In the previous post I have discussed how limited proteolysis aids in protein’s fold boundaries determination and identification of the minimal crystallizeable fragments or domains. An important factor controlling protein crystallization is surface contact arrays between one molecule to another. Under most circumstances crystallographers can’t know which residue participates in surface interaction and whether this modification will aid in crystallization at all. For this reason lysine methylation, initially developed for isotope labeling of proteins, is a purely empirical method that is another avenue to check when protein doesn’t crystallize.
Lysine methylation technical tips
For this modification you will need formaldehyde and dimethylamine-borane complex (aka ABC), the latter serves as the reducing agent and is appropriate for methylation conducted at close-to physiological conditions (pH ~7.5). The protein solution should not contain any other free amino groups (such as present in Tris buffer) and thus buffer exchange should be considered (or better, start fresh from a new purification with HEPES or phosphate buffer). Since the reaction might generate several protein species, the best way to maintain homogeneity (which is crucial for succesfull crystallization) it is recommended to conduct a size exclusion chromatography after completing the reaction. Fix your protein solution to 5-10 mg/ml with accordance of 20-molar access of formaldehyde to number of lysines in the protein (remember that not all lysines will be exposed to the surface and thus not all reduced). In case of massive aggregation, lower the protein concentration up to 1 mg/ml.
1. Mix 20ul of 1M ABC for every ml of protein solution by gentle mixing.
2. Add 40ul of 1M formaldehyde and gentle mix the solution.
3. Incubate at 4°C for 2 hours.
4. Iterate steps 1-3 one more time.
5. Add 10ul of 1M ABC as a final reaction and let the reaction finalize overnight at 4°C with gentle rocking.
6. Next day, quench any remaining formaldehyde by exchanging the buffer to Tris.
Verification of methylated proteins
One way to validate the efficiency of the methylation is to use Matrix assisted laser desorption/ionization – time of flight analysis (MALDI-TOF) or the better accurate Electrospray ionization (ESI) technique. Since every successful fully methylated lysine will contribute 28 Dalton to the protein’s molecular weight, its mass over charge value (m/z) will shift sufficiently to be observed (especially if the protein contains many lysines).
This empirical technique has made a significant breakthrough in protein crystallization for several proteins such as SycD, SelA, YopN, SycN and YscB complex and many others. If you have experienced hard time crystallizing your protein, you might want to explore this relatively cheap and easy protein modification.
Share your protein methylation success stories!
Isn’t it time to take control over your many projects? Try out Labguru for free!