Hi Amit,
like Artem wrote below, even despite poor solubility, soaks with dried
compounds work well (also in our hands).
We adapted it from the work of T. Barthel, J. Wollenhaupt and Manfred
Weiss. Also colleagues from industry report good results.
We add compound dissolved in DMSO to the crystallization well, let DMSO
evaporate overnight (37° incubator), then put mother liquor on top of
the dried compound and then the crystals. If all compound would dissolve
again, final concentration would be 10-50 mM. Soak time from 1-2 h to
overnight.
This worked for poorly soluble compounds where we did not succeed with
soaking in the presence of DMSO.
Hope that helps!
Best,
Guenter
Dear Amit
As David already pointed out, all proteins are different and it's hard
to say in advance what amount of DMSO may work (or not).
An additional concern is that DMSO can also interfere with ligand
binding (cases from my personal past history), especially if these
inhibitors/ligands are on the weaker side.
Solutions:
Despite its very high boiling point (189C) DMSO can in fact be
evaporated from a small sample of your inhibitor, resulting in more or
less solid inhibitor sample that can be re-dissolved in the same DMSO
(but higher concentration), some other solvent, or perhaps directly in
the protein solution. The latter is sometimes the only way to do this
- I used to set up drops of DMSO solutions, then evaporate the DMSO in
high vacuum (heating helps) with a cryofinger, then set up protein
drops on top. This of course requires access to a lyophilizer or
something similar.
If you have a vial of your solution you can freeze-dry DMSO with
water, by first diluting the sample then freeze-drying it. Also water
can sometimes crash the substance out (if not water, then perhaps
Ether or another solvent where your inhibitor does not dissolve) which
makes it easier to redissolve (but there will be a loss of course).
Find a friendly chemist nearby and ask then to put your sample in a
speedvac on 'high BP' setting
Notably, if you're "blessed" with an inhibitor that has the general
solubility of a Sony Walkman, once you get rid of the DMSO, you may
find out that the damned thing does not want to dissolve in anything
else, including your protein solution. This happens a lot during early
discovery phases when compounds are not very active (micromolar) and
also poorly soluble (also micromolar). This is by far the most
frequent cause for failing to co-crystallize (or soak) a ligand of
interest. Very frustrating. Some success can be achieved using high
DMSO or DMF (DMA also can be good) in your crystallization, or by
phase transfer catalysts like Cyclodextrin(s) or appropriately
formulated micelles. All of which can also mess up crystallization,
needless to say.
Best of luck in your endeavors!
Artem
- Cosmic Cats approve of this message
On Wed, Oct 2, 2024 at 2:51 PM amit gaur <cdriamitg...@gmail.com> wrote:
Hi everyone,
I am trying to crystallize a protein with a drug molecule. The
protein concentration is 15.5 mg/ml, the drug stock concentration
is 10 mM, and the drug is dissolved in DMSO. I am adding the drug
to a final concentration of 1 mM in 100 ul of protein, and the
DMSO volume is 10 ul for Co-crystallization. I want to know how
much DMSO is permissible during co-crystallization with the drug
and if DMSO can poison crystal formation. I have not been
successful in getting crystals with inhibitors till now, but I
obtained crystals of protein without DMSO, and those diffracted to
2.5A.
Thanks,
*Dr. Amit Gaur,*
*Research Scientist*
*Center for Biotechnology and **Interdisciplinary Studies,*
*Rensselaer Polytechnic Institute,*
*1623 15th Street, Troy, NY, 12180*
*
*
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