Le Samedi 2 Avril 2016 19:15 CEST, Gyanendra Kumar <[log in to unmask]> a écrit: 

Hello 
We made exacly the same kind of co-crystallization protein+peptide.
We noticed that the intermolecular interactions in the packing could be strong enough to prevent occupying all binding sites (Burnouf et al.,J Mol Biol. 335(2004)1187)

Also, practically, E. Ennifar introduced ITC as a very efficient tool to prepare the material for crystallization: instead of letting the titration go to completion (i.e. reaching a stoichiometric ratio significantly above 1), you stop it just above 1 and retrieve the materiall in the cell for concentration and crystallization [Da Veiga et al., "Isothermal Titration Calorimetry: Assisted Crystallization of RNA-Ligand Complexes." Methods Mol Biol. 2016;1320:127-43. doi: 10.1007/978-1-4939-2763-0_9]
This allows one to get the minimum and sufficient amount of ligand. Also, analytically, this reveals whether 100 % of the  macromolecule can bind the ligand (if the concentrations are correct). If significantly less than 100 % can bind the ligand, then you know that all attempts of crystallization will lead to a mixture of molecular species and, likely, to poor crystals or no crystals. At the extreme limit, you may well discover that your macromolecule/ligand pair do not interact significantly.

I hop this will be useful.
Philippe Dumas

 
> Hi Nicola,
> 
> I would suggest that you keep increasing the concentration of the peptide
> as long as it results in crystals appearing in your drop. If your peptide
> has low affinity for the protein, it may take a lot of it to see it in the
> crystal structure. Try 10, 20, 50, 100 fold higher concentration of the
> peptide. Also, shoot your 1:5 ratio crystals, it may already be there. In
> some cases, if the crystals have large enough solvent channels, the
> morphology of the crystals may not change significantly upon binding of the
> peptide.
> Congratulations on having a fast crystallizing protein at hand. That makes
> it much easier to try new things and test your hypothesis at a faster pace.
> Also, you can try variation in length of your peptide, if increasing the
> peptide concentration doesn't give you the complex. May be, addition or
> deletion of a residue or two will result in better binding and you will
> have your complex.
> 
> with best wishes,
> Gyan
> 
> 
> On Fri, Apr 1, 2016 at 10:58 AM, Nicola Evans <[log in to unmask]>
> wrote:
> 
> > Hello all, I have a question regarding co-crystals with peptides, please
> > do share any experiences you may have had. In my group we are trying to
> > crystalise 15KDa protein with a ~1KDa peptide (related to a binding
> > partner). We initially set up apo crystallisation and protein:peptide at a
> > 1:1 molar ratio. We had crystals in both with very similar morphology
> > (plates),  the protein:peptide mix crystals diffracted well and gave us a
> > good structure, but no peptide was bound, however the purely apo tray gave
> > crystals with much poorer anomalous diffraction. It looks therefore like
> > the peptide is helping the protein to crystalise in a more ordered form,
> > but maybe the concentration isn't high enough to reach saturation. ITC
> > indicates that this peptide does bind to our protein.
> >
> > We have set up further crystallisation with a protein:peptide ratio of 1:5
> > and have again grown crystals, however the morphology and plate profiles
> > look exactly the same as before. In my experience with compounds this is
> > quite common (and of course plates are a common morphology) but would you
> > expect with the larger peptides to see this? Or would you expect something
> > different? If not in morphology in the cell dimensions of the crystal? Is
> > it worth collecting a dataset for these crystals to see if the peptide is
> > bound?
> >
> > Some more information is that the crystallisation is very fast
> > (concentration dependant), within minutes to hours. The peptide is also
> > co-purified with the protein.
> >
> > Thanks in advance for any help.
> >
> 
> 
> 
> -- 
> Gyanendra Kumar, PhD
> St. Jude Children's Research Hospital,
> Department of Structural Biology,
> 262, Danny Thomas Place, MS-311
> Memphis, TN 38105
> Phone: 901-595-3839