I am trying to form a complex of two proteins, one of which requires Arginine (0.5M) in the GEC buffer for its stability. Has anyone obtained crystals of protein complexes in the presence of additives?
Thanks & Regards
Sunil Singh
Hi Sunil,
You can add arginine or any other compound needed to stabilise the interaction, but for an intrinsically stable complex you probably will only need to provide co-factors (metal ions like Ni2+ , Zn2+, etc... or FAD, NAD, etc...) if they are needed for function and structural integrity, rather than arginine. Arginine is sometimes used as an additive in crystallography to provide a highly ionic buffer that stabilises negatively charged proteins. These proteins are also generally stable in buffers with high salt contents that provide the correctly charged environment, but high salt conditions often inhibit crystallisation (NaCl is a chaotrope), which is why arginine is used to circumvent this problem.
You can definitely try and mix your two proteins in an equimolar fashion (or in whatever ratio they are expected to interact: 1:1, 2:1, 3:1 etc..) by using the buffer of protein A ... or protein B, or an intermediate buffer that works for both ... and then set up trays and hope for the best.
However, as Sebastian suggested, it's better if you co-purify your two proteins before you set up crystallisation trays to a) ensure both proteins are in exactly the correct ratio and b) to make sure they actually interact at all. I would suggest you test this with a variety of different buffers using a SEC column. An analytical SEC column is ideal for this, as they are only a fraction of the size of a large purification SEC column, you can use them with very small amounts of protein and a whole run only takes around 20 to 30 min. A good buffer will produce a single sharp peak (protein A+B) and less ideal buffers will give you several peaks (corresponding to protein A+B, protein A and protein B). You can further test the resulting samples with dynamic light scattering (DLS), which will tell you if your complex is behaving as a monodisperse protein in solution (polydisperse protein is more likely to precipitate and less likely to crystallise). Once you have established the best buffer you can then carry out a large-scale purification with a purification-size SEC column and then set up trays.
Tobias actually has a point. X-ray structures of complexes are extremely informative if you need atomic resolution for details of the interaction, but they are also quite challenging to obtain. If you fail to grow crystals or they simply won't diffract, etc... it's easier to solve the structures of the two proteins separately and then obtain a good cryo-EM map or SAXS envelope and fit the two X-ray structures into the density of the cryo-EM or SAXS data to make a hybrid model. Also, if you're only interested in the overall shape of the complex and you don't need the atomic resolution, then SAXS or cryo-EM would probably be much easier and quicker anyway.
Dear Sebastian and Tobias,
Thank you for your suggestions.
Dear Antonio,
Thank you so much for providing such insightful suggestions. I will definitely try to optimize the buffer condition. i have a E2-Ub pair and i want to form a complex of this with one E3- Ligase which requires Arginine for its stability. I need to mix these two proteins to form a complex. Therefore i was wondering that Arg might inhibit the interaction of E3 Ligase and E2-Ub. i will add these two proteins in the presence of Arginine and set up crystallization conditions. I will further change the Arg concentration in the buffer as you have suggested.
Thanks& Regards
Sunil Singh
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