Substitution to glycerol during crystallogenesis

From: Toby Longwood
Date: 3 April 2012 12:49

Dear all,

My question is related to a sample preparation.

I'm working with a complex that can be stabilized with glycerol (at least 10%) during purification. The use of detergents does not help. After purification, the sample is homogeneous (EM) and can be concentrated (3-4mg.mL-1) . I already set up many drops, changing several conditions (pH, salt...) but nothing conclusive appeared.

I know that crystallogenesis in presence of glycerol works (Sousa, Acta Cryst (1995), ...) however, because of the aspect of the drops (precipitates that seem close to the nucleation phase), I suspect that the glycerol can be one of the limiting factors of the protocol.

Has anybody else been already confronted to the same problem? Does someone know if there is an alternate additive to glycerol?

Thanks in advance for suggestions/help

With best wishes

Toby

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From: Florian Schmitzberger

Dear Toby,

I don't think there is a basic problem using glycerol in crystallization. Glycerol will affect the vapour pressure (if it is not present in the well/precipitant solution) and 10 % glycerol is ~ 1.3 molar concentration. During equilibration the drops may increase in volume, decreasing the protein concentration. Thus, when using glycerol I think it is generally beneficial to start with a high protein concentration. Perhaps, you can concentrate your protein sample further.

I have on several occasions observed immediate precipitation upon mixing protein solution (containing glycerol) and precipitant solution; drops then cleared up after a short period of time (and crystals eventually formed). In this case, the crystallization experiment starts in the supersaturated zone, and "moves" towards an undersaturated concentration, traversing the (metastable) zone where nucleation and crystallization can happen (rather than the other way around, which seems the more traditional approach with crystallization by vapour diffusion). 

Enrico Stura published a recent article, describing an effect of glycerol on crystallization. Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11 :2755–2762. "

You could replace glycerol with ethylenglycol or a small molecular weight PEG (e.g. 400), which may also have a stabilizing effect on your complex. 

Regards,

Florian

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From: Debasish Chattopadhyay

We use ethylene glycol and glycerol mainly to reduce nucleation (or showering of crystals).  However, we also found that these two additives may not be interchangeable, that is effects of these reagents were markedly different on crystallization behavior of a particular protein.

Debasish

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From: Enrico Stura
Glycerol is known to be able to reduce nucleation. This might be countered
by an increase in protein concentration.
Vera, L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth & Des. 11: 2755–2762.

Enrico.

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From: Edward A. Berry

Florian Schmitzberger wrote:

Dear Toby,

I don't think there is a basic problem using glycerol in
crystallization. Glycerol will affect the vapour pressure (if it is not
present in the well/precipitant solution) and 10 % glycerol is ~ 1.3
molar concentration. During equilibration the drops may increase in
volume, decreasing the protein concentration. Thus, when using glycerol
I think it is generally beneficial to start with a high protein
concentration. Perhaps, you can concentrate your protein sample further.

It sounds like you are encouraging the OP to crystallize by reverse
vapor diffusion. Unless there is reason to believe that diluting the
droplets will push the protein toward saturation (salting-in region?),
it would seem better to skip the reservoir, seal the wells and crystallize
by batch method. Even more sensible, (as you parenthetically hint) add
the same concentration glycerol to the reservoir solution to compensate
and crystallize by normal forward vapor diffusion.
And if you are lucky you can use the glycerol-containing reservoir
solution as artificial mother liquor if you need it for mounting
crystals or mixing cryoprotectant solutions.

Still, it makes sense to use higher protein or PEG to counteract the
tendency of glycerol to increase solubility/decrease nucleation.