Aggregated protein for crystallization

From: Raji Edayathumangalam
Date: 21 February 2012 23:21
Hi Folks,

As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience.

After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein.

Thanks.

Raji

--
Raji Edayathumangalam

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From: Jacob Keller
Hey, it could be that you just have a big oligomer--any support for
that in the relevant literature? A 10-mer would probably beat out an
s200, no? Do you have any other way to ascertain the oligomeric state?

Jacob

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From: Phoebe Rice
I probably it depends on whether you've got gunk or a functionally relevant oligomer in that void volume.  Is it active?

RecA and Rad51 both form open-ended head-to-tail oligomers and yet they still crystallize.

=====================================
Phoebe A. Rice

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From: Puey Ounjai
Hi,

Your idea does sound really crazy but actually Jacob had made a very good valid point.
Question is do you think your aggregate still functioning or not, if not, can you revive them in vitro and how effective is your refolding process if you are going to refold them?

You may want to take a look at structures of some bacterial pore-forming proteins. Many of them can be expressed as large inclusion bodies in heterologous host of which we call "aggregate". I know it sound fishy but after refolding and proper proteolytic activation, those proteins retain the ability to induce mortality to their host cell as good as the real one.

Anyway, good luck with your endeavors!
Puey

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From: Bernhard Rupp (Hofkristallrat a.D.)

Well, depends on what 'aggregated' really means. If it implies reasonably
weak oligomerization interaction - and it
might not be too strong given that the oligomers remain soluble - a
chaotropic crystallization agent (on the
extreme end certain high salts, consult Hofmeister for chaotropicity) may
rip such soluble
aggregates apart or at least get them into a conformationally reasonably
well defined state.
Crystals do appear/transform even from precipitates on occasion.  CD will
tell you about
the (secondary structure) folding state, not the aggregation state, DLS/MALS
would give an estimate for
and distribution of the aggregation state. With light scattering, you can
also do some systematic experiments
exploring what might reduce the aggregate size.

I think soluble, defined secondary structure,  and a lot of it, is already a
good sign.

BR

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From: Shya Biswas

Hi,

Did you try using a different column like Superose 6? This column works well to separate large molecular weight proteins including oligomers. Ideally if your solution is not cloudy (coming out of void volume) those are not aggregates those might be oligomers. 

HTH,

Shya

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From: Allan Pang
Not sure if it will be helpful... but my protein is not the most stable protein, in fact, it does aggregate over time (most likely due to its 'sticky' nature).

However, I still get crystals.  The problem is the crystals are among the gunks and precipitates.

Your case might be different since my protein does not elute out at void volume. Perhaps, try work faster?  Sometimes protein aggregates over time rather than immediately.

Allan

--
Allan Pang

PhD Student

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From: Savvas Savvides
Dear Raji
Running a blue-native gel with lanes in the presence and absence of a reducing agent could prove quite informative. DLS could also return a quick result on the particle distribution in your sample. In that case I would measure samples as fractionated from the superdex200 and compare the measurements after centrifuging the same samples at 100k x g for one hour.

Best regards
Savvas

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From: Pius Padayatti
some more thoughts,
Do a cryo-EM imaging, it will be ideal than DLS.
if the particle sizes are uniform i would think your protein in that state
might be useful.
cheers
Padayatti

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From: Pius Padayatti
This was meant to Raji,
So here it goes to all.



---------- Forwarded message ----------


Hi Pius,

I have done exactly that. I have one protein eluted at void volume of S200
column. The MALS experiment estimates more than 100 copies of monomer in the
aggregate. Against my belief, the protein crystallized and diffracted to 2.3A
and the resolution was improved to 1.6A later. It turns out that the
crystallization buffer breaks the aggregate to dimer and crystallized it from
there. I used the lower concentration of the crystallization buffer to run the
sizing column and the protein was eluted at reasonable elution time for dimer
even though the profile looks ugly and the purified protein is not
crystallizable any more. I guess the aggregate somehow protects the folding of
the protein and releases the protein slowly to the protein crystal in the right
buffer condition. So I think you should setup crystallization trials with your
aggregate protein. We cannot search hundreds of conditions for running sizing
column. So why not let crystallization trials find that for you?

Good luck.

Zhen

Marasco Laboratory

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From: Ho Leung Ng
     If you haven't done so already, I would screen buffer conditions
(pH, salt concentration, glycerol, strongly reducing conditions,
ligands, detergents) by DLS to see if you can reduce aggregation. You
might get lucky by setting up crystallization plates, but chances are
you won't get very useful information from them, especially if your
aggregated protein is soluble.

     There are fluorescent dyes sensitive to aggregation state such as
ANS (anilinonaphthalene-8-sulfonate) or Nile Red. I have not used them
myself and would like to hear if others have found them useful for
screening buffer conditions.


Ho

Ho Leung Ng

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From: Bernhard Rupp (Hofkristallrat a.D.) 

> You might get lucky by setting up crystallization plates, but chances are
you won't get very useful information from them, especially if your
aggregated protein is soluble.

I seem to fail to understand how crystallization plates would give
information in the not-special case of protein aggregates NOT being soluble?


BR

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From: Ho Leung Ng 

I should have been more clear. If your protein is insoluble aggregate,
you can use crystal screen results to get an idea of what buffer
conditions favor solubility (and hopefully monodispersity). An example
is described nicely in Collins et al, Acta Cryst F 61:1035.

Ho

Ho Leung Ng