Let us know the ligand solubility in buffer solution used for the complex
formation!
The fact that your adding 0.05mM of even 10mM ligand in your solution does
not mean that you got that concentration in solution.
It is very likely often that the ligand bonds to protein the protein is OK
but the concentration (solubility) of the ligand is not high enough.
You need a ligand solubility (concentration) approximately 10xKd +[protein]
Kd = dissociate constant of protein-ligand complex
[protein]= Total protein concentration added in solution
George
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of John
Newitt
Sent: Friday, February 6, 2015 4:34 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] Protein-Ligand Crystallization
Hi Monica,
Your Tm results don't suggest any binding of your ligand to the protein.
Your ligand concentrations are quite high so I am not convinced of even weak
binding. The destabilization that you are seeing at the highest
concentration may be due to ligand precipitation causing protein
denaturation/unfolding.
If available, I would search for other ligands for co-crystallization.
Alternatively, confirm the ligand binding by some other technique before
investing a lot of time and protein in crystallization screening.
John
Sent from my iPad
> On Feb 5, 2015, at 8:43 AM, Monica Mittal <[log in to unmask]>
wrote:
>
> Hi all,
> I am working to crystallize a protein-ligand complex. I did a preliminary
melting curve analysis for the protein in the absence and presence of 2
ligands (dissolved in protein buffer). I kept the other controls as buffer
an a known standard to confirm instrument performance. All expts done in
triplicates.
> Now the results are like : Tm of protein alone is 56 deg, Tm in the
presence of Lig1 conc. of 0.05mM, 0.1mM, 1.0mM and 10.0mM is 56.2, 56.1,
56.0 and 53.5 respectively !! Tm in the presence of Lig 2 conc. of 0.05mM,
0.1mM, 1.0mM, 4.0mM and 10.0mM is 56.2, 56.1, 54.9, 52.3 and 50.6
respectively !!
> Although the effective delta Tm for both is different at higher
concentration, but both are kind of making protein less stable. So i was
wondering, will it be difficult to co-crystallize them !! Any suggestions in
this regard are highly appreciated !!
>
> Thanks
> Monica
|