This masterclass is about the largest proteome-scale investigation of membrane protein solubilization to date. The class is based on research conducted by Dr. Merino and scientists from the University of Cologne.
You’ll learn how copolymers can solubilize and stabilize membrane proteins without the need for traditional detergent-based methods and enhance your understanding of near-native membrane protein assays.
Yeah hello everyone I’m very excited to moderate this interesting seminar today on synthetic co-polymer Nano disks enabling NE native membrane protein essays uh first I will introduce myself really quick uh my name is Mario lard as you heard I’m a postdoc at eth syc at The Institute of molecular systems
Biology and I’ve previously completed a postdoc at the University of Washington Seattle so my own research centers on the development of mass spectrometry based proteomic technologies that permit proteom by characterization of cellular signaling and protein regulatory networks using these strategies I investigate the wiring diagram of the
Cell and try to identify and reverse changes that occur during aging and disease and so me myself I came into contact with Cube biotech because I’ve been using some of their excellent products for phosphopeptide enrichments also for protein biochemistry and proteon I in general but um the people at cute
Biotech do much more than that they’re also experts for membrane protein research and they provide services and produced consumable in this area um specifically I know that they assist in protein preparation biophysical Target characterization and also structural determination by electron cryon microscopy along this line apparently they offer the largest portfolio of
Synthetic polymers that host cell resulting in higher protein stability and activity yeah now if this all sounds interesting to you you’re at the right place because we have Dr Philipe Marino here um Philipe is the head of cube biotex cryoem division where he oversees several of their structural biology
Services and is also a key part of Cube’s R&D team Philipe became an expert in cryo em through his post-doctoral training at the max Blanc Institute for molecular physiology in Dortmund and then a project leader at the max Blanc Institute for biology in tubing and his combined expertise in atomic modeling
And cryoem has provided him with a unique skill set allowing him to tackle challenging structural biology problems such as those typically presented by membrane proteins and with this I’m very excited to hand the mic over to Philipe please take it away yeah thank you very much Mario for
For for your kind introduction so what I want to talk to you today about is um basically one of our most beloved technology platform which is this copolymers to basically study membrane protein in as as near native environment as possible so as you just heard we’re we’re a German company we are situated
In monheim this is in between cologne and and and dldo we live here just right next to the Rin River right in the creative campus in the city of monheim we are interested in in in protein purification and our core interest basically is membrane proteins
So we are we are a CR so that means that we are providing Services of mem protein purification and characterization for other companies or academic LS and during our services we’re usually generating new tools and new strategies for purifying and studying membrane proteins so whenever we see that
Something is working really well right or we are designing something that is very important for protein purification for example we’re going to put it in our shop also as a as as a as a product right which is something that you can also go and do in CU biodex web page
Right you can directly purchase the products that we’re using every day to to do our own internal research so basically our our services are centered in as I told you in in in membrane protein expression purification and characterization right so and we at the moment are going are doing
Everything from the construct design up to basically structure determination or preparation for any Downstream applications we also can do some other assets like for example Affinity determination to small molecules using um thermophoresis we can do spr measurements and things like that so the the core of our R&D part is centered
Basically on let me show the pointer here so on strategies to solubilize the best way possible the membrane proteins then how to purify them and then to how how to make them ready for any Downstream application that you might have not only structur determination this is of course my my
Most the part that I’m most interested in because it is the part that I do most right but of course we we’re doing any other sorts of of biochemical characterizations of these proteins right so the question is of course why membrane proteins so they are about
Between 25 and and 30% of the human genome are membrane proteins right so most of the proteins are going to be anyway soluble proteins so why concentrating on this very particular set of proteins and although they are not so many in in number right they are very important for for cellular life you
Can see here for example in this cartoon right they are sitting at the edge of the cell and this is basically the first barrier that the cell puts with the environment right this is how the Environ the cells understand what’s around them get signaling from other cells interact with their with their
Surroundings they anchor to their substrates for example so they are very important ways in which cells can know where they are and what to do and of course in eukariotic cells they are not only at the plasma membrane but they are also inside in any membranous kind of compartment including the mitochondria
For example and things like that so they are they control what goes in and out of organel and and the cell itself so they are quite important and you might not be surprised that although they are only about 30% of the of the proteins that a
That a that a genome has they are more than half of the targets of the currently approved farmaceuticals so for example you have that 33% of the current drugs this was in 2017 right and not so long ago we’re targeting gpcrs another almost 20% we’re targeting ion channels
And this is because they are really really important but the problem is that they are some of the most challenging kinds of proteins to to characterize so for example if you if we start the cartoon here with the with the cell that I that I showed you before so what you would
Typically do for for characterizing these proteins is that you need to isolate them right and have them purified so what you do basically you take your cells you break break them most often mechanically with a sonicator for example or a French press then you get these membrane
Pellets so you you can you can get the membrane disrupted right then you can collect the membranes and these membranes are going to have your protein along with all the other membrane proteins that are in the organism so for purifying them what you need to do is to
Take them out of this environment right and get them a single units otherwise you’re canot purify them so what we’ve done for the last I would say 40 to 50 years is solubilizing with detergents so what the detergent is going to do is to completely remove all the lipids that
Are present there in the membrane and substitute this lipidic environment by a membrane by a detergent mle once you have that then you can you always have to put in your in your in your buffers and in all your applications you always have to you always have to have a little bit of
Detergent present right because otherwise his Mel would fall apart then you can PU ify your protein and if you think that it’s really important to have the lipids around then you can just take your protein and reconstitute it into something that mimics differently the the membrane environment so for example
You can use a MSP based Nano disk to put lipids back inside or you can use the old generation amples that is a polymer that would wrap around your protein and substitute basically the the detergent or you can bring them back into a fully liing environment and then put them
Inside Myles the problem is that even though you bring them back into a lipidic environment you are always going to have to do this stripping naked process in between right so you’re going to lose a lot of protein the detergents are going to interfere with what you’re
Doing later right so this is this works of course this keeps being done because this detergent solubilization is very efficient and uh some proteins are happy in detergents right but many others aren’t you might think well maybe we can skip all this process and and and then go directly to the reconstitution and
This is more or less what I’m going to talk to you about there are all technologies that can do this in one step like for example when you use saposin but the problem with that is that you still have to put detergent into the sample for the proteins to be
Scooped out of the protein of the membrane so you’re always having this detergent around which we want to which we want to avoid right so the but what I want you to to get from this is start um at some point your proteins are going to be completely stripped from their native
Environment and we usually do not want to do that to proteins so um this is just an example um that is also very close to me because this is an example from cryan right there’s many other similar that you can find in the literature this was a a
Project that we had together with MP Dortmund a few years ago and this was a cry characterization of a of an iron channel from from drosophila this is called slow right and if you look very carefully at the at the methods of the paper R you’re
Going to find that at some point for the last step during the verification we had to take the lipids from the insect cells and put them back into the sample in order for the protein to be happy and if you look in the reconstructions for example you see that there’s all these
Magenta kind of densities that are that were interpreted by by by by toas the person that did the as a lipid densities right so actually what happens when you do not take this step and you do not put the lipids back into into your preparation this is kind of the Reconstruction that
You get and then you see that in the area where the lipid with the transforming region should be there is basically nothing ordered so the proin is really unhappy in just certain it needs to have these lipids there and what we’ve done like a few years later
Was to just as a part of our QC just checking out that all our cryo pipeline was working correctly we just took the same sample purified by a completely different person reconstructed by a completely different person and we still see that those limbits are exactly in the same position so this is a
Reproducible thing that we see with this protein right and uh this thing to this uh this interaction seem to be really important and of course you don’t have to take our experience Sol as a as a as an example there’s many other papers like the ones I’m showing you here that
That show you that not only lipids are important for keeping the structure of the protein they are also involved in maintaining or determining specific functions because you’re not only going to have specific lipid protein interactions there is also kind of a more passive effect of the of the lipids
Around your protein but that are going to create specific shapes that are going to be kind of tailored to your protein right and then you’re going to want to have the the right lipid composition to promote the correct function in a way so we have basically uh this kind of uh
Levels of nativeness if you want in the in the in the different preparations that you can have right so the most the least native I would say is detergents ampols maybe are a little bit better although they would be comparable to detergents the king of Kim I would say
Is MSP based Nano dis because these give very nice reconstructions but you still have to go through this process of stripping your protein naked and then putting the lipids back again what we like better and this is our core technology here in in CU biotech is synthetic copolymers and in
Synthetic copolymers you’re going to end up with something that looks very similar to an MSP based Nano disk but there’s some key differences that I’m going to show you in a second that we think make it a significantly better technology as any of the other available Technologies right so they were introduced around
2010 right as this they were called Mass this was the first polymer that was introduced it’s a polymer made by malayic acid here and styrene and what they discovered in the original paper was that these polymers so you have repeating units of this to a molecular weight of like 10 kilodaltons or bigger
And then these polymers would go into the membrane and incorporate and then self assemble into an anod disk and this polymer itself would scoop out basically the protein out of the membrane but the import part is that they would scoop out the protein from the membrane together with the native lipids of the
Protein so this is from a from a different paper I think this is from 2014 and then you see that these are basically now thin layer chromatography not to check the protein but to check the lipids present in the sample and then these are different steps in the
Purification until you get to the very to the purest part to to O your protein right in its Nano desk and then you can see that basically there’s different kinds of lipid head groups there so the lipids are still there right the protein is isolated and is relatively happy so
This is exactly what we want so fast forward to now and basically these are the four main families of polymers that are available or or the main families of polymers that are available you have the original SMA that it’s malayic acid with tyrine around 2014 or 15 uh the group of
Sandrella introduced these dma polymers which is the iso buyin with malic acid so you don’t have the ring anymore and a few years ago this new one was introduced right that it’s not not malayic acid but it’s acrylic acid so you have basically less charge in the
Backbone and then a few years ago the group of Manuel aans introduced this new generation amphiboles that are commercially called Ultra solute and basically the main difference with the original anol is that these ones have a different s here and they now have the capability of solubilizing proteins just
As the other polymers so these are basically the main polymers that you’re going to see in the literature today so now how a PR purification would work with these guys so basically up until the step for example is exactly the same so you collect your your membranes but now you put the polymer
Inside and instead of having to solubilize and reconstitute and everything this is going to directly create native nanodisks from your protein and all the other proteins that are in the cell right and then from this point forward basically you can think of your protein just as a soluble
Protein so you never again have to put H polymers inside detergents or any additive whatsoever other than your buffers salts and the typical things that you would use for for membrane for soluble protein work right and then you can purify your protein and then you have your protein again in a nanodisk
But the advantage of this nanodisk is that it has the lipids that are preferred by your protein and not the ones that you put back into it and it was never naked right so the idea is that with these polymers you can preserve the local environment and preserve F function and
Stability of the proteins which is kind of an issue especially with with membrane protein work right the problem and why these polymers got a bad I would say people got the wrong idea perhaps about this this polymers is that this is just an example from the
Literature as well this is how a typical um solubilization try of a of this is beta 2 adrenergic receptor for example but most proteins would look like this you have solubilization in DDM and you have this very fat band right you say like okay solubilization work really
Well and then you compare it with in this case with dma right and then you see that the solubilization efficienc is significantly lower than DDM so many people thought like okay these polymers are really not working so nicely but the systematic characterizations of the polymers against detergents are not
So common right or they were not done so often right so other than individuals individual examples of particular proteins we don’t know really how badly if at all these polymers perform compared to for example state-of-the-art detergents so what we did was to say okay you know what let’s do this
Experiment where we get to this part where we have all the proteins solubilize so we collect the membranes we throw the the polymers inside but instead of purifying single proteins we did this with hex cels right so to have an idea of how this is performing on the
Human proteum membrane proteum and then we directly sent it to a to an lcms to do mass spectrometry and try to see how many membrane proteins are we really getting out with these polymers and now we took advantage of the of the very large amount of polymers that we have
Now in the in in our shelves right so we went to the Shelf we took all the polymers that we had in 2021 and then we tried all of them and then we compared them against typical detergents both aggressive and not aggressive to see really how these things were were working
Out so uh right so the first thing we saw like the the negative control is just hey buffer is here then we have two controls of mild detergents and aggressive detergents right so this would be our pos positive control and hopefully you would expect that our polymers would end up somewhere here in
The middle right so we are not counting here right now how much of membrane proteins in Mass you’re taking out but rather how many different membrane proteins you can identify in the in the solubilization itself and I’m going to come back to the so now this is about
The versatility of the polymers and I’m going to come back to how efficient they are extracting the the membrane protein in a in a second so what was perhaps most surprising we were expecting these things to land kind of in the middle right we knew that they weren’t going to
Be as efficient as detergents necessarily what happened so this is the the line that is marked here what happened is that for example New Generation ampol end up basically to be being as versatile as detergents both ionic and non-ionic this which was a surprise right we we have come to accept that the
These Ultras solute amphiboles are basically the star solubilizers they they are they are very efficient but also smas are performing really well and even the ones that have the lowest solubilization capacity which are dmas they are still significantly better than the negative control and then they have a very respectable solubilization
Capacity so they turn out to be very versatile and the advantage here is that if you look at the at the axis is that these are thousands of proteins that we’re proving at the same time this is not a single example but we see that they are able to solubilize thousands of
Different proteins from a from a human cell so what we did then afterwards was to we know that they can solubilize several proteins at the same time so what we did was for each of the polymers get kind of a a solubilization profile let’s say protein a is
Solubilized with this efficiency B with a different efficiency and so on and so forth for these thousands of proteins right and do principal component analysis with that and we wanted to see if basically in the Pro the detergents and and the and the polymers we’re going to end up in different places of
The of the principal component space or not so in other words we wanted to know if is there a solubilization fingerprint to these things or are they all being kind of a general solubilizers and the first thing that was kind of interesting was that when you look at detergents
They all clustered together ionic and non-ionic detergents end up more or less in the same space except for Digitonin and we know this wasn’t so surprising because Digitonin is a very mild detergent so it’s not necessarily comparable to these other ones what was interesting was to see the Rapa buffer
Here this is a buffer used for for M spectrometer usually and the the this buffer is generally not conducing to to to Native preps so it’s really aggressive it’s going to get everything out right but at the expense of a of of the structure when we compared it to how the
The polymers look like this was very interesting to see because all the families of polymers basically clustered differently in this space right so DM end up here as is in a different place and Ultra solute in a different spot as well so where they end up in space is
Wasn’t so interesting to us as to see that the families are kind of clustering together so it’s really that families of polymers have different solubilization fingerprints basically and we thought like okay maybe this can Be this can be due to specific um characteristics of the proteins so maybe more hydrophobic proteins are solubilized by a certain polymer better or more negatively charged are solubilized by a different polymer better so this is exactly what we check next we checked whether these differences were coming from any that we
Could calculate directly from the sequence but as you can see for example this is the isoelectric point of the proteins that we are uh identifying basically in our Mass back runs and either when we look at the trans membran regions or the full protein sequences we do not see any difference in these
Violin plots for any of the polymers so I don’t think it’s they are almost identical for for all intent and purposes we didn’t observe any difference in in isoelectric point nor or in the hydrophobicity of the proteins so the only difference is of course between the the negative control which
Is heis and the rest of the polymers but this is kind of expected right so we are not seeing any differences in something that we could kind of trivially calculate from the from the sequences so if it doesn’t come from these characteristics then where does it come from so I think it’s still
Determined by by the protein itself but if it doesn’t come from the primary sequence then it has to come from a tertiary sequence ter structure sorry which means that it comes from the protein structure right so what we think is happening is that this I borrowed this from a from
Molecular Dynamic simulation uh paper and what they are showing here is basically that for they tried this for different proteins right and they had a mixture of lipids that were uh the mixture of lipids that you would have in a normal human cell and they saw exactly for different proteins you have these
Areas where specific uh lipids get enriched or depleted around the around the protein so this is for for fully saturated lipids this is for polyunsaturated lipids and this is for cholesterol and then you see really that in some areas cholesterol is going to bind really tightly to the protein this is the upper
Lifet in the lower lifet you see a different pattern right so it’s really like the protein is tailoring its own lipidic environment or there’s this specific interactions that are going to fit the the the shape of the protein and this is going to end up in a local specific lipidic environment
In a local lipidic fingerprint for each of the proteins so we think that this is what the polymers are sensing and unfortunately this is not something that we could calculate trivially right from from the from the proteins themselves so the take home message would be really if you want to
Find the best polymer for your protein then you have to screen but the good news is that you’re are going to find probably one of the polymers that really solubilizes your protein and the protein is really happy after solubilization right so up until now I told you about the versatility of these
Of these polymers right so how how many membrane proteins you can different membrane proteins you can get out of this uh of the cell but of course we try to calculate also raw raw efficiency right so how much material are you getting out of the out of the cell
And this is basically kind of a measure of the total amount of signal that you’re getting from the from the samples so you see that and we did this experiment now not at saturating concentrations of the polymers but we really did a a little a little gradient
Right from zero from 0.1% to 2.5% to see exactly which ones were more efficient right and how much you’re getting out of the cell and maybe if some polymers really need higher concentrations than others so you see that for element G and DDM already 1% is really high right and uh
You can see now that with amphipols you get a very similar a very similar profile and you see that the scale in all the graphs is exactly the same so you’re getting not only kind of the same saturation so 1% is enough but also at 1% you’re getting
Almost as much as you get in detergents so it’s really an not only versatile but very efficient compounds to solubilize your membran proteins we see that mass are a little bit less efficient is also is basically what we knew from before and some of them really
Like this SMA 200 takes a little bit longer to to to saturate you need a bit higher concentrations deas I think they kind of saturate at 1% as well but at the expense that you’re not getting as much protein out of it and AES are also very efficient particularly the 650
Which is very small so we think it’s very aggressive solubilizing the the membrane proteins and then you get performance similar to here with anoles so this was cool because now we see that they are vers as versatiles as detergents and some of them are actually as efficient as
Detergents so this is how basically the raw data would look like now so what you have here is a very large amount of data so basically this is the details of the experiment I just showed you so we have five concentrations for each of the for each of the compounds right and
Each row in this um in this heat map is basically one protein so we have here like 1800 proteins that could be at least detected in one of the compounds right and then we group them also by the number of transmem passes that they had
So you can see really that it’s not only single pass transmembrane proteins but you have two up until 10 I think the biggest one in this data set is like like more than 20 so it’s really they can not only solubilize small membrane proteins but we can they can solubilize
Big membrane proteins and I think this is one of the advantages or the main advantages that you would have over MSP based Nano disc the size of the Nano disk is going to adapt itself you don’t need to use differently sized polymers into it you as you would need different
Msps for your for your Nano dis of different proteins you just mix them with them with the with the sample and then it’s going to take out the appropriate the appropriate Nano disk out of it so and what I wanted to highlight here is that you can see that
Compared to element year DDM Ultra solute 17 and 18 and also these ASD show very similar profiles so of course this is a lot of data if you want to zoom in a little bit this is how everything looks like right so LG and DDM are working very efficiently with most of
The samples there are some some some proteins that you can even detect in in the negative control because maybe they got uh prote Iz during preparation or maybe they are just Loosely attached to the membrane but you have basically similar to element G and DDM the two ultra solutes are working really nicely
But you have many proteins here that are not for for example in the negative control and then you see them in Demag glycerol for example they maintain some of the smas right so it’s a I think it’s we’ve come to a point in the lab where we think
That if we can get a protein expressing cells a construct that expresses really well then we’re going to get that protein purified with a with the with polymers so it’s the purification of the of the protein has is not the bottleneck anymore but it’s really the production of the protein in in the
Cells right so this is a lot of valuable information so how can you access that so of course we have the preprint that that has all this data right but we also have created a solubilization database so for example if you’re interested in a prodin in particular this one here for
Example you go to our website to the solubilization database put that information there and this is the kind of answer that you’re going to get if we could detect it in our in our in our tests then you’re going to get a link to your protein right together with the
Solubilization profile of that particular protein so you can say okay this one I’m going to probably solubilize or try to solubilize with this MK here or I’m going to try this Demag glucosamine because I’m interested in that particular backbone or something like that so the solubilization database it’s already it’s available basically
For everybody to use you can you can just go to the link here at the bottom of the screen and then you can check if you can see any information on your protein of Interest so right now I just talk to you about uh raw solubilization right so
What I hav touched on is whether or not these proteins are happy whe whether or not these proteins are are are functionally solubilized which is kind of an important thing right so this is our basically our Workhorse right now this prot is called PX4 is a trimeric
ATP receptor it’s related to to pain so it’s a pharmaceutically very interesting right so the protein itself weighs like 120 kilodaltons and with the Nano it’s a little bit more of course right and it’s a it’s involved in neuropathic pain so this is the kind of pain that you feel
Not when somebody hits you right but it’s the kind of pain when you that you feel when you have a separate nerve for example so it’s not so biologically useful anymore and there’s a lot of necessity to to drug this so this is how a solubilization campaign basically would look like with
P2 X4 so we see that the protein gets very well solubilized with several AES Ultra solute 18 works really nicely and one of the mass works really nicely as well so this is um the proteins and this is the Western blood for the raw attack that we’re using always to purify our
Membrane proteins and in this case we decided okay let’s go on with ultr solute 18 because it’s one of the best polymers that we that we have so this is how a purification of PX4 would look like right so you see that this is the control that we have in
The DM CHS then you see that the peak is not so homogeneous as you would like the peak in Ultras solut in looks really sharp really nice this is what you want to see for a for for a purification of a protein and then this is some work that
We’ve done together with El a company doing um asms and this is a silver staining gel so no it’s not anymore Kumasi and then you see that in silver staining you have your the P2 X4 solubilized in Ultra solute 18 it’s really really pure so there is nothing else than P2 X4 even
Better than what you get in in Ddm so this is this was very encouraging so we’ve uh tested this in the the stability of this uh of of PX4 in different in polymers compared to detergents so this is a temperature ramp that we did in our in our pan right so
This is in nanot Temper pan so this is a DLS part measurement particularly and then you see that the different temperatures there is a certain variability in the size distribution of the particle but it’s really not that large difference when you do the same with the DDM CHS you see that already at
45 degrees Celsius the protein really starts to Aggregate and then it’s irreversibly aggregated into into something that is not useful anymore and we are very excited about this kind of results because this tells you that if the protein is really stable up to 40 degrees then maybe you don’t need to do
Your assays at 40 degrees anymore but you can really do them at 37 and maybe this is something that is really important for you especially if these things are going to go at some point back into the into the body right and it’s important also that you
Have that we can find this Superior stability compared to detergents because some some applications are really going to need that so if you need to raise antibodies for example then you want something that is going to survive a little bit longer in the in when when you’re kind of immunizing an animal for
Example so this is this is really nice to see so as I told you we are we partner up with this company doing Affinity selection spectrometry so there’s a few things that you need to do to identify basically compounds for for proteins right so you are using your protein to
Pull different compounds through a through through a through a matrix right so this is basically a small uh size exclusion chromatography if you want and then you need your protein to go really clean through it so this is the first thing that they tried and this is what one of the
First um encouraging results that we that they got is that always with in all the matrices that they tested Ultra solute 18 was outperforming detergent so the detergent seems to interfere very much in the in the recovery of the protein after passing through the Matrix this was not the case for for ultr
Solute 18 or to a significantly less degree to ultra solute to to DM and when they tried to actually measure the affinities of a known compounds right then they got again these are preliminary results right but they got something that was very similar to what is known in the literature for
These compounds this one gets a little bit of unspecific binding there in the end and this is because there are some issues with the solubility of the compounds themselves because this negative control should be basically here at the bottom so it’s more of a technical issue that an issue with the
Protein pree itself necessarily but this was of course very encouraging to see so we have something that is that lives longer and it’s functional as well which is good to see again so I’m I’m an electron microscopist right so I’m a structural biologist and then what we
Try to do also is to find better ways of getting membran proteins into into structures right and this is when we found a minor bottleneck you see on the on the left you see the protein in in DDM right so these are 2D classes of that protein they all look very nice
When you look in the right this is the same protein in Ultra solute 18 so two things that you might notice one of them is that the the belts here so the native Nano disk is significantly smaller than the belt uh that you get with with DDM we
Knew that that was going to happen because the particles look smaller in the DLS as well the problem is that most of the protein comes from the top so you you see these two ones are top views and then you can hopefully see that the details in the 2D glass are very crisp
So the protein seems to be happy but we’re seeing it from a single side and then you see that in these histograms here so this is a histogram of how many particles are looking for spec from a specific Direction so everything is kind of in this direction where you see in
The DDM case they are kind of around the protein and this is a becomes a technical issue for this is why we could not solve a structure with ultra solute but we know that the protein is happy because you see very high resolution details in the classes
So this is how the structure is looking like right so the pree is very nice itself so we have basically a known antagonist here bound to the to the soluble part plus several glycosilation around the protein so this is of course work in progress that we’re doing right
Now but uh not to get dis encouraged by this so these are all the structures that have been solved by other people not by us in the literature with a with with Native Nano disks right so you you have several cases where you can get even below three anstrom resolutions so
This is very encouraging in many of these cases you can see lipids in the preparation so this is also very encouraging and if you can see the dates this is something a field that is really emerging right now so the oldest and the oldest paper is from 2018 so structors
Keep coming up every year right and then the important thing is that we have proteins Al from eoli from other bacteria and from eukariotic sources so basically you can do this with proteins from any source and then you can in principle get high resolution structure for all of
Them so right the last thing I wanted to to to touch on before before ending the seminar is that maybe some of you thought like okay if this polymers really can incorporate into membranes why do we need to break the cells first and then collect the proteins right can we just mix the
Polymers with the with living cells or with with any cell right and then the memory would get solubilized directly so we ask ourselves the same question and the answer is yes you can do that so basically this is now with the hexels right so this is RI of buffer the the
One that I mentioned before that usually is used for mass spectrometry and this is a buffer that we have called galiz so it’s basically based on our polymers as well this is one minute after mixing with the with the polymers and then you see that already so the experiment was
We mix the our buffer with the cells right then we pellet everything that is INS soluble and then we run a gel of what’s left over right so if there is no breakage of cells then there should be no proteins here or almost no proteins but you see that basically there is
Comparably as many proteins as you have in the ri buffer and if you incubate this for a bit longer half an hour then you get really lots of proteins starting to appear whereas in the control right where there was no no compound there it’s almost completely empty so you can
Really break cells like that and as you can see here so if you count the number of cells that you could see under the microscope right after a minute you already have a significant drop from the control and after 10 minutes you have basically lies more than I would say
2third of the cells with our galiz dissolve uh buffer but of course we didn’t only make one to brake cells we made it all another one called galiz stabilize that mix the cells and also solubilize your membrane proteins all on the flly right so you mix these things
With the cell and then you’re going to get basically your soup of membrane proteins and soluble proteins directly there in a kind of a not native but not near native kind of environment and the advantage of this is that you can break your cells and then
You don’t have to use this very harsh treatment that you would do for example by sonicating them so or putting very strong detergents into it and the advantage of that would be that you if you’re after something that is a very Lile complex for example it would
Not break apart with gentle I as it would break apart with a detergent based uh liis buffer and we see this being applied for example in the case that you have let’s say you don’t want to purify your proteins to do dell screening right but you want to do dell screening in
Lysate so you would take gentle I mix it with your cells the cell would open then you throw your Dells and then you would purify your protein directly right and in a very gentle Environ in a very gentle environment kind of maximizing your chances of getting a view of the
Native fish St of the of the of the cell right so we created these two buffers right and then you can you can get them also right now they are basically the same except that galiz dissolve will not extract your membrane proteins so it’s only going to be for the Sol soluble
Part of the cell but with stabilized you’re also going to get the membrane proteins into into native nanodisks so with that I would like to wrap up and tell you that remind you of what I would like you to take home today right so showed you and hopefully
Convinced you that these are very versatile tools to solubilize and stabilize membrane proteins that they all have their own solubilization fingerprint wi so it’s very important that you screen the the polymers and choose the one that best fits your needs I showed you that they offer improved stability compared to to
Detergents which is a a very welcome characteristic of this uh of these polymers but what’s very important here is that more is not always better so not you are not necessarily going to end up choosing the the polymer that gives you the best solubilization efficiency but
You have to tailor fit them to the downstream application depending on what exactly are the needs of the technique that you’re going to use afterwards and these polymers can also be used to lies eukariotic cells so then we we’re very excited about this new development and I encourage you if you
Have a project that this that needs this to to try it out so with that I would like to thank you for listening to me today and I would just to H acknowledge basically some of our partners right so this protein solubilization my spectrometry work was done in collaboration with cat
With Stefan Müller and John V lakman from second in University of cologne and the Affinity selection work was done with the in collaboration with ELD so you can on the left upper side of the screen is a is a link to the to the preprint about the micrometry work so if
You want to have a look then go there and then please check it out and with that I would like to again thank you for your attention and I welcome your questions yeah that was definitely very nice informative seminar so please put all your questions in the little type box on
The right and also if we don’t have time to answer the question the cube biotch team will later follow up on your questions um maybe I can start with the first question so I mean you said that you um screen like all the different polymers to find the best for the
Specific application uh is there also a possibility to just use a mix of all the different polymers or is that basically yeah that’s a that’s a good question so we we’ve been exploring that I I don’t know to which extent you’re going to be able to separate them
Afterwards so if you have a mixure of different polymers I don’t know if you’re going to end up with something that has always a mixture of the polymers or different uh combinations of native Nano disks embedded into it but that’s that’s definitely something that we are that
We’re trying to find out if whether or not we can mix them to get better better solubilization or things like that yep good and I was also wondering so you talked about um asms um would it also be possible to use this polymer for uh Co imuno purification applications
Where you um basically identify protein lians of of membrane receptors or something like that absolutely absolutely so we We I think we we’ve done that before well it def it’s definitely possible it’s just so one of the main problems I don’t think for a coip would be an issue so some tags Affinity tags don’t work so well with with polymers so for example his tags are not going to work so
Greatly but I’m guessing if you’re doing Co imuno precipitation you’re going to be pulling them out with an antibody and if you do that then yes absolutely and I think it would be significantly better than doing it with the with detergents okay yeah definitely interesting application um yeah here we have a
Question are there any drawbacks of using co-polymer instead of detergents right so the one of the issues with the with the copolymers is that they have all these carboxilate in the backbone so they will interact very badly with the with the with the veent ions right so many of
Them precipitate after you add let’s say more than 5 Millar um magnet for example then you would need to choose there’s several that don’t have that problem by so you would just need to choose one that uh that doesn’t have that problem deep Mas for example are very good for that um
Any other drawbacks is that as I told you so nickel NTA doesn’t work really well with them so you have to choose an appropriate tag we favor our Ro tag because it’s an antibody based purification it works really nicely but other than that I think it just it’s
Meant it’s all about Tailoring your the polymer that you use to the specific needs that you have so some of them absorb at 280 right so if you need a if you need to measure something at that wavelength in your application then you should choose a polymer that doesn’t do
That right and things in that of that sort okay and then another question um would be uh there are many membrane proteins that were either missing or very poorly solubilized in the human membrane prodium experiment is it possible that co-polymers will fail with some of these targets yeah that’s an
Interesting topic so yes we could so in in the database of course there’s going to be many pro many human proteins missing because either they are not expressing in hexels so that’s a that’s that that’s entirely possible or what we saw is that when you compare the presence of a certain
A membrane protein in the in the mass spectrometry data with the number of transcripts that you have for that for that particular protein in in HEX cells there is a direct correlation so we saw that the main issue with identification of proteins in our he hexel experiments
Is that the proteins are just not in high enough copies but for overexpressed proteins like you would do for for a protein purification we have never encountered a single a single examp example where this is not solubilized by by by a copolymer okay yeah please keep on asking question put them in the
Chat um here we have another one what should I consider when choosing the appropriate co-polymer for specific application right so as I was saying for example if you need to do something analytical for for calcium binding for example then you would take one of the polymers that doesn’t bind that or doesn’t interfere
With the Vons or if you need very strongly to measure at the 280 absorbance then you would take something that doesn’t have a styring so it doesn’t interfere with your measurement so but it all depends maybe it’s always good to have a few after you find out which one
Solubilize the best then to have a few that you try against your Technique and then you say okay this is one that’s giving me the best result okay and I was actually wondering is it possible um when you have your um proteins in these Nano discs with your
Polymer can you polymers can you freeze them and for long-term storage and reuse them later we I guess this is dependent on each protein so some proteins are going to survive that better than others we haven’t seen a specific case where you a freeze th cycle with end up in the
Protein being kind of a the nature or something but I guess this this is something that you definitely need to try per project I wouldn’t I wouldn’t do it blindly yeah um and one question here in the chat is would the gentle Li buffer work on other kinds of cells right
So no well yes so it it Works in in me in in in in animal cells it definitely works so we have SE seen it working in tiny cells so insect cells and on Hex cells these both work equally well it does not work for bacterial
Cells so so if you have ANUK eukariotic application then definitely it works if you have a bacterial application we’re working on finding a solution for that but for the moment it does not work for for bacterial cells okay um and one more question that I had for your big proteomic screen where you
Tested the different polymers uh could you see any enrichment for different membrane types for example some polymers are better with your membrane proteins or something like this yeah that’s a that’s that’s a good question um this was one of the things that we wanted to explore so because if there is a
Specific preference for a for for proteins for for the lipidic environment of each protein then you would expect that maybe some polymers are better for for mitochondria for example some are better for for GGI the the problem is that since we were working with the total membrane lysates I don’t know to
Which extent the membrane asymmetries were kept you know because you break the cells and then everything is pulled together and then everything is solubilize in in in nanodisks and then H the nanodisk themselves can exchange lipids so I think I think there should be an effect like that I don’t think in
Our experimental setup it was possible to see that okay um and maybe one more question so were you also planning to do any lipidomic experiments that maybe the polymers capture different lipid types yeah so this this is definitely in the in the pipeline so there’s some other
People that have done that you can you can basically for each of the proteins or for each of the particles that you have you can try to to find out exactly which lipids come with it you can see it in other people’s experiments that then you get different lipid compositions for
Different proteins right but we we haven’t done the experiment yet definitely one of the things that we want to try because it’s important to see exactly that the proteins come lipid with their lipids and and it’s really its native environment okay and then in general for these Nano disks are they pretty uniform
In size or or is there a pretty big spread um so when you soluble when you make Nano discs from just membranes the size of the Nano disk is more or less uniform and it depends on the lipid to polymer ratio so you get different sizes depending on how how
Many lipids per polymer you have with the with the proteins themselves I think to a certain extent the protein dictates the size of the Nano dis that you’re getting I think a few polymers are going to kind of arrange in a way that it gives the size that the protein
Needs okay so the assumption is that usually you capture only one protein within a dis yes yes so within the I mean a one complex I would say so something tightly bound I guess you by chance are going to end up with a with you could end up with
Two proteins in the same Nano disk I think doing purification you’re going to get rid of these things just because uh they don’t have the right size and usually there is a size sizing step in the purification process but that’s I’ve seen that as well in C sometimes in in
MSP based Nano so even though they are supposed to have the a fix size you sometimes have two proteins inserted in the same Nano this because you cannot really control that way but it happens every now and then okay and um do you think we’re you’re done with uh finding new
Um synthetic polymers for these type of applications or is there other type of polymers that still could be discovered we are always incorporating new new polymers into the into the into the portfolio so very recently we made a collaboration to introduce a new type of a smas developed by B kumman for
Example um yeah so uh they keep coming modifications to the backbone or modifications to the to the side chains that make them more or less a hydrophobic right or that they are neutrally charged instead of a instead of a negatively charged right so these ones have proved to be proven to be very
Popular lately so definitely improvements are to come okay um yeah I don’t see any more question for the moment but yeah if you still have a question put it in there and the team will follow up uh with it and I think yeah if if you don’t want to say anything anymore
Filipe yeah so just to thank everybody again for attending and then if you have any questions just drop us an email and then we’re going to do the best to to answer them