[ccp4bb]: Responses to a protein requiring high salt for solubility

["tommy wang" <wch@post1.com>]

***  For details on how to be removed from this list visit the  ***
***    CCP4 home page http://www.dl.ac.uk/CCP/CCP4/main.html    ***

Hi everyone, this is a compilation of all the responses I got for my
question below. Thanks to all the contributors and I hope it will be of use
to everyone here as it did for me.


I have a protein that requires 0.5 to 1M NaCl to be soluble at 10mg/ml. My
question is whether I should bother screening this protein and whether there
are any protein crystals grown using protein buffers at such high salt
concentrations? I am aware that ammonium sulfate is used at high
concentrations to grow many crystals but are there crystals successfully
grown from proteins that require high salt concentrations to be soluble?
Please share your experience. Thanks



In some ways this is a very good situation to be in.......with a salt
soluble
protein, it is very easy to drive it out of solution simply by lowering
the
salt concentration (through very slow dialysis) or by continuing to a much
higher salt concentration where it is no longer soluble.  One particular
example would be the serpin, PAI-1, which is most soluble at +AH4-1 M NaCl but
crystallizes at 1.8-2.0 M NaCl.

-Tom Stout


          I don't know whether this will answer your question, but
sometimes changing the buffer ( keeping the same pH) , can drastically
bring down the amount of salt required to keep the protein in
solution. You could try out a different buffer if nothing else works.


best wishes
ganesh


I've had similar experience with three homologs from a thermophile -- they
all needed fairly high +AFs-NaCl+AF0- (0.25 to 1.0 M) to stay in solution at 10
mg/mL.  My advice is to just try concentrating in the high salt and see what
the standard screens give -- in my case several did crystallize from PEG.
You might also try to determine more systematically the +AFs-NaCl+AF0- requirement
for a given target protein concentration (e.g., 10 mg/mL).  I did this by
dialyzing a more dilute protein solution in various +AFs-NaCl+AF0-, concentrating to
the correct volume for the target protein concentration, then measuring
concentration by UV.  Using a protein solution near its actual solubility
limit may improve the results in the screen.
The salting-in (by dilution or dialysis into lower salt) suggested in other
e-mails also worked for some of my homologs -- try reverse vapor diffusion
using one drop of concentrated protein in high salt over pure water in
hanging drops to start.  Good luck+ACE-

--

David R. Buckler


Lipovitellin requires 1M NaCl (or other salts as long as the ionic strength
is above a certain level) to remain soluble in aqueous solution.  Even
higher
concentrations of NaCl were used to obtain crystals.  No other additives
were
needed.

Best regards
Jim T


Hello,  We recently published a dioxygenase structure (Titus et al, Nat.
Struct. Biol. 7, 542-546) where, for solubility purposes, we maintained the
protein stocks in 0.5 M NaCl or 0.5 M Amm. Sulfate.  The protein
crystallized nicely using 1.5-2.0 M amm. sulfate as a precipitant.  Dave
Timm

If you need such high salt concentrations to keep this protein soluble why
not try salting in for growing crystals?
Regards Arjan



You can produce crystals by both salting in AND salting out - so you
might get a result using a batch method and equilibrating against a
+AF8-lower+AF8- salt concentration. I'm sure there are quite a few examples
of this .. I think that the cyanobacterial photosystem 1 is
crystallised in this manner.

S.M.Prince


One obvious thing to try first is to slowly reduce the salt
concentration. Using this method I crystallized Aminopeptidase A from
E. coli, which precipitates if the salt concentration (NaCl or KCl) is
lower than about 0.8 M. In the case of this protein vapor diffusion was
o.k., e.g. the dilution of the protein as the drop grew bigger did not
prevent the formation of crystals. Otherwise you might want to try
diffusion across a membrane.

Good luck,
Norbert

Yes, I've grown crystals at high salt concentrations, but usually in the
crystallisation reagent rather than in the protein buffer. I prefer to
take as much salt out as possible before crystallisation. So if it isn't
soluble at 10 mg/ml, I crystallise at a lower concentration. Some of my
experiments are at 4 mg/ml or lower. I've even known people to crystallise
at 1 mg/ml. If you look in the literature you will find a wide range of
concentrations being used for crystallisation.

Hope that helps.

Sarah

Just wondered if you had tried +ACI-salting out+ACI- your protein?? It is used
when proteins require salt to be soluble. To try and crystallise, you
reduce the salt concentration to just the right ammount so that your
protein is supersaturated. Hopefully crystals might grow+ACEAIQAh-

Ducruix  Giege have a short bit about it in their book+ACEAIQ-

Its worth a try anyway+ACEAIQ-
HTH
Lou

It is not that unusual with your protein - sometimes some proteins require
high salt for solubility - My opinion you could try screen but do not try
all of them check carefully the composition.
In past I have crystallised one protein in presence of 3.8M urea - that
protein has had the same problem.
I can tell you one more approach successive salting out experment may also
help you.
Next you could also try different organic precipitant such as ethanol,
iso-propanol etc.

Ajit



One obvious thing to try first is to slowly reduce the salt
concentration. Using this method I crystallized Aminopeptidase A from
E. coli, which precipitates if the salt concentration (NaCl or KCl) is
lower than about 0.8 M. In the case of this protein vapor diffusion was
o.k., e.g. the dilution of the protein as the drop grew bigger did not
prevent the formation of crystals. Otherwise you might want to try
diffusion across a membrane.

Good luck,
Norbert


If you need such high salt concentrations to keep this protein soluble why
not try salting in for growing crystals?
Regards Arjan


You should never give up on an experiment without first trying it.  I
have found on several occasions that 'the general rules' do not always
apply to science - especially crystallography +ACE-  If you at least try the
experiment and find that it doesn't work then you can definitely stop
there, but don't give up before you start.
There is absolutely no harm (and probably you will be rewarded) to try
to crystallize your protein in solutions containing the high conc. of
salt.  Although I have not myself crystallized protein in such a
concentration of NaCl, I know of other groups who have, in particular a
group in Japan whose crystallization conditions for a protein called
HBP23 included a reservoir consisting of 10+ACU- PEG6000 and 2M NaCl +ACEAIQ-
So now do you think that 0.5M is a high concentration to be
crystallizing in ??
The reference for that experiment is

Hirotsu et al, (1999).  PNAS, 96: 12333-12338.

Hope this helps (and keep trying)

Magnus S. Alphey


My protein crystallizes in 15-25 +ACU- PEG of various sizes in the presence
of 600 mM NaCl.

In my case I have a lot of saltbridges in the crystal contacts.

I would absolutely bother to screen it. With various salt
concentrations.

Stefan



0.5 to 1M NaCl is NOT a high salt concentration - and yes there are
crystals grown at high salt concentrations - 2 to 3 M AS is no that
unusual (ionic strength +AD0- 3 to 4 ) whereas 1 M Nacl has an ionic
strength of 1.

Amo Pahler


I grow my crystal at 100mM NaCl.But i have also tryed at
1M NaCl.Na crystal grew, and diffracted to almost same extent as
the 100mM NaCl grown crystal.

S. Dhatta


If you require high salt for your protein to be soluble, it is likely that
your protein comes out of solution if you dialyze against a buffer with
lower NaCl concentrations.

This could, in principle, be used for crystallization, e.g. in dialysis
buttons. Other approaches are possible (classical hanging drop setups,
where the reservoir contains a buffet with lower NaCl, and e.g. small
concentrations pf polyethylene glycols).

We have successfully crystallized a protein this way. Solving the
structure remains difficult, however+ACE-.

F.M.D. Vellieux