Thank you to all who responded. I find it amazing how generously helpful
the crystallography community is.
Lots of good ideas.
Yvonne
_________________________________________________
-----Original Message-----
Sent: Tuesday, August 07, 2007 12:17 PM
To: [log in to unmask]
Subject: [ccp4bb] highly soluble proteins
Our lab is trying to crystallize a highly soluble (100+ mg/ml) protein
with a molecular weight of 35 kd.
The protein was screened against 1536 conditions at 20 mg/mL. Most drops
were either clear or produced "bubbles" (often oily looking). The few
that had precipitate contained high concentrations of K3PO4, cobalt, or
zinc. We have tried repeating some of the bubble conditions at 100+
mg/mL and are still getting clear drops or bubbles.
Is there something about highly soluble proteins and/or secreted
proteins and/or proteins with unusual portions of their sequence that
needs to be considered in order to successfully crystallize it?
I am considering trying "salting out" using dialysis, and also adding
ligands/inhibitors. The protein is in 50 mM NaCl plus 50 mM buffer.
I welcome thoughts and suggestions on crystallization ideas,
publications, etc.
Thank you
Yvonne
____________________________________________________
Try adjusting the pH close to the pI to reduce solubility.
______________________________________________________
For what it is worth: I've worked with a few highly soluble
proteins....(150-200+ mg/mL) I'd start by raising your protein
concentration to at least half of the solubility limit. I hope your
protein is also highly expressing, since the high protein concentrations
will eat through supplies rather quickly! You can also get some
additional advantage by altering the precipitant to protein ratio: so,
rather than setting up 1:1 drops, set them up as 1:3 with the 3X being
precipitant. Finally, a desperation tactic can be to spike the wells of
your drops with highly concentrated salt: this will act as a desiccant
and draw water out of your protein drops -- effectively serving to
concentrate it. I would do this after your screens have equilibrated
for a few days--week(s) if you are not seeing any precipitation
occurring.
_____________________________________________________________
Effects of Zn and Co may be non-specific, aggregative kind of binding and
ppt formation, and may not be very helpful. Phase separation (bubbles)
seems
normal at such high []'s
I would try (a) making SeMet protein since most Se-labelled proteins are
less soluble than their native conterparts, but this depends on Met content
and (2) reductive methylation of Lys, as this should also decrease the
polar
character and therefore solubility. Again, depends on your Lys content.
______________________________________
I would suggest the technique of surface entropy reduction. In this
server you can find all the references and informations about this
methodology: http://nihserver.mbi.ucla.edu/SER/intro.php
____________________________________________________________________
Something you might want to try is reductive methylation of lysine
residues.
This procedure reduces the solubility of proteins,
by typically 30-60% and - in many cases -
people have managed to crystallize proteins
which were otherwise intractable.
Reference:
Walter et al. (2006) "Lysine methylation as a routine rescue strategy
for protein crystallization"
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17098187
In this study we took 10 proteins which
did not crystallize and applied reductive methylation:
In the end, three of them gave high-resolution structures.
_________________________________________________
Hi,
How homogeneous is your sample?
Are your proteins glycosylated?
If so, you may try to deglycosylate them (PNGase F, EndoH, etc...).
You can also try to use detergents (for example beta-OG as an initial
additive to the drop).
Try changing the pH.
In which buffer is your protein sample?
You can try to change it to for example to something like 10 mM Tris-HCl
pH 7.5, and then repeat screening with and without detergents.
Try different temperatures (4, 12, 20, 30 °C).
_______________________________________________
We had a similar situation. This might not be applicable to you, but
cutting off our his tag reduced the protein's solubility, and we then
got some excellent crystals.
________________________
Reductive methylation of amino groups (lysines and the N-terminus) is
a fairly routine chemical modification that should drop the solubility
of your protein. An easy-to-use protocol can be found in Walter et al
(2007) "Lysine Methylation as a Routine Rescue Strategy for Protein
Crystallization." Structure, 14:1617-1622
_______________________________
May I ask if you have tried using The Solubility Tool Kit? I have
attached a
PDF of the datasheet that accompanies the kit.
Protein exists in solution as a salt and different protein salts have
different solubilities. You can effectively do a salting-out experiment
with
the solubility tool kit which will allow you to screen your protein against
different salts.
What you will find with the solubility tool kit is that as you go across
the
Hoffmeister series your protein will become less soluble.
I would suggest that you try and increase the concentration of your
protein.
You don’t mention how you determined the concentration of your protein.
_____________________________________
Have you tried different buffers and NaCl concentrations the protein
solution?
Not sure if that helps, but I recently got crystals from a extremely
soluble protein. As you did, I screened lots (>1000) of different
conditions with a robot. In the end were I got Xtals was at 100 mg/ml,
at 4C and no salt in the protein solution. same condition with salt in
protein solution or at 18C gave nothing. The crystals are growing out of
from very strong phase separation/ oily drops after 3-4 weeeks. I
reproduced the crystals at 4 C with protein concetrations ranging from
100-150mg/ml.
______________________________________
i would lower buffer to 10-20 mM (its bit high and will affect the pH) and
remove salt. you may want to try various metals and high salts if those
give
the precipitate.... and keep the concetration at least to 50mg/ml.
of course you can mutate the protein too (there is a paper on mutating
surface charges to alanine for crystallization
or another one on methylation)
_______________________
First i think that your protein concentration is to low... A common rule
is : protein concentration is good when 50% of your conditions are
precipitates and 50% are clears drops. If most of your drops are clear,
i think that you must increase your protein concentration (a screen from
Hampton can help you for the concentration of your protein: PCT screen).
For your oily bubbles, it often appear when PEG is in the condition,
it's just phase separation.
An other suggestion is that your buffer concentration is too high... If
your buffer is a strong buffer, the conditions buffers can be too weak
versus your 50mM. By the way, real pH in your condition may be something
between the pH of the condition and the pH of the protein sample and not
the real pH of your condition. So you don't have a real access to all
the pHs of the screens.
In summary, reduce your protein sample buffer concentration (to 25 or
10mM) and increase your protein concentration (to 50, 100 mg/ml or more).
____________________________________________
this is what I received to a similar question.
Never forget NMR ..........
********************************************************
Kornelius,
You might want to try a buffer with a pH lower than the pI
of your protein.
The C-terminal domain of gp10 from bacteriophage T4 was
crystallized in
Gly-HCl at pH 2.0-3.0. These were the only conditions that
gave crystals. The
calculated pI of the protein was 4.0 and in reality the pI
was close to 3.0.
The protein was extremely soluble and could be concentrated
to 100 mg/ml. The
crystals diffracted beyond 1.2A resolution.
Here is the reference:
Leiman PG, Shneider MM, Mesyanzhinov VV, Rossmann MG.
Evolution of bacteriophage tails: Structure of T4 gene
product 10.
J Mol Biol. 2006 May 5;358(3):912-21.
Petr
On Monday 22 January 2007 10:27, you wrote:
> > Dear all,
> >
> > I'm sending a summary of useful advices which I received
on
> > my email concerning the crystallization of a very acidic
> > protein. I would like to thank all the people who
> > responded!
> >
> > Have a nice day!
> >
> > Kornelius
> >
> >
> > There are an number (WT & mutants) of X-ray structures
> > published on xylose isomerase from A. missouriensis (see
> > e.g. 1XIM).
> > This is a highly negatively-charged protein with a pI of
> > 3.2-3.5.
> >
> > RNase P protein is quite basic (20-25% Arg/Lys).
> > Crystallization conditions (Stams et al., Science v 280 p
> > 752, 1998) are not particularly informative for your
> > problem, although notably it could only be crystallized
at
> > 3 or 24 mg/ml. DLS revealed that the protein was a
monomer
> > or dimer, respectively, in solution under these
conditions.
> >
> > Before cocrystallization with another protein, I would be
> > inclined to try crystallization from high [salt] or in
the
> > presence of polyamines.
> >
> > *****************************************************
> >
> > This is a difficult problem. It reminds me of the
opposite:
> > when you have a protein with many positive charges and it
> > is meant to interact with a negatively charged polymer
> > known as DNA. When you omit the DNA, frequently you
cannot
> > crystallize the protein presumably because the repulsive
> > positive charges keep the protein from assuming the
correct
> > conformation. Along that thought, you might try to find
> > (more) positively charged particles to counteract your
> > protein charges. I cannot think of positively charged
> > polymers very quickly, but they must exist and/or it must
> > be possible to make those. Maybe (arbitrary thought) you
> > could try positively charged detergent molecules?
> >
> > You write that you have apparently decent CD data
> > confirming you protein folding. Do you have information
on
> > the protein aggragation? (I would somehow not encourage
> > dynamic light scattering, it is a pain in the neck.) Size
> > exclusion chromatography or analytical
ultracentrifugation
> > could help in assessing this.
> >
> > I am asking about these things because, if you had
> > confirmation of the aggragation state (notably the
> > knowledge that the protein does NOT aggragate), then you
> > could try to use SAXS to determine the global shape and
> > perhaps the positions of the individual domains. It would
> > also tell you if the protein is fully folded, or
partially
> > folded (which would be of great importance for
> > crystallization). It would also tell you how these
> > parameters change as function of environmental parameters
> > (pH, ions present, additives), so you might
experimentally
> > determine which conditions/additives help your protein to
> > be 'best behaved' for crystallization.
> >
> > *****************************************************
> >
> > Have you run your protein sequence through the FoldIndex
> > server (http://bip.weizmann.ac.il/fldbin/findex) to see
if
> > it is even predicted to be completely folded? When you
have
> > a protein with many charges, those charged areas are
likely
> > not to be folded, but just hanging out into solvent
(since
> > their interactions will be very favorable).
> >
> > *****************************************************
> >
> > The problem with these highly negatively charged proteins
> > is that they are extremely soluble. It is hard to get
them
> > out of solution. You mentioned you tried concentrations
up
> > to 50 mg/ml. This does not surprise me.
> > Ten years ago we managed to crystallize a halophilic
2Fe-2S
> > ferredoxin and determined its structure. The protein was
> > crystallized from 4 M phosphate, pH 7.
> > It was the only salt that brought the protein out of
> > solution.
> > The reference is
> > F. Frolow, M. Harel, J.L. Sussman, M. Mevarech, and M.
> > Shoham. (1996) Insights into protein adaptation to a
> > saturated salt environment from the crystal structure of
a
> > halophilic 2Fe-2S ferredoxin. Nature Structural Biology
> > 3:451-457.
> >
> > *****************************************************
> >
> > We have worked with a highly basic protein that refused
to
> > even precipitate at concentrations lower than 100 mg/ml.
> > What finally worked was to co-crystallize it with
> > monoclonal Fab that were available from collaborators.
> >
> > You might consider trying favorite additives for DNA
> > crystallization,
> > e.g., cobalt hexamine, spermine, spermidine, etc.
> >
> > *****************************************************
> >
> > We managed to crystallize a halophilic protein (very
> > acidic) in its presumably natural medium (3M NaCl) +
around
> > 2M ammonium sulfate. On the other hand, we completely
> > failed (so far) with other halophilic protein around
these
> > conditions and many others. Have you checked the
> > proteolytic digestion pattern of your protein ? Could
there
> > be some flexible regions the prevent crystallization ?
> >
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