[3dem] Any experiences with SMA extracting proteins in native membrane?

Timothy Dafforn T.R.Dafforn at bham.ac.uk
Thu Jun 28 09:31:33 PDT 2018


Hi Alka,
how are you..?
As you have divined, proteins in SMALPs often bind weakly to NiNTA resin. We think that the histidine tag binds back to the carboxylates in the polymer.. That's why we bind in low imidazole. There is also a kinetic effect on binding with it often being slow (we suspect that if the tag is bound to the polymer it may free up transiently making it available for binding to the resin) which is why we say bind overnight by batch. 
The addition of salt helps to break the unwanted interactions with polymer and is definitely worth a try. Another option (although more work) is to switch to a strep-tag which works very well (too well.. you sometimes need way more ligand to knock it off the resin).
If it is still a problem we have just published a new positively charged polymer (SMI) which should have no interaction with the His tag. It works like SMA. Of course it is a lot newer so not so thoroughly tested.
regarding increasing the concentration.. why do you want to go higher?
Cheers
Tim

Tim R. Dafforn 
Professor of Biotechnology
School of Biosciences
University of Birmingham 
Edgbaston
Birmingham 
B152TT
0121 414 5881


> On 28 Jun 2018, at 4:22 pm, "aagrawal at tetragenetics.com" <aagrawal at tetragenetics.com> wrote:
> 
> Hi all,
> 
> I've been using SMA (initially powder, now the Xiran Na salt of SMA2000 from Polyscope, after pHing a 2X solution to ~8.0) to solubilize ion channels in Tetrahymena thermophila. I've gone up to 3% where I start seeing some solubilization of  my proteins (I've tried 4 different ones), but almost nothing is binding to Ni sepharose (from GE). Binding is done in the absence of imidazole, at 150 mM  NaCl, in batch overnight.
> 
> Any suggestions on how to improve solubility and binding? Is the protein that solubilizes thought to be of higher quality than the protein that doesn't? For binding, I've seen suggestions such as increasing salt, diluting the sample before binding, and removing free SMA by dialysis before binding. It's a very cool system - any direct experience would be highly appreciated.
> 
> Best,
> 
> Alka Agrawal, PhD
> Senior Scientist
> TetraGenetics, Inc.
> 91 Mystic Street, Arlington, MA 02474
> P 617 209 4018 M 617 710 8983
> E aagrawal at tetragenetics.com
> www.tetragenetics.com
> 
> ----------------------------------------------------------------------
> 
> Message: 1
> Date: Tue, 12 Jun 2018 20:09:59 +0000
> From: Timothy Dafforn <T.R.Dafforn at bham.ac.uk>
> To: "Magdalena.Schacherl at caesar.de" <Magdalena.Schacherl at caesar.de>,
>    "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: Re: [3dem] Any experiences with SMA extracting proteins    in
>    native    membrane?
> Message-ID:
>    <AEE414A76439ED4DAF3FC1A02C638295012895F41D at EX11.adf.bham.ac.uk>
> Content-Type: text/plain; charset="windows-1252"
> 
> Hi all,
> We developed the SMALP system in 2009. It is a lot milder and does leave behind lipids that we think help stabilise the proteins. Proteins in SMALP can be frozen and even freeze dried into a powder and sent by post! As Magdalena mentions there are a number of polymers out there now. The basic SMA ( or Xiran, trade name for SMA from Polyscopes) comes in two flavours with 2:1 and 3:1 styrene:maleic acid ratios. We have shown that there is very little difference between these. If anything the 2:1 is marginally better. The DIBMA (sokalan) seems to be harder to work with and the particles are less stable. However it is less sensitive to divalent cations. We have recently published work in styrene maleimide which is positively charged and not at all sensitive to divalents.
> We have done bits of work on EM, notably collaborating with Steve Muench in Leeds to get a sub nanometer resolution structure of AcrB. But nothing like the structure of complex III mega complex in SMA in Nature recently. Truly awesome!
> Very happy to help anyone with this.
> Cheers
> Tim
> 
> 
> -------- Original message --------
> From: Magdalena.Schacherl at caesar.de
> Date: 12/06/2018 20:24 (GMT+00:00)
> To: 3dem at ncmir.ucsd.edu
> Subject: Re: [3dem] Any experiences with SMA extracting proteins in native membrane?
> 
> Dear Teige
> 
> we have quite intensely used SMAs for the extraction of a highly abundant protein from native membranes. Compared to extraction with detergents, SMA-extraction seems to be much milder and preserves the active state of our target protein, possibly mediated by the preservation of the native environment (lipids, binding partners). As with nanodiscs, it is probably wise to try several protein/polymer combinations ? unfortunately no ?magic bullet? here.
> 
> We have tried XIRAN 30010 (that we got as a free sample from Polyscope ~100 mL) and Sokalan CP9 (also a free sample from BASF ? smallest sample size is 1 liter!).
> 
> We have dialyzed the solutions against the desired buffer, as the pH of the original solutions (ca. 30% v/v) is 10-13!.
> 
> XIRANS are well behaved and after dialysis you reach the desired pH. In contrast to that, Sokalan never reaches the desired pH and stays one pH-unit above that (despite >24 h dialysis and several buffer changes) ? we are not sure, why this is the case. Possibly it has some buffering capacity by itself??
> 
> When using SMAs/DIBMAs be careful with higher concentrations of divalent cations (> 1 mM), as they might change the solubility of the polymers themselves and lead to precipitation. This is less an issue for Sokalan. Also the pH-range is limited (we used pH 6.5 and 7.5), but you may have to try it out.
> Also binding of your protein to NiNTA-matrices can be an issue in some cases.
> 
> We also always omitted glycerol in the extraction step, but this is no must (compare the recent AcrB structure). The extraction parameters have to be established for every protein/polymer combination. We used a 0.25-8% (v/v) range for 30-90 min for initial small-scale tests. The efficiency was a bit lower than with detergents, but it is all about quality, right?
> 
> We are using ash-free filter paper for blotting/plunge freezing, as the residual divalent cations may lead to precipitation at the very last step ? but I would consider this to be rather ?voodoo magic?. We never saw any ?weird? behavior of SMALPs in plunge freezing using holeys (with Vitrobot and Leica blotters).
> 
> In contrast to detergent-solubilized protein, boiling of SDS-samples with XIRAN-solubilized proteins is easily possible without inducing the ?laddering effect? and thus artefacts. Sokalan-solubilized protein smears in gels. We tried the CH3OH/CHCl3/H2O-extraction method (which is in principle a Wessel-Fluegge protein precipitation) as suggested by Sandro Keller, but in our hands it did not work so well. This might be a protein-dependent behavior.
> 
> In the end we went for nanodiscs in our project, as the SMALPs appeared somewhat roundish/blobby in cryogenic conditions (our target protein is rather small with ~250 kDa and it is quite unclear how large the SMALP-disks really are). For larger membrane proteins this might not be an issue.
> 
> 
> A huge knowledge base is found under: http://www.smalp.net/. There you can find some of the tips I have mentioned above and citations of most SMALP-related publications.
> 
> There was a SMALP community meeting recently with contributions from most of the experts in the field: http://www.smalp.net/SMALP-2018-agenda.pdf
> 
> All the best,
> Magdalena
> 
> 
> --------------------------------------------------------------
> Dr. Magdalena Schacherl
> Structural Dynamics of Proteins
> 
> phone +(49)228-9656-278
> e-mail  magdalena.schacherl at caesar.de
> 
> research center caesar
> center of advanced european studies and research
> - an associate of the Max Planck Society
> 
> Ludwig-Erhard-Allee 2
> 53175 Bonn, Germany
> www.caesar.de
> ________________________________
> Von: 3dem [3dem-bounces at ncmir.ucsd.edu]" im Auftrag von "Matthews-Palmer, Teige Rowan Seal [t.matthews-palmer14 at imperial.ac.uk]
> Gesendet: Dienstag, 12. Juni 2018 18:04
> An: 3dem at ncmir.ucsd.edu
> Betreff: [3dem] Any experiences with SMA extracting proteins in native membrane?
> 
> Dear 3DEMers,
> 
> I only recently became aware of SMA co-polymers for solubilising membrane proteins in a ?native bilayer? environment https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593810/ - and to be honest was a bit shocked I hadn?t heard of this option before.
> Supposedly you can directly solubilise your target with some amount of native membrane, which seems an improvement over MSP nanodiscs and salipro which require you to transfer your detergent-solubilised protein in to the lipid construct.
> 
> I wonder how many of you have experience with this. I?d love to hear anyone?s first hand experience and any caveats that may be hard to find in the literature - like solubilisation efficiency, dependence on your target?s density in your membranes, any difficult behaviour with plunge freezing on holey support foils? Importance of testing different co-polymer average sizes to suit your target?
> 
> Thanks & best wishes,
> Teige
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> ------------------------------
> 
> Message: 2
> Date: Tue, 12 Jun 2018 20:51:46 +0000
> From: Garry P Morgan <Garry.Morgan at Colorado.EDU>
> To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: [3dem] review of Leica plunge freezer?
> Message-ID: <053FEC51-EF31-4C55-BB57-8E8DA2BF5062 at colorado.edu>
> Content-Type: text/plain; charset="Windows-1252"
> 
> Hi,
> Is anyone willing to share their opinions on the Leica EM GP or GP2 plunge freezer?  i?m curious if it gives more consistent results than a Vitrobot Mark IV.  or if people are happy with the unit overall.
> thanks,
> Garry
> 
> ------------------------------
> 
> Message: 3
> Date: Wed, 13 Jun 2018 03:22:48 +0000
> From: Craig Yoshioka <yoshiokc at ohsu.edu>
> To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: [3dem] Relion benchmarks on Volta
> Message-ID: <B8588F41-5C19-4F8B-B314-B36BD3788015 at ohsu.edu>
> Content-Type: text/plain; charset="utf-8"
> 
> Hi all,
> 
> Does anyone have any Relion benchmark #s on Volta GPUs?  I?m also interested in multi-GPU gains using NVLink.
> 
> Thanks,
> -Craig
> 
> 
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