am struggle to crystallize glycosyltransferase enzyme which is organize in two domain N-domain has GT-A fold and C-domain represent rossmann like fold (Add-on) , no way to do truncation because C-ter. contain 100aa, then if truncation take place will have only the N-Ter. which is GT_A fold that have been done by other , so anyone can give an idea , trick to got full length structure?
Dear colleague,
First of all, you must be sure of extreme purity, for getting a typical crystallization of your protein if this data will be used for protein structure, for example in an X-ray analysis. The process before getting the crystal is essential for further protein stability where some ions play a role for stabilization or 3D functional array/structure. You should also verify the REDOX environment for selecting a reducing/oxidizing appropriated environment for S-S bonds that stabilize protein. Extracellular proteins are frequently stabilized through S-S bonds (for exhibiting biological activity in an oxidative environment) and reducing environments are not favorable for them; while intracellular proteins usually works in reducing environments (free -SH are usually catalytically active, even S-S bonds also contributes to intra and inter polypeptide strand stabilization).
I don’t know certainly if you are requesting this not from your question but from your comments. If not clear, please do not hesitate to contact me again for helping you, if I have the answer.
Hi Wafa,
Crystallization is more an art than science. I would require more information from you before I can suggest possible approaches which may help you in crystallizing the full-length protein.
1) What is the length, in amino-acids, of the full-length protein?
2) Is there a long linker between the N-terminal GT-A domain and the C-terminal rossman domain? Do you have ligands( inhibitors/activators/ or single substrates in case the reaction is bisubstrate) that can be added to the protein to increase its stability and restrict its conformational variability, if any?
2) Have you done DLS to make sure that the protein is homogenous? what is the oligomeric status of the protein?
3) What methodology are you following to obtain crystals? The two most popular methods are hanging drop vapor diffusion and the under oil micro batch crystallization methods. Both these methods rely on screening a lot of conditions that are grid variable combinations of precipitant, salt and pH (premade screens are commercially supplied by Hampton Research). As additional variables, you can attempt crystallization at variable temperature, varying protein concentration and, if possible, microgravity. In case you obtain very poor quality crystals, you can also attempt seeding by sitting-drop method.
Dr. Bharath thanks for ur consideration , my protein length is 404aa , loop between two domain approximate 15-20 aa ,i usually crystallize in complex with UDP and Galactose separately.
DLS (attached )showed protein is homo-dimer , but when done NH2 cross-linking reagent (EGS, SB3) indecate that protein make a polymer more than 250KDa, in fact there two band on the SDS-PAGE. For crystallization usually use sitting drop for initial screening and hanging for optimization , the problem all crystal i got was twin salt crystal,
i used to crystal different protein conc. but the protein some time it changed the state , i mean purification profile some time as oligomer, but most of time i got monomer profile but i got very sharp and homogeneous peak in both (oligo and monomer) from CD knew that turn is 29% it may influence the crystal growth ??
my protein is metal dependent do i need to add metal in purification buffer .. i use 20mM Tris-8.0 100mM Nacl as purification buffer. or do i need to add DTT?? protein has 6 Cys. do u think it will make sense??
Hi Wafa,
Thanks for your detailed response. Assuming per residue mol. w of 110 daltons, your protein's M.W is ~44 KDa. Since you see monomeric peak, dimeric peak and hexameric peak with the various techniques you employ, it seems like the protein is highly heterogenous. I think that addition of 2mM DTT in your lysis buffer while purifying will help avoid the formation of high-order non-specific aggregates due to cysteine oxidation. Further, you can also try adding 5-10% glycerol in your lysis buffer. What screens are you employing to obtain the crystals? Usually SaltRX from hampton screen or any condition with a lot of salt in it would give you salt crystals. Further, make sure that you spin your protein at 13,000 rpm for 10-15 minutes and use the supernatant to setup crystallization. Also, is it possible that you express and purify the individual domains separately before attempting crystallization? The above approach would essentially let you work around any heterogeneity because of the long linker that you have. Further, researchers have demonstrated that additives are sometimes very essential in preventing aggregation. presence of NaCl, EDTA, DTT and glycerol have been shown to be helpful in improving aggregation and heterogeneity. Here is an example
http://www.ncbi.nlm.nih.gov/pubmed/15802222
I was also wondering whether you see micro crystals? If yes, you can differentiate between salt and protein crystals by adding small quantities of protein staining dye. Protein crystals will absorb the dye while salt crystals would be refractory to dye uptake. Once you make sure that you have protein crystals, use them to seed fresh crystallization attempts.
I hope the answer helps!
Hi Bharath
i really do appreciate ur guiding .. i consider ur point , but the thing that i do not have two peak, i always get one peak whether for monomer or dimer i don't know the problem does protein changed it's state or problem regard to size exclusion column it's self.? know got crystal in this condition ( 10% 2-propanol 50mMMgcl2 5% PEG400 0.1M MES PH 6.5) i use HR2-110 Hampron research ..
have tried many time to add 2mM EDTA but doesn't work because my protein is metal dependent has 2 DXD motifs.. again thanks a lot :)
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