S200 10/300 analytical column

What molar ratio would be best to try? 1:1 or something else? Do i need to add more phospho-protein (32 kDa) than the binding partner with a groove (35 kDa).

The protein should be all right at the present condition for solution experiments. However, the stability might be insufficient because the crystallization process takes days to finish. Namely, the protein might need stronger stability. There are two parts we might need to consider.

  1. The stability of the protein from degradation. For example, try SDS-PAGE for every day from the first day of purification. Moreover, The days between the final screening and the days waited, typically 5-10 days. If there is an additional band(s) that appears, you probably need better condition, reducing agents, protease inhibitors, and so on.

  2. The sequence is probably known, but not shown here. Thus, there is another possibility that the condition is fine for the protein stability. However, the intrinsic dynamics of the protein might be a factor we cannot neglect. For example, we cannot have a 20-residue long loop hanging around in either one of the proteins. If your team have not made secondary structure prediction of the two proteins, it might be necessary to clear out the issue by analyzing the sequences to see if there are any dynamics problems.

  3. The binding of the two proteins might be okay now. However, the exchange of the two might cause an issue in a long-term crystallization. Typically more than uM (Kd) affinity is required. If the team thinks that this might be a problem, a condition screening might serve a good way to find conditions for the higher affinity between the two.

Both the structures are known we are trying complex. The Kd determined by us is 4.36 µM.

This affinity might be insufficient. Though, there are successful cases that have this type of affinity.

Structures were known (assuming X-ray crystallography was used instead of NMR) indicated that 1. and 2. probably are not the problems. However, adding two proteins together always make the crystallization more complicated. (dynamics problem can worsen) We probably cannot completely rule out the possibility.

Crystallization is hard. Prediction can hardly be done. Thus, to test more conditions is probably the next to do.

Dear Sunil,

is NMR an viable option?

Did you try to take the proteins after the crystallization attempt (i.e. after few days together at whatever conditions you keep them) and ran the gel filtration to see, if the phosphate and the complex are still intact?

What about mutagenesis of the amino acid into Glu? That is used to mimic the phosphorylation and permanently switch the protein ON (or OFF, depending on what effect the phosphorylation has). In case your complex would not be long-term stable enough (as suggested in above paragraph), this could provide some help.

Hi Hluska

What do you mean by "What about mutagenesis of the amino acid into Glu? That is used to mimic the phosphorylation and permanently switch the protein ON".

Can you elaborate more about it.

I do get precipitate in many of the drops. I check many pdb entries where people keep the complex concentration between 8-10 mg/ml for setting drops.

I am new to complex stuff. Most importantly we see very good symmetric peak on gel filtration with 0.8 to 1 ml peak shift for the complex (S200 10/300 analytical column).

Any more things i can try out. Any suggestions. I am trying right now linking the two proteins by Glycine linker by recombinant DNA technology.