen in the crystal structures. You might look there for a good method. Good Luck, Mike Thompson ----- Original Message ----- From: "intekhab alam" <faisal...@gmail.com> To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, August 9, 2010 4:37:45 AM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] monomer-dimer 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 -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
