Hi Ruben,
Timing is everything - We are just going through the proofs of a paper entitled "What's in a drop? Correlating observations and outcomes to guide macromolecular crystallization experiments" by Luft, Wolfley and Snell to appear shortly in Crystal Growth and Design. In putting this together we found a number of useful references related to the phase separation phenomena - temperature may be a very useful variable to try. To quote the relative paragraphs and hope we don't start a huge discussion (with references to the figures in the paper removed);
".... There are protein-rich and protein-poor liquid phases. Protein concentrations of 400mg/mL have been measured in the protein-rich phase, a concentration comparable to that found in crystals. (1) Experimental and theoretical studies demonstrate the formation of immiscible liquid-liquid (L-L) phase separation in the metastable region of the phase diagram forms only where there are short range, and/or highly anisotropic interactions between protein molecules, with further experimental evidence that demonstrates this region is connected with conditions for growing crystals. (2)
When the temperature of crystallization is near or below the formation temperature of a metastable, immiscible L-L phase separation, at high levels of supersaturation, experimental data and numerical simulations support a two-step, non-classical nucleation process.(2) In this mechanism a protein-rich liquid phase first forms. Nucleation takes place from this phase followed by initial growth of the nuclei sometimes into the protein-rich and other times into the protein-poor environment. Haas and Drenth(2) suggest that this growth mechanism can lead to fewer crystal defects and more rapid crystal growth as molecules in the concentrated liquid protein phase that surrounds the crystal are not driven to the surface of the crystal by diffusion and therefore misaligned molecules can be more readily exchanged. Literature also supports that it is not the higher protein concentration within the coacervate droplets or the molecular fluidity that may initiate nucleation but rather an interface effect between the dense liquid of high-protein concentration in the droplet and the immiscible surrounding liquid of low-protein concentration.(3)
When a L-L phase separation is observed ... if one phase is protein-rich and the other protein-poor, then the system is very close to conditions that have the potential to produce crystals. If the protein contains tryptophan residues, then the presence of a protein-rich phase can be verified using UV fluorescence, .... Crystals will sometimes form from the dense liquid phase without intervention; .... As is the case with metastable conditions, this protein-rich immiscible liquid phase can be used for seeding.(4) The other useful and effective option to induce crystal formation is to drive the system towards a higher level of supersaturation, the labile state, using temperature. The rationale for this approach is to increase the attraction between protein molecules by decreasing the temperature.(5) However, this process will be dependent upon the solubility properties of the protein/solvent. Protein solubility is dictated by the combination of the protein and its chemical environment. The same protein can have increased solubility at higher temperatures in one chemical environment, and lower temperatures in a different chemical environment. If the protein/solvent is more soluble at higher temperatures and L-L phase separation is seen in the drop, then decreasing the temperature will drive the system towards a higher level of supersaturation. The opposite applies in cases where the protein/solvent exhibits retro-solubility, i.e. the protein is more soluble at lower temperatures. In this case the experiments would be moved to a higher temperature environment, or set up at a higher temperature in a replicate experiment."
1. Kuznetsov, Y. G.; Malkin, A. J.; McPherson, A. Journal of Crystal Growth 2001, 232, 30-39.
2. Haas, C.; Drenth, J. Journal of Physical Chemistry B 2000, 104, 368-377.
3. Vekilov, P. G. Crystal Growth & Design 2004, 4, 671-685.
4. Bergfors, T. J Struct Biol 2003, 142, 66-76.
5. Dumetz, A. C.; Chockla, A. M.; Kaler, E. W.; Lenhoff, A. M. Biophys J 2008, 94, 570-583.
Hope this helps,
Cheers,
Eddie
Edward Snell Ph.D.
Assistant Prof. Department of Structural Biology, SUNY Buffalo,
Senior Scientist, Hauptman-Woodward Medical Research Institute
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631 Fax: (716) 898 8660
Skype: eddie.snell Email: [log in to unmask]
Telepathy: 42.2 GHz
Heisenberg was probably here!
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Ruben Van der Meeren
Sent: Thursday, January 13, 2011 4:56 AM
To: [log in to unmask]
Subject: [ccp4bb] Phase Separation
Dear all,
I'm trying to crystallize a small, soluble part of a protein (~15kDa, 152AA). I did some standard screens (Crystal Screen I & II + Index screen) with a protein concentration of 25 or 45mg/mL in an 1:1 (0.75µL)96 well set up. In most of the conditions I got phase separation (mostly PEG conditions)! Precipitation was formed in conditions with salt. I did not have phase separation with the control (buffer only, see below). For so far I know my protein was soluble up to a concentration of 60mg/mL (I didn't went higher). Its predicted to have a lot of beta-strands (according to CD-spectra and secondary structure predictions).
So here are my questions:
- What is the molecular basis of phase separation? I mean what is going on at molecular level? I would suspect that my protein is not soluble in a PEG environment, is this correct?
- What can I do to prevent my protein or buffer (?) going into phases? Is it temperature dependent? Are there additives I can add? Do I need to lower the salt concentration?
- Are there examples (some of your personal experience) where phase separation was a good thing?
For your record: the protein is in a 150mM NaCl, 20mM HEPES pH7.5 buffer and the pI is 5-6. It is cloned with a his-tag (but cleaving the his-tag didn't change much).
Best Regards,
Ruben
____________________________________________________________________
Ruben Van der Meeren
Ghent University
L-ProBE, hoogbouw, verdiep 5
K. L. Ledeganckstraat 35 9000 Ghent (Belgium)
E-Mail: [log in to unmask]
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