Recently i was trying to get the crystal complex of two proteins, one is 76 AA length,anther is 44AA length. I am not sure whether the two proteins can bind tight enough to form a complex just through mixing them in the mole ratio 1:1. According to the size exclution chromatography figure, there was only one peak after mixing and the retain volume of this peak is less than two single proteins. I also ran SDS-PAGE(15%), unfortunately, the smaller protein ( the 44AA one) ran out of gel,so i am mot sure whether they form a complex or just a misture in the ratio of 1.
For smaller protein try detection by gradient tricine gel as Paul suggested; some time people use peg ppt of the complex and then set up for crystallization screening.
One way to test whether the proteins form a complex is to use chemical crosslinking. If the proteins can be crosslinked with a low crosslinker concentration, it suggests that they form a complex. The protein concentration can be varied to see at what concentration the crosslinking occurs.
If the SEC says it is a complex then it is. I would concentrate the peak fractions and set up crystallisation experiments.
You should be able to show all three chromatograms in the same image. If it were actually not a 1:1 complex then you would see two peaks in the mix chromatogram. If the complex was weak then you should see three peaks.
Maybe you can characterize the interactions between these two proteins while you are trying to obtain the crystals of the complex sample. Dissociation constant of the binding process (determined using ITC etc.) may give you some clues.
By the way, just remind that NMR is an alternative method to determine structures of small proteins (including complex structures), as small proteins are usually too flexible to crystallize.
Good luck!
hi, pengfei Ding,Thank you for your suggestions, I have get the ITC data,and the ITC data shows the kd is about 100μM, suggesting the complex combining not very tight.now i am considering using the NMR to determine the structures, but i almost know nothing about solution NMR. so if I ran into problems during expriments, can I ask you for adivice directly?
Hi, Lan Huan,
For proteins with 76AA and 44AA, NMR is a good method to determine their structures if you can get enough proteins (the typical NMR sample contains 0.1-1 mM protein in 400 μL and it will help a lot if you can get the 15N/13C isotope labelled samples).
Considering the binding is too weak (~0.1 mM), I am not sure whether you can get the complex structure using NMR. However, you can characterize the binding interface by NMR titration experiment and identify the residues involved in the binding process. If this is the case, you can only get a complex model based on the experimental data (using HADDOCK etc).
Sure we can discuss this directly~
One thing I'd like to add is that complex with Kd of 100μM may also be crystallized, as I read that "it was found in a number of studies that weak interactions may manifest themselves in highly condensed pre-crystal solutions and crystalline states"
However, the same author said "in the course of the crystallization procedure weakly bound complexes have a chance of being sacrificed (disassembled) in favour of nonspecific contacts if this results in the formation of crystal packing that is more suitable from a global energy point of view." and "weak interactions with Kd>100 μM have a considerable chance of being lost during the course of crystallization" thus "a complementary noncrystallographic study should be always conducted when inferring structural aspects of weakly bound complexes."
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