the following content is provided under a Creative Commons license your support will help MIT OpenCourseWare continue to offer high quality educational resources for free to make a donation or view additional materials from hundreds of MIT courses visit MIT opencourseware at ocw.mit.edu [Applause] ribosome called Rybak's and experiments that were done to characterize it and determine whether it did a better job at allowing the tRNA to suppress the Amber stop codon and then we'll be transitioning into module 2 so we'll be starting protein folding today and be spending the next couple of days on that area and thinking about some of the macro molecular machines and other proteins involved so just as a recap where we left off last time was thinking about incorporating unnatural amino acids using a new ribosome and so we discussed how an orthogonal ribosome was designed that binds two orthogonal mRNA and then the idea was can this orthogonal ribosome be improved the immuno genesis and selection to get a new orthogonal ribosome wear suppression of the amber stop codon by the tRNA is favored over termination by release factor one so we talked about this issue with release factor one causing truncated protein phenotypes right and so where we left off was in a series of three types of experiments to look at how well this ribose works so we looked at protein yield to ask is it translating polypeptide as well as the starting orthogonal ribosome and then the next experiment we were looking at and where we left off with was experiment 2 which was the question of amino acid miss or incorporation okay and so what was done just in recap is that a GST maltose binding protein and the P fusion protein was designed okay and recall that this protein had a protease cleavage site okay and that the GST portion contained cysteine and MBP does not contain cysteine okay so the idea was to use radio labeled cysteine as a probe for miss incorporation of cysteine and Tamil toast binding protein okay so in terms of the actual experiment what was done right imagine that we express this fusion okay in the presence of the radio label cysteine we use a protease okay and in this case it was thrombin this protease gives us two fragments GST plus MVP okay and these two proteins have different size so we can separate ok so just doing SDS page okay and ask where do the proteins migrate on the gel and then where do we see radioactivity okay and so the data are shown here that were reported by the authors so what are we looking at we're looking at on the left the Coomassie stain for total protein and then on the right we're looking at radioactivity here okay so what do we have here in terms of the lanes they've done some labeling to help us so they'll toast binding protein is running up here GST is running down here and we have four different conditions okay what do we see we have in lane one ryback's is what's used in lane two we have a system where it's the initial orthogonal ribosome that was not evolved right so that's our point of comparison in lane three we have the native ribosome only and in the four lane for a control for no gene so what do we see don't know change translate yes so why do we come to that conclusion they're all doing the same thing right what we see on the left here and the Coomassie stain is that in lanes one through three we see a band for MVP and they're all similar in terms of intensity and likewise for GST we see a band all similar intensity so it looks like in all cases a similar amount of protein is being synthesized and that this protease cleavage worked in a comparable manner the native ribosome is not going to bind to the orthogonal mrna so you need to give a plasmid that has a ribosome binding site compatible with the native ribosome and yes it's possible that native ribosome won't work as well or reverse I say reverse is more likely that one of these mutants won't work as well but that doesn't appear to be the case here so what does the gel on the right show so where do we see radioactivity mostly at GST but do we expect background or make the background comfort right so we see a strong band here right and this is at the same places where we see GST so that's a good indication right we know that there cysteines in GST so we should see a band here so what about background and what about MVP have some of this incorporation identity is very much must compare with just you but a little bit an MDP participation okay is that band MVP dude do we know that definitively in this gel there's many species in these gels right there aren't it isn't just two bands one for GST and one from MVP right so where do these other bands come from right could it be from the initial protein purification and there's some contaminants could it be a result of the thrombin cleavage and maybe thrombin cut at some places other than just this cleavage site so that's preferred but maybe it can cut some other places right so there's other species in the gel and cysteine comes up in other proteins so it makes sense that there'd be some background just say this protein wasn't 99 100 percent pure right or maybe from cleavage there in terms of this little band is that MVP or not a little hard to say where they place the arrow indicates maybe not right but what could you do to find that out yeah so maybe a Western blot if there's an antibody you could run something less concentrated you could do mass spec you know there's options if you wanted to track that down but the bottom line is if we compare the radioactivity here to what's seen here there's much more with GST which is what we would expect and so through some further analysis they conclude the error frequency is less than one in ten to the three for Raible X at least for cysteine right so what's the limitation of this experiment from the standpoint of missing Corporation which is what I asked you to think about in closing last time only testing for sistemas incorporation so we don't really know about other amino acids right so this is good news but there's other information we're not getting from this experiment there so is it possible that other amino acids are Mis incorporated it would take some additional experiments to get at that so the last experiment we're going to look at is asking the question does ribose actually do a better job than the progenitor or a ribosome so what we want to look is at the efficiency of suppression of the amber stop codon right so last time we talked about how one limitation of the Shultz method is that there's a truncated phenotype right because our f1 enters the a state rather than the tRNA so in order to look at this they continued to use this type of fusion but instead of having a protease cleavage site here they stuck the amber stop codon in between GST and MBP okay so they made two different class modes so GST a plasmid encoding GST the stop and Maui so this is the gene name for MVP so if this gets translated the question is do we get the GST MVP fusion or GST right because if the tRNA does its job polypeptide synthesis will continue and we'll get the fusion protein if release factor one ends up here at this position we get termination and only GST and they also made an additional construct so in addition to asking what happens if there is a codon for one natural amino acid what happens if there's - so recall with the Shultz method what we saw last time is that when an attempts were made to incorporate two unnatural amino acids than one polypeptide the efficiency went down to below one percent there so what if two of these are here again the question is these get translated do we get GST MBP or GST okay and as we can see from the jel we can differentiate pieces by size okay so in addition to comparing these what was done is in both sets of experiments there was a comparison of the Schultz method we discussed first and Rybak's here okay and just in terms of size so GST MBP is about 70 kilotons okay and GST is about 20 kilotons 26 so easy to separate on the gel so what are the data and we'll just and what was the unnatural amino acid employed so they ended up using an unnatural amino acid called BPA which is a benzophenone still a cross linker okay and so I'm going to write up some details that came out from the jela and the board because there's a lot of things to navigate in that gel okay okay so effectively what are the comparisons we want to make so in lane three the method we have is the Schultz method there's one codon for incorporating one unnatural amino acid and what they see is they get about 24% efficiency of full length fusion protein in Lane five we have the Schultz method with two here and we get about one percent efficiency in Lane seven analyzing the orthogonal ribosome Rybak's and one and what seen is sixty-four percent efficiency so quite an improvement there and then in Lane nine what we have is ribose with two and an efficiency of twenty-two percent here okay so here we're looking at the wild-type ribosome okay and here we're looking at the orthogonal ribosome orthogonal mRNA okay with the two mutations we saw before so these values come up from quantification of the data here right so you can convince yourself by comparing the band's for GST so resulting from Shadid phenotype translation termination and the bands for the fusion protein GST MVP okay so showing that there was successful suppression of the amber stop codon here right so what are the major conclusions right the major conclusion is that at least with this system what we see is that ribose has minimized this truncated peptide phenotype compared to the wild type ribosome okay and then it's been possible to basically diverge the decoding properties of the orthogonal ribosome from the endogenous cellular machinery so this percent here it that's the percent of the total Express protein let's delete the other you know 99% would have been the GST on the year yeah I mean so here for instance if we take a look at lane let's compare lanes three in lane five right so here's lane three and what do we see so in here we have one incorporation of one natural amino acid and we see that there's a band for the fusion protein and there's a band let me make sure I'm in the right lane I mean three and a band for GST itself right and the intensity of this band is greater than the intensity of that band and you can imagine doing quantitation whereas if we look at Lane five where we're trying to incorporate two by this method we see a band for GST and what do we see up here very little okay if we look at Lane seven we're seeing 64% Lane seven here right we see that we have this band for the GST MVP fusion and a weaker band for GST alone there so percent efficiency percent percent of the total so there's other things happening in this field so the schulz method and these orthogonal ribosomes are two examples and one thing that came up after this work with Rybak's was to design ribosomes that can use quadruplet codons rather than triplets so a lot of creativity and things to look up if you're curious but with that we're gonna close the translation module we will not leave the ribosome it will keep popping up throughout modules two and three but we're gonna move into what happens to a polypeptide as as its leaves the ribosome so how does it get its native fold and so what happens to nascent polypeptides you know emerging from the ribosome and how do polypeptides fold and so there's reading posted for module 2 on stellar and listed here one required paper which is a really wonderful review that came out about two years ago so let's think about folding and as a point to thinking about that let's think about our ribosome okay and there's some emerging polypeptide chain so the nación polypeptide so what happens to this polypeptide and the first thing to keep in mind is something we need to think about is where is this polypeptide destined to go is this a polypeptide that will be in the cytoplasm is this a polypeptide that will become a membrane protein or part of the secretory system and so we can think about cytoplasmic protein ok versus membrane proteins or in eukaryotes the secretory here and so we're going to focus this module in terms of thinking about what's happening in the cytoplasm we might touch upon this if there's time but I think there won't be so the cytoplasmic proteins are folded by chaperones that they can come into contact with as they're emerging from the ribosome or also after the polypeptide is released to extracellular matrix proteins these categories I actually don't know Joanne weird to extracellular matrix proteins will they think of me inside the so mean and they can shut so do you should go talk to man shoulders you look at collagen put in your play yeah okay so these interact with a player called signal recognition particle which allows for targeting to the membrane or endoplasmic reticulum and eukaryotes okay and then folding folding can happen here so we're gonna be focused in the cytoplasm just realize there's other machineries involved for membrane proteins so here's just another view of our ribosome we saw this early on in the ribosome unit and want to think about this exit tunnel and the emerging polypeptide chain okay so it's the 50s subunit and as we discussed before this exit tunnel is long and it's also quite narrow and it's lined by both ribosomal RNA and proteins and I note a few of you asked about the hydrophobic residues of proteins that line this tunnel after lecture two and the thing to keep in mind is that it's not all hydrophobic there's also RNA there there will also be other residues and something just to think about like can water molecules get in there as well so we see for instance there's two proteins L 4 and L 22 that line part of the tunnel we have protein L 23 at the exit but there's also a lot of RNA there so don't don't forget that so a question just to address early on teen folding occur in the exit tunnel and I'd say this has been a bit of a controversial question over the years and there's been camps arguing both possibilities yes or no I think the thing to keep in mind is that the dimensions are limited right and although we can imagine some conformational flexibility and dynamics in this exit tunnel it can't undergo some tremendous change to say accommodate something like ubiquitin that you saw early on right that that doesn't just make sense so is it possible for some alpha helical fold to occur in this exit tunnel presumably there is some work that indicates there's folding zones and the exit tunnel so maybe some folding happens there but really the main conclusion is that most folding occurs outside of the ribosome and after the polypeptide emerges from the 50s here okay so if we're thinking about most folding of polypeptides as occurring in the cytoplasm for cytoplasmic proteins what we need to think about is that environment and we learned in the introductory lectures or had a reminder that the cellular environment is very crowded right so we have this issue of macromolecular crowding and in thinking about that we need to ask the question how does this emerging polypeptide fold to its native form in this type of environment what type of machinery is there to help protect it how is Miss folding avoided and intermolecular interactions that are non-productive avoided so where are we going to go in this module we're going to look at protein folding from both the in vitro test tube perspective and also from in the cell and so in thinking about protein folding in vitro we'll discuss some of the seminal study so Anthon scens hypothesis and folding of ribonuclease a eleven fellas paradox which brings us to thinking about energy landscapes and also touch upon some of the experimental methods that are employed and then in terms of machines we'll think about largely post-translational protein folding in the cytoplasm so grow al grow yes DNA k and j we'll also talk about a protein called trigger factor that associates with the ribosome and those nascent polypeptide chains okay so these machineries fold soluble proteins not membrane proteins and I'd also like to point out and again we may or may not get to these systems depending on time but in addition to these chaperones and macro molecular machines involved in folding there are classical enzymes that are really important and these include enzymes that say isomerize prolene also dial oxidases and isomerases there so what are our questions for this module so why and how our proteins folded and in terms of how in the lab versus in the cell classical enzymes and macro molecular machines what happens when proteins are Mis folded how does protein folding relate to disease what methods are employed to study these phenomenon and for the case studies we'll look at in terms of cytoplasmic players we want to understand really what are the structural properties of these different chaperones and their partners how do their structures relate to function how do they help peptides obtain the native fold and how good is our understanding of these systems will see in the case of DNA KJ it's actually pretty difficult to know what they're actually doing here and really what is the experimental basis for our understanding if we just take an overview of folding and misfolding and this is diagram for a eukaryotic cell and from many many different types of studies what do we see so we see that some sort of bio molecule called chaperon keeps coming up again and again so these are proteins that assist with folding or unfolding disaggregation right there's many possibilities for the trajectory of a protein here right so here we see a nascent polypeptide emerging from the ribosome and imagine that some folding intermediate is released so this is not fully at the native fold but it's somewhere along that pathway what might happen right here what we see is some chaperones allow this intermediate to form a native protein right but look there can also be unfolding and this could work its way back this native protein could unfold to a Miss folded state right we can think about remodeling and maybe there's chaperones involved in taking this miss folded state back to an intermediate that's on a productive path way what happens here maybe there's some trouble and rather than reaching its native fold this intermediate ends up aggregating okay it forms some sort of protein aggregate and maybe that can form oligomers or some sort of amyloid fibril like what we hear about with alzheimer's disease here we see their chaperones that can be involved in having disaggregates activity and they can help in breaking down these aggregates and getting back to to some productive place here okay so there's inherent complexity here and many players and relationships between protein misfolding and disease just to be aware of right so we typically think about the protein fold providing function and protein misfolding can result in improper function and there's many different types of improper function right it could be lots of fun it could be gain-of-function it could be formation of some sort of aggregate that's deleterious to the cell for one reason or another and if we just take a look in terms of human diseases that are associated with protein misfolding what do we see so there's examples out there like Alzheimer's Parkinson's familial ALS and mad cow so Alzheimer's disease is associated with formation of a beta plaques in the brain in Parkinson's there's a peptide called alpha synuclein that aggregates in familial ALS also called Lou Gehrig's disease there are single point mutations in an enzyme called superoxide dismutase that results in Mis folding and some negative consequences there and then Mis folding of the prion protein so a lot of these in terms of neurological disorders so in addition to fundamental studies there's significant interest in understanding protein misfolding from the standpoint of disease and prevention and I'll just note sometimes questions about natively unfolded proteins come up and those are outside of the scope of our discussions today but be aware there are proteins that are natively unfolded you saw a little bit about of that with some of the ribosomal proteins right that had those unfolded extensions going into the interior so in terms of thinking about protein folding in the test tube where we're going to begin is with anthem fins hypothesis and his seminal experiment on protein folding so anthen sin is responsible for the thermodynamic hypothesis of protein folding and he performed seminal experiments on a protein an enzyme called ribonuclease a and so what Amundsen hypothesized is that in terms of a protein shape or fold it's the primary sequence so the sequence of amino acids that dictates this final shape in aqueous solution so whatever that primary sequence is it dictates basically the array of possibilities and the thermodynamically most favorable result so what was the experiment and FinCEN did to probe this here what he did is look at denaturation and refolding of ribonuclease a okay so this enzyme Queens RNA single-stranded it's a hundred and twenty four amino acids in length and in the native form it contains four disulfide bonds here okay and since there's four disulfide bonds there's eight fifteens in the primary sequence right so two cysteine side chains can come together to form a disulfide okay and so if we think about a cysteines forming for disulfide bonds there's many possibilities in terms of how those cysteines are matched in the linkages so different regio isomers right over a hundred possible combinations of these eight cysteine to give four disulfides okay and only one regio isomer so one of these combinations is the native form here so one out of over a hundred so these native disulfide linkages that are formed endogenous li are required for activity so what was anthem sins experiment the experiment he did was to take native ribonuclease a and I'm gonna just sketch that so imagine we have the four disulfides okay here so first step one he reduced it so he added a reducing agent to reduce these disulfides we'll talk a little bit about more what that might be in a minute and so the end result is rather than having these disulfides we have eight free cysteines so free meaning not in a disulfide indicated by sh okay so this is reduced okay and so over the course of this Anthon s'en developed some assays to monitor for activity of this enzyme and what was found is that there is a loss of activity next step okay how did in nature in okay so at nature in is some chemical like urea or guanidinium that is going to disrupt the fold of the protein okay and in this case he used urea right so as this is sketched this is still folded but the dye sulfites are gone okay so what's the result here we get some unfolded polypeptide with the cysteine somewhere so this is denatured so we have no disulfides no native fold okay and inactive it can't cleave the single-stranded RNA okay so we've succeeded in destroying activity and destroying the fold of this protein what did he do next so the next experiment steps in this experiment was to ask okay if we start with this unfolded polypeptide that's completely denatured and there's no disulfides can it return to this native form by removal of the removal of the denature int and then allowing it to oxidize so imagine here we work backwards and step 3 you know remove remove the de nature and so how that might be done dialysis is a way to die Eliza way the de nature in and then what happens if we allow this to oxidize so for instance air oxidation here so what he found is that in this order of steps so that nature and sri moved and then the protein is allowed to oxidize that greater than 90% of the enzymatic activity was restored so you have this you know denatured polypeptide and dilute aqueous solution and work your way back and you can restore this activity question is still the same or similar and results in terms activity for that very yeah so how do we how could we confirm if the fold is perturbed what might be a method to do that circular dichroism so that's one possibility did he have that available that's another question that right that will give you a readout on Alpha helix II or beta sheet and that's one one possibility right you can imagine other possibilities maybe it would run differently on you know some form of column there as a possibility there was a loss of activity here yeah was it a hundred percent or less than a hundred percent I'm not sure about that detail Joanne jus you should before they deuce it's down there wait protein with a lot of disulfides in it they may not be accessible to read Upton mm-hmm I mean often it's done you add them together to get here right and he did a lot of experiments as well with additives so and then definitely in this direction my understanding is he performed this both ways so effectively if the denature it's removed first and then it oxidizes versus oxidizing it and then removing them in nature it and when it was that later scenario that the dye sulfites are allowed to form first the end result was a sample that had negligible activity less than 1% right and so from that you can imagine why because if this was allowed to oxidize right it's not pre-folded to allow the correct disulfides to form here entirely on its own yes oh isn't that incredible for this case I don't know and depending on the polypeptide it can vary you know from a short period of time we have examples in my lab where maybe in 30 minutes you can see the properly folded form today's or even faster than that depending like seconds two days depending on on the protein and the size yeah but that's what's really incredible about this experiment just beyond the details of the ordering and what happened is the fact that he could take this 124 residue polypeptide that needs to have four specific disulfides and just in dilute aqueous solution without any help you know - they're here it could come to its native fold right so this was support of this hypothesis that it's you know the primary sequence of a polypeptide that can dictate shape and if these polypeptides are allowed to fold under dilute conditions where intermolecular interactions aren't a problem they can achieve the thermodynamically most favorable result and he did plenty of additional experiments - in terms of putting additives in and asking how do these perturb the results here so what did he actually have to say from his experiment in his words the results suggest that the most that the native molecule is the most stable configuration thermodynamically speaking and the major force and the correct pairing of South hydral groups and disulfide linkages is the concerted interaction of sidechain functional groups distributed along the primary sequence so this primary sequence dictates the array of possibilities so in thinking about that that brings us to the paradox of the menthol here so he was thinking about this problem of protein folding and just thought well imagine we have one polypeptide with hundred amino acids so smaller than ribonuclease a what if each amino acid had only two possible confirmations what does that mean in terms of possibilities we have two to the 100th here so if that polypeptide were to sample every possible conformation during folding taking just a picosecond per transition the time required to fold the protein would be what and based on his back-of-the-envelope work here it would be ridiculous larger than the time of the universe right and that tells us that just can't be in terms of how we think about this here right so each amino acid can't adopt its shape independently that's just not working on a biological time scale so how do we think about this we can use energy and landscapes here so thinking about tumbling through hoes and valleys and so basically we can depict protein folding and this is an example say in a test tube where there's some ensemble of starting unfolded or partially folded structures and these are of higher energy and there'll be some sort of stochastic search and basically these forms will give us an ensemble of partially folded structures and ultimately converge to a native structure here that's of lower energy here for that okay so we have an ensemble of many denatured proteins that needs to make its way to the native form and just looking ahead a bit to our discussions of chaperones these proteins that assist in folding this is another view of an energy landscape but it's taking chaperones into account so we have energy here and this depiction is from the assigned reading it basically divides things up in terms of like productive intramolecular contacts versus intermolecular contacts lead to situations like oligomers and aggregates and fibrils so up here at high energy we have unfolded or partially folded species and what we see here bless you is that these chaperones are helping to allow these partially folded States to reach a native state by helping getting over these these barriers and the chaperones do not want to have the proteins going in this direction here two species that are potentially deleterious and result from intermolecular contact so oligomers fibrils and aggregates here what are some methods in terms of experimental methods for folding there are many that can be employed so you just need to take studies by a case-by-case basis commonly used fluorescence whether that be native emission from a protein so if you imagine you have say a tryptophan emission can vary depending on where it is in a protein methods like fret we just heard about circular dichroism which tells us about secondary structure NMR FTIR stop flow and there's a large field and computation in theory looking at protein folding as well what are some methods to denature a protein so here we saw urea used there's many others whether that be heat or pH and denatured protein means unfolded protein in the context of the lectures in this course so often studies in vitro start from using an unfolded protein sample and then you look at how folding progresses what are some lessons from in-vitro folding studies just to keep in mind one every proteins different - and even proteins that seem similar are very different you know so maybe they have the same secondary or tertiary structure some small peptide but when you try to fold them they may require different there are multi-dimensional energy landscapes like what we saw on the prior slides you can often see intermediates along the folding process and in dilute aqueous solution as anfinsen hypothesize primary sequence dictates fold just to note to anyone doing experimental work why do we like to use the ice bucket in the cold room when working with proteins and enzymes many native proteins are only marginally stable under physiological conditions so we can think about a delta g of denaturation per amino acid right so what this means is use your ice bucket and the cold room when working with your samples just closing thinking about protein folding in vitro verses and vivo do studies and vitro really enhance our understanding of what's happening in the cell just some observations to keep in mind so on the bench top folding can occur over a tremendous time skill from nanoseconds to hours I have here it can can be days depending on your peptide and conditions here the studies are generally performed in dilute buffer and in the absence of any additional protein so you have some pure polypeptide that you want to fold or study and that's what you work with and it's found that small proteins will often fold without assistance here so they don't need helpers in the cell how do we think about the rate of folding so from one point of view the rate of folding is limited by the rate of polypeptide biosynthesis and how quickly that polypeptide is emerging from the ribosome right if you're thinking about a nascent chain and we can think about the concentration of peptide coming off the ribosome which is often quoted as low micromolar and as I mentioned earlier we really need to keep in mind that this cellular environment is very crowded with many different biomolecules and player okay and as a consequence of this crowding there's many proteins that help in folding especially the chaperones we'll see that a number of these chaperones protect the polypeptide that needs to be folded from this environment here so a take-home is that just spontaneous protein folding in the cell is error-prone if it were to happen and that inter and intra molecular interactions are a big issue and so these chaperones are available to help overcome these issues here so where we'll begin on Monday is looking at trigger factors grow L grow yes and DNA KJ as an overview and then we'll work our way through these different systems for protein folding in the cytoplasm there you