Hi
I have expressed and purified 16 kDa protein with N-terminus His-tag and C-terminus polyLys tag. I measured the 1D NMR spectrum and found this protein has folded structure and also found the protein forms intermediate of monomer and dimer (or higher) because of its broad peaks. The protein is highly basic and interact with SEC column and showed larger elution volume the expected. And now I can not use ultracentrifuge and dls. Is there any additive to improve NMR spectrum? and any good method to test its oligomer size?
In addition I tried several additives including 0, 75, 150 mM NaCl, 10 % glycerol, 0.2 % Triton X-100, 0.2 % CHAPS and 10 mM DTT. 0 mM NaCl showed the best S/N ratio and the presence of 0.2 % detergents showed slightly diverged peaks. And 10 degree showed better S/N ration than 20 degree.
Is this oligomerization mediated by hydrophobic interactions?
Hi,
well storage of proteins at low temperatures around 4 degree Centigrade could prevent dimerization and oligomerization of proteins up to certain extent.
the additives you used are quite reasonable.
Well done......... good going
Dear Kiyohiro,
are you sure that your protein is forming dimers? Peak broadening can be caused by other things (measurement parameters, resolution of your spectrometer, temperature, conformational flexibility, etc.). You can use the rotational correlation time to confirm this, as described in this paper:
Article Dimer interface of the effector domain of non-structural pro...
What's the pI of your protein? If it's really that basic the molecules should bear a strong positive charge and repel each other. You can increase this effect by making sure that the pH of your buffer is at least one, if possible two numbers away from the pI of your protein.
To get better resolution in the NMR you can try different temperatures or if your Protein has any cofactors or substrates, adding them to the buffer will often stabilize the conformation (or a particular one from a set of conformations).
Another simple way to confirm the oligomerization state is to chemically crosslink the sample followed by SDS-PAGE analysis (so oligomers will not be separated by SDS). There are various reagents available https://www.thermofisher.com/de/en/home/life-science/protein-biology/protein-labeling-crosslinking/protein-crosslinking/crosslinker-application-reference-guide.html
You can even do this in vivo to see if the dimer is the natural form of your protein. There's a huge number of protocols available online (e.g. this one http://www.fgsc.net/neurosporaprotocols/How%20to%20cross-link%20proteins.pdf), the most commonly used reagent would be glutaraldehyde (crosslinks side-chain amines on the surface) as it's very cheap and easy to use.
Kind regards,
Lorin
With regards to the protein interacting with the SEC column, what buffer do you have during the SEC run? Reason I'm asking is since you state you tried 0, 75 and 150 mM NaCl as additives, indicating that you might not have any salt in the buffer? SEC without salt in the buffer often leads to the protein interacting with the column material in a non-specific manner.
I would recommend to run the SEC with 150 mM, 300 mM and 750 mM NaCl in the buffer, to see whether the equilibrium between monomer and dimer differs with ionic strength. For NMR you of course want to have as low salt as possible, but this would give you info regarding the nature of the oligomerisation.
Hi Faryal and Lorin
The protein I'm measuring is the 12 kDa and might fold small globular structure. Because the overall finger print region of the spectrum is same as we expected from secondary structural predictions. And unfortunately this protein is totally new and I don't know the substrate.
The PI of protein of interest is 10.0 this is enhanced by poly Lys tag at c-terminus of the protein (original PI is 8.0-9.0). This tag (SEP tag) is necessary for protein solubility enough for NMR analysis and crystallization. And the buffer solution is 20 mM MES pH 6.0 only and I think this is enough considering its high pI.
The NMR we used is 600 and 800 MHz NMR and a lot of protein structures were solved by them with same measurement parameters. So I don't think those are the reasons of peak broadening. And we are now establishing the in vivo analysis system.
And I tried 10, 15, 20, 25 degree for NMR measurements and found 10 degree showed the best S/N ratio and even I think lower temperature such as 4 degree would show better spectrum. In addition storage at 4 degree showed no precipitation but it is slightly precipitated at room temperature.
And I appreciate your idea of crosslinking followed by SDS-PAGE, I want to try as soon as next sample will be prepared.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus