Crystalization in low PH

From: Sam Arnosti
Date: 7 November 2011 05:19


Hi everyone

I have a protein that is extraordinarily stable at PH=3.0 or even 2.0.

I want to crystallize it in the  low PH and compare the differences between the crystals in regular PH and low PH.

I was wondering how people set up the boxes in low PH, as usual buffers are mostly less acidic.

Regards

Sam

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From: Boaz Shaanan


Hi,

I'm sure there are proteins that were crystallized at low pH but I can't remember which. The best thing is to go to the BMCD database: http://xpdb.nist.gov:8060/BMCD4/index.faces
and query it with the key pH (look into advanced search).

 Cheers,

             Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

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From: George M. Sheldrick

Tendamistat (1OK0) was crystallized at pH 1.3 and diffracted to 0.93A.
George

--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany


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From: Craig A. Bingman


I'm not convinced that you need a conventional buffer at pH 2 or 3.  At pH 2, the hydrogen ion concentration is 10 mM.  If you want to use something else, the second pKa for sulfuric acid is around 2.  The first pKa for phosphoric acid is slightly higher than 2.  Lactic acid has a pKa close to 3.  Formic acid has a pKa just under 4.  Most of these numbers were in an appendix in the first chemistry text you ever used.  <wink>  These numbers imply pretty strongly that most crystallization screens emphasizing common salts will require determined modification to hit these low pH values, because many stabilizing anions in the Hoffmeister series will be partially or completely protonated at these pH values.  PEG and organic screens will require a smaller hammer to retrofit.

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From: Enrico Stura


I have crystallized in PEG with citrate at pH 3. If you want to go lower
I would suggest maleate:

effective pH range       pKa 25°C        buffer
1.2-2.6                               1.97       maleate (pK1)
2.2-6.5                                3.13      citrate (pK1)

Enrico.

--
Enrico A. Stura D.Phil. (Oxon) ,   
Room 19, Bat.152,                 b


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From: Heping Zheng


I remembered that people had crystallize a series of
streptavidin-2-iminobiotin structures at a low pH. If it might help, check
the following PDBIDs:

2RTD
2RTE
2RTI
2RTK
2RTL

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From: Ed Pozharski

Not clear if you already have crystals at "regular pH", but if you do,
you may consider direct transfer to lower pH.  Of course, crystals may
dissolve, which you could possibly prevent by cross-linking with
glutaraldehyde.  Three caveats:
a) If lattice is incompatible with lower pH, even with cross-linking the
resolution may sink to essentially useless levels
b) I have no idea if the cross-linking will not be disrupted at really
low pH, perhaps someone else can comment on that
c) the 3rd reviewer can always say that lattice forces could have
prevented a conformational change.  But same goes for direct
crystallization at low pH (but caries less weight).

--
"I'd jump in myself, if I weren't so good at whistling."
                              Julian, King of Lemurs

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From: Katherine Sippel


I had a protein that loved pH 3 if it was holo (citrate, ammonium bromide, and PEG) or pH 4 when apo. Those were microbatch conditions rather than vapor diffusion. If there is no indication of gross structural changes you might consider different pH soak on your crystals rather than finding a new condition. Citrate-phosphate buffers are great for a broad range of pHs (2.2-8) and you don't have to worry as much about changing chemical environments.

Just an idea,

Katherine

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From: Gloria Borgstahl


Glutaraldehyde works best at low pH