Kinase crystallization

From: CHAVES SANJUAN, ANTONIO
Date: 30 January 2012 10:06

Dear all,

I am trying to crystallize a protein kinase without any success.

I suspect about its characteristic catalytic loop. I have already prepared different constructs, different expression vectors, and different mutant proteins (pseudo-phosphorylated, active, inactive?). I have also tested some ligands as ANPpnp (non hidrolizable nucleotide), ADP (product) and manganese (cofactor).

I am thinking in trying to cocrystallize it with a general kinase substrate (a peptide, a small molecule...). Does any one have any experience or suggestion?

Thanks in advance.

Sincerely,

Antonio
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From: Artem Evdokimov

It is a fairly common issue with kinases. Among other options you may want to try a generic kinase inhibitor (there are several good ones just look at pdb structures for ieas) and if this does not help then you could attempt to clamp the motion down via an inter-lobe engineered disulphide bond...

Artem

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From: Schubert, Carsten [JRDUS]

Staurosporine come to mind as a general kinase inhibitor. I also second Artem's suggestion that ligands make a big difference, we had several cases of kinases which required ligands for crystallization success. Also make sure you eliminate any floppy ends which may interfere with packing.

 

Good luck

 

                Carsten

 

 

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From: Debreczeni, Judit

Does your kinase autophosphorylate by any chance? -- That can produce differently phosphorylated species and affect crystallisability. You can detect it by e.g. mass spec, and tackle it by dephosphorylating the protein prior to crystallisation or by coexpression with a phosphatase.

 

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From: George Kontopidis

if the ligand binding site is exposed to the solvent a bound ligand may help.

if the protein has is flexible domains and ligand fix it in one conformation, a bound ligand will help even more.

All the above assuming that the purity and the concentration of the protein are high.

 

George

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From: Pius Padayatti


a comment to add about phosphatase (PP2A, C class serine theronine)
buying phosphatase for crystallization trials was kind of not
practical( very high prices )
so we tried expressing and purify it from overexpressed in bacterium
with yields very low.
Also the purified phosphtase co-purify some proteasaes that led to
proteolysis of the samples.
what is the experts here experienced in such conditions?
Judit, Is there a better source? cheaper and reliable?

thanks