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  • ELIZABETH NOLAN: So where we're going to begin today

  • is continuing with our discussions

  • of the substrates for groEL, groES, and analysis

  • of the data.

  • And after that we'll talk about the DNAK DNAJ chaperone system

  • here.

  • So recall last time we left off with the question

  • of the groEL groES substrate.

  • So inside an E coli cell, what are the polypeptides

  • that are folded by this macromolecular machine?

  • And so there was the pulse chase experiment,

  • there was immuno precipitation, and then analysis.

  • And so in this analysis, we talked

  • about doing two dimensional gel electrophoresis, and then

  • trypsin digest and mass spec of the various spots.

  • So where we left off were with these data

  • here and the question, how many polypeptide substrates

  • interact with groEL in vivo, so inside an E coli cell?

  • And what we're looking at are the various gels

  • for either total soluble cytoplasmic proteins

  • on top at either 0 minutes--

  • so at the start of the pulse--

  • recall that these cells were treated with radio

  • labeled methionine, and then there

  • was a chase for a period of time when

  • excess unlabeled methionine was added.

  • So here we're looking at total soluble cytoplasm proteins

  • 10 minutes into the chase.

  • And then at the bottom, what we're

  • looking at are the polypeptides that were immunoprecipitated

  • by treatment of this cell lisate say

  • with the anti groEL antibody.

  • So the idea is this antibody will bind to groEL,

  • and if polypeptides are bound those

  • will be pulled down as well.

  • So it's kind of incredible this experiment worked.

  • There was a bunch of questions after class

  • in terms of the details of this immunoprecipitation just

  • to think about is it a groEL monomer,

  • or is it a groEL heptamer?

  • How tightly are these polypeptides bound?

  • How do they stay bound during the course of the workup?

  • Where's groES?

  • These are a number of questions to think about

  • and to look at the experimental to see about answers.

  • So where we're going to focus is right now

  • looking at these gels.

  • And so what we need to ask is, what do we learn just

  • from qualitative inspection of these data?

  • So on these along the y-axis we have molecular weight,

  • and along the x-axis the PI.

  • So if we first take a look at the total soluble cytoplasmic

  • proteins at zero minutes and 10 minutes, what do we see?

  • Do we see many spots or a few spots?

  • Many spots, right?

  • And we see many spots both at 0 minutes and at 10 minutes.

  • So the E coli genome encodes over 4,000 proteins--

  • roughly 4,300.

  • And if one were to go and count all of these spots, how many do

  • we see?

  • It's on the order of 2,500.

  • So they detected on the order of 2,500

  • different cytoplasmic proteins on these gels.

  • What do we see in terms of distribution

  • by molecular weight?

  • Is it a broad distribution, or narrow distribution?

  • Broad, we're seeing spots of all different molecular weights,

  • so from low to high on this gel.

  • What about PI?

  • AUDIENCE: It's also broad.

  • ELIZABETH NOLAN: We also have a broad distribution

  • in these gels, right?

  • So we see polypeptides of low through high PI

  • on this scale from 4 to 7.

  • So now what we want to do is look at the gels obtained

  • for the samples from the immunoprecipitation and ask

  • what do we see, and is that the same or different from what

  • we see for the total cytoplasmic proteins up here?

  • So if we look at the data here which are the polypeptides that

  • were obtained from immunoprecipitation at 0

  • minutes, what do we see?

  • So do we see a few spots, a lot of spots?

  • AUDIENCE: It's still a lot, and it's still distributed

  • over a pretty wide range.

  • ELIZABETH NOLAN: OK, so let's start with the first point

  • Kenny made, which is that we have a lot of spots,

  • and I'd argue that's true.

  • In this gel, we see many spots where each spot indicates

  • a distinct polypeptide.

  • Do we see the same or less than here

  • for the total cytoplasmic protein?

  • AUDIENCE: It's less.

  • ELIZABETH NOLAN: We see less, right?

  • AUDIENCE: And they seem more concentrated.

  • ELIZABETH NOLAN: Yeah, just wait a second.

  • Right, so we see less, and that's a good sign

  • because an antibody was used to pull down

  • some fraction of this pool.

  • So about how many are here?

  • They found about 250 to 300 polypeptides there,

  • so about 10% of these cytoplasmic proteins

  • were found to be interacting here.

  • So on the basis of the experiment,

  • we can conclude these are polypeptides

  • that interact with groEL here.

  • OK so now Kenny has a few additional observations

  • in this gel.

  • What are those?

  • So how are these polypeptides distributed?

  • And we'll just focus on C for the moment.

  • So in terms of molecular weight, what do we see?

  • AUDIENCE: It's all scattered pretty wide range

  • of molecular weights.

  • ELIZABETH NOLAN: And so we have a wide range,

  • and where is that range and how does

  • that range compare to here?

  • So I agree, but look at the subtleties.

  • AUDIENCE: Most of them are above 8 kilodaltons?

  • ELIZABETH NOLAN: Yeah, so let's roughly say in the range of 20.

  • So if we look at the bottom part of the gel versus the top part

  • of the gel here, and we compare that

  • to the bottom part of the gel here

  • and the top part of the gel here,

  • we see some differences that aren't just

  • the total number of spots.

  • Rebecca?

  • AUDIENCE: So it's like the ones that are smaller--

  • so the spots that respond to the smaller proteins,

  • they seem to be more highly charged.

  • ELIZABETH NOLAN: More highly charged.

  • Yeah, so let's first stick to the size.

  • So we're seeing that in the bottom region of this gel where

  • we have lower molecular weight species,

  • we see fewer of these here than here.

  • So why might that be if there's less

  • polypeptides with molecular weight

  • smaller than 20 kilodaltons?

  • Steve?

  • AUDIENCE: If you just consider the total number

  • of possible confirmations of protein

  • can adopt or peptide to adopt as a exponential function

  • of its size, larger proteins are more

  • likely to have more non-productive

  • folding pathways.

  • So it's just less likely to have something that needs

  • a chaperone at a smaller size.

  • ELIZABETH NOLAN: Right, so maybe these smaller polypeptides,

  • they need less help.

  • Their domain structure is more simple.

  • For instance, they're easier to fold,

  • and other machinery can take care of that here.

  • And then if we look at PI, what do we see?

  • So how is the distribution in terms of PI?

  • AUDIENCE: Large molecular weight proteins are pretty evenly

  • distributed, but the smaller ones have more of a charge.

  • ELIZABETH NOLAN: Yeah.

  • How do you use the word charged?

  • AUDIENCE: Sorry, I was looking at the scale.

  • They actually have a PI closer to 7.

  • ELIZABETH NOLAN: Yeah, just like you heard in recitations 2

  • and 3, pay attention to the scale and what kind of charge--

  • if you're talking about charge, you

  • have negatively charged and positively charged amino acids.

  • So where in that regime are you?

  • But if we look at these areas here,

  • we see a wide distribution.

  • And maybe when they're smaller we're

  • seeing some more over here, but then ask yourself,

  • is 22 an outlier there?

  • So what can be done in terms of these data?

  • This is actually an analysis of the gels looking

  • at total proteins and groEL bound proteins

  • for the total percentage in terms of PI

  • and in terms of molecular weight.

  • And so you can compare.

  • And so what we see is that overall, and look a bit closer,

  • that PI distributions are quite similar.

  • Molecular weight we see some differences.

  • We also don't see that many proteins

  • that are greater than 90 kilodaltons being

  • folded by this machine.

  • And then again, why might that be?

  • We learn that the chamber can accommodate polypeptides up

  • to about 60 kilodaltons, so maybe they're

  • just too big here.

  • So what are the identities of these proteins here?

  • So this is where the trypsin digest and mass

  • spec comes into play.

  • So you can imagine extracting the spots,

  • digesting them with the protease trypsin,

  • and then doing mass spec analysis to find out

  • the identities and comparing that data to databases

  • of E coli proteins.

  • And so from that, of the 250 to 300 proteins that they

  • identified in these immuno precipitation gels,

  • they were able to identify 52 without a doubt.

  • And what are some of those 52 proteins?

  • So I've just highlighted a few examples.

  • What do we see?

  • So here's our friend DFTU as one example.

  • We see subunit of RNA polymerase, ferritin,

  • and certain rhibosomal proteins.

  • So just thinking about these proteins and their role

  • in translation, in RNA polarization,

  • ferritin is an iron storage protein.

  • What do we think?

  • What are our thoughts about these proteins?

  • They're pretty important, right?

  • Imagine if EFTU you couldn't adopt its native confirmation.

  • There might be some major problems.

  • And recall when I introduced groEL,

  • groES, we learned that they fall into the category

  • of chaperonin, so they're essential for life.

  • So that makes sense in terms of seeing some of these proteins

  • as being very important.

  • And what about structural motifs?

  • It's then we see, OK, these are the 50 proteins we identified,

  • what are their structural features,

  • and what does that tell us about this chaperone?

  • The conclusion is that overall, the proteins

  • identified have quite complex structural features.

  • So these can range from complex domain organization

  • to beta sheets, including those that are buried

  • and have large hydrophobic surfaces here.

  • And so we can speculate that maybe some

  • of these hydrophobic surfaces interact

  • with the groEL-applicable domain to have these polypeptides

  • enter into the chamber.

  • Here, was there a question?

  • AUDIENCE: Well, I was going to ask, I don't know for ferritin,

  • but I know that you need a lot of ferritin molecules

  • to form the thing.

  • But all of those are also--

  • and again it's only 4 out of 52, but they're

  • all proteins that exist in relatively high abundances.

  • So could you also be making the argument

  • that proteins that are more likely to have

  • high concentrations, and therefore

  • a higher probability of aggregating

  • just because it's a prime molecular reaction

  • could favor binding to groEL?

  • ELIZABETH NOLAN: Yeah, I even thought about it

  • in terms of they certainly are abundant.

  • It could be, I just don't know.

  • AUDIENCE: The experimental setup also biased it

  • towards more abundant proteins.

  • ELIZABETH NOLAN: Yeah, so could that

  • have happened in the experimental setup?

  • It's a possibility.

  • So we learned that what EFT was about 10%

  • of all rhibosomal proteins.

  • So that's something also to keep in mind,

  • and a good thought there.

  • So what else can we learn?

  • One more observation from these experiments

  • before we move on to DNA K J. So recall last time

  • when we talked about the actual pulse chase experiment,

  • they took samples at multiple time points.

  • And so why did they do that?

  • You can imagine doing this analysis

  • not just at 0 minutes and 10 minutes,

  • but at a variety of time points and ask,

  • if we compare gel to gel and we compare spot to spot--

  • so going back, these spots are labeled many of them in here--

  • we can ask the question, how does the intensity

  • of that spot change over time?

  • And what does that tell us about the interactions

  • of that polypeptide with groEL?

  • So just for example, here.

  • Example, just imagine at time equals

  • 0 we see some protein or polypeptide x.

  • So then what happens at, say, time equals 2 minutes?

  • If we do not see it, let's consider two options.

  • Do not see x, maybe we conclude that x dissociates quickly

  • or folds relatively easily.

  • Imagine if we do see x after 2 minutes here,

  • maybe the conclusion is x is not yet folded here.

  • And then we can imagine doing this at different time points,

  • and they went out to 10 minutes here.

  • So maybe if we see x at 10 minutes,

  • the conclusion is x is difficult to fold.

  • And too, we want to think about these time points

  • also from the standpoint of what we

  • saw in terms of the residency time of a polypeptide

  • in the groEL chamber.

  • So we saw from the various models

  • that that's somewhere on the order of 6 to 10 seconds.

  • So there can be multiple binding and release events that occur.

  • So in this paper, what the authors did is trace the spots

  • and compare the intensities of the spots over time.

  • And you can do a little exercise from these gels looking

  • at spots they circled and just ask qualitatively,

  • what's happening to the spot?

  • Is the intensity staying the same?

  • Is it being reduced?

  • So for instance, it's easy to look at spot number

  • 22 here at 0 minutes versus spot 22 at 10 minutes.

  • And what do we see?

  • Does it look the same, more intense, less intense?

  • Less intense, right?

  • What about spot number 12 at 0 minutes versus 10 minutes?

  • They look quite similar by eye.

  • So you can imagine doing this type of exercise

  • through each gel and actually doing it quantitatively

  • using some instrumentation.

  • So what do they see?

  • Effectively in this, they divided the data

  • into three groups based on certain trends.

  • And that's shown here where what we're looking at

  • is the relative intensity change versus time.

  • So you can imagine at some time point

  • that spot has a maximum intensity that they've

  • put at 100.

  • So we see the three groups here.

  • And the question is if we look at these as groups,

  • what do the data show?

  • So in group one, we see examples where the spot at time equals 0

  • is at a maximum, and then the intensity of that,

  • so spots decrease over time.

  • And the other thing we see is that at some time that

  • isn't very long, the intensities go to approximately 0.

  • So we're not seeing these polypeptides bound any longer.

  • And then effectively what we want

  • to ask is do these polypeptides have any similar features?

  • And what the authors observed is that the polypeptides falling

  • into this group showing this behavior

  • are smaller than 60 kilodaltons.

  • And as shown here, they're seeing them completely released

  • over the time course of this experiment,

  • and in general within the first 2 minutes.

  • So what does that correspond to?

  • How they interpreted this was that these polypeptides are

  • either binding groEL once or have several rounds of binding

  • and ultimately reached their folded state

  • in this relatively short time period.

  • So how does group two differ?

  • Looking at these data, what do we

  • see in group two that's different from group one?

  • [INAUDIBLE] Yeah, we're seeing the relative intensity

  • never go all the way to 0.

  • So here we've gone to 0, here we see 20% to 30% as the cutoff.

  • So how are these data interpreted in this work,

  • and what are the identities of these polypeptides?

  • So similar to group one, these polypeptides

  • are also all smaller than 60 kilodaltons.

  • And how this behavior is interpreted

  • is that even after 10 minutes, there's

  • some fraction of these polypeptides that are still

  • associated with groEL.

  • So they haven't reached their native fold

  • and are remaining bound.

  • What's going on in this group here, group three?

  • This behavior is very different.

  • AUDIENCE: You see peak intensity is a little bit later

  • than the rest of them, and they also

  • don't go to 0 after 10 minutes.

  • ELIZABETH NOLAN: Yes.

  • So these proteins are interacting with groEL

  • because they were pulled down, but it

  • looks like they're interacting at later time points.

  • So we see this growth in terms of increase

  • in intensity over time, and then they go down.

  • And here we see 40% or higher.

  • So they are not readily dissociating,

  • binding at longer time points.

  • So one question here is are these dead end species?

  • And within this work, the authors

  • did some additional controls which

  • there's some detail in the notes I'll post in lecture.

  • But effectively asking, what happens

  • if we add in groES, what happens if we add in ATP?

  • Do we still see these species or not?

  • And some of them were released under those conditions there.

  • So in summary, what we see from this

  • is a method to look at chaperone substrate selection

  • in the context of a cell.

  • We see that groEL folds proteins over a range of sizes,

  • but not really the small ones.

  • So under 20 kilodaltons not so much,

  • and over 60 kilodaltons not so much here,

  • and that these polypeptide substrates

  • have complex native folds.

  • So where we're going to close the chaperone unit

  • is with looking at the machinery DNA K J.

  • And so we'll introduce that system

  • and then look at a similar series of experiments

  • where the substrate scope for this chaperone system

  • was evaluated.

  • So if we go back to the overview from the start

  • where all of these players were introduced,

  • this is where we are now.

  • So we're looking at DNA K and its co-chaperone DNA J.

  • So these are downstream of trigger factor.

  • What do we have for DNA K and J?

  • So these are heat shock proteins.

  • DNA K is an HSP 70.

  • So 70 kilodaltons, and HSP 70s are ubiquitous.

  • So just to note, they're involved

  • in a variety of protein quality control functions.

  • So we have folding, as we'll talk

  • about in the context of today's lecture in this module,

  • but even rolls that range from protein transport to assisting

  • with protein degradation occur.

  • So here we have HSP 70 for DNA K and HSP 40 for DNA J.

  • So in this system, DNA K is the chaperone

  • and DNA J is the co-chaperone, and DNA K is ATP dependent.

  • So it's monomeric.

  • So with this system we don't have a chamber

  • like we have with groEL, groES, and it's ATP dependent.

  • DNA J is the co-chaperone here.

  • So what happens in terms of this system?

  • So effectively DNA J, the co-chaperone,

  • scans hydrophobic surfaces of proteins or polypeptides,

  • and it associates with them so it binds.

  • And then what DNA J does is it delivers

  • non-native polypeptides to DNA K.

  • And then how we think about DNA K

  • is that DNA K binds and releases unfolded polypeptides.

  • And this is another case where there

  • can be multiple cycles of binding and release.

  • So DNA K will bind to a polypeptide that

  • has an unfolded region, there'll be some period of time

  • that that complex exists, and then DNA K will release it.

  • And so in terms of where it likes to bind,

  • these are typically six to nine amino acid

  • segments that are hydrophobic.

  • So it likes residues like leucine and isoleucine.

  • And statistically, this type of region

  • occurs about every 40 amino acids.

  • And for these segments, just to note

  • that there's a range of binding affinities.

  • You can imagine there's a variety of possibilities here.

  • And what's found from studies is that the KD

  • of DNA K for various polypeptides

  • can range from about 5 nanomolar to about 5 micromolar,

  • so by several orders of magnitude.

  • In terms of size of polypeptide, it

  • stated that DNA K has some preference for polypeptides

  • on the order of 20 to 30 kilodaltons,

  • but it can bind larger ones and it

  • can bind polypeptides greater than 60 kilodaltons,

  • as we'll see later.

  • So in this system there's another player

  • that we need to think about, and that's this GrpE, or grip E.

  • And what we have here is a nucleotide exchange factor.

  • So any f, and it's also a thermal sensor.

  • And what GrpE does is that it regulates

  • DNA K binding to a substrate by inducing ADP release.

  • So what we'll see is that the ATP and ADP bound

  • forms of DNA K have different affinities

  • for these polypeptide substrates.

  • So what we're going to do is look

  • at the structures of the components of this system

  • and then look at the cycle.

  • And so if we consider DNA K, so we think of this protein

  • as having two different domains.

  • So there's an N terminal domain and a C terminal domain.

  • And in this end terminal domain what

  • we have is the nucleotide binding domain, NBD.

  • So this is where ATPase activity occurs, and this

  • is about 44 kilodaltons here.

  • There's a linker region, and then the C terminal,

  • and we have the peptide binding or substrate binding domain.

  • This is 27 kilodaltons.

  • So here if we think about this part

  • just in cartoon form, what's observed

  • is that there's a cleft for binding ATP or ADP.

  • So ATP or ADP binds here, and this

  • is also where the nucleotide exchange factor

  • GrpE will interact, because that's

  • its job as a nucleotide exchange factor is to help with that.

  • So basically we have GrpE here.

  • What we see in this domain, it's often

  • described as being a beta sandwich

  • plus an alpha helical latch.

  • And the idea is that this latch closes in the presence

  • of the polypeptide.

  • So effectively, if we look at this as a cartoon--

  • and we'll look at actual structures in a minute--

  • this peptide binding domain can either be in an open form,

  • and this is the latch.

  • You have the alpha helical part, here's the beta sandwich.

  • And if there's some polypeptide to bind, what happens

  • is that the latch closes and the polypeptide is bound here.

  • So this is the closed form, and this pocket is hydrophobic.

  • And that makes sense based on what

  • we know about DNA K liking to bind hydrophobic stretches.

  • So let's look at some structures of DNA K. I present

  • two slides of structures here, one from the assigned review

  • and this other version.

  • And I'll just focus on this one for here.

  • So here we're looking at the domain organization.

  • What we have here is the nucleotide binding domain.

  • So here's that clef for ATP binding.

  • Here we're looking at the peptide binding domain.

  • So the beta sandwich region is in green,

  • the alpha helical latch is in yellow,

  • and we see that there's a model polypeptide here,

  • and this is in the closed form.

  • Here's another view of DNA K with a peptide bound.

  • So we see the beta sandwich, here's the alpha helical latch.

  • This depiction here from the review

  • is showing the closed and open states, and closed and open

  • is referring to the green area here.

  • So don't get confused with the nucleotide binding domain

  • and how these are shown.

  • So what we see here, again, there's

  • a bound polypeptide in this peptide binding domain,

  • and here there's no bound polypeptide.

  • And we see that now this alpha helical region

  • is sticking up there.

  • So what about DNA J?

  • You consider DNA J, we're just going

  • to focus on the domain organization and just

  • a more simplified view than what's on the slide.

  • We have two domains for DNA K binding,

  • and then for peptide binding.

  • So DNA K is going to go out there and find some polypeptide

  • that needs the help of DNA J. It's going to bind

  • that polypeptide and deliver it to DNA K. So effectively,

  • it interacts both with the polypeptide substrate

  • and it also acts with DNA K when delivering this polypeptide.

  • So just to point out DNA J is part of an HSP 40 family,

  • and these are quite diverse.

  • I just illustrate that from the range of different sizes,

  • so from about 100 to about 2,000 amino acids.

  • And all of these HSP 40s have what's called a J domain,

  • and in this more detailed depiction here

  • it's indicated these 70 amino acids at the N terminus

  • are the J domain, and they're important for interacting

  • with DNA K or another HSP 70.

  • So what about GrpE?

  • This nucleotide exchange factor, so GrpE is a homodimer.

  • And if we just look at one monomer,

  • and then I'll show you the structure.

  • So in '97 a crystal structure of GrpE with DNA K nucleotide

  • binding domain was published, and this

  • is what came from that.

  • So just use your imagination, maybe I'll draw this a little

  • differently.

  • Basically what we see with GrpE is that there's a beta sheet,

  • and this is the C terminal region.

  • And then what we see here is an extended alpha helix.

  • And this is the end terminal region.

  • And this is just a cartoon of the monomer.

  • So what happens is that the GrpE homodimer uses

  • one of the beta sheets of one monomer

  • to insert into that ATP binding clef here of DNA K.

  • And when that happens, it forces it open there.

  • So let's look at the structure, and this

  • is something that actually puzzled me for quite some time,

  • but there's been a recent update.

  • So this is a crystal structure of GrpE homodimer.

  • So we see one monomer in blue and one monitoring green bound

  • to an end terminal nucleotide binding

  • domain of DNA K, which is shown in pink.

  • And so we see the beta sheet region of each monomer,

  • we see the extended alpha helix.

  • The C terminal end is here, the N terminal end is here.

  • And I note that not shown in this structure

  • is there is an unfolded region after the end here of GrpE.

  • And so we see this nucleotide binding domain interacting

  • with one of the beta sheets.

  • So a one to one stoichiometry.

  • So the idea, as we'll see when we go forth with the cycle,

  • is that GrpE is inserting the C terminal beta

  • sheet into the nucleotide binding class of DNA K.

  • And this happens for the ADP bound form,

  • and it facilitates ADP release.

  • So what's going on down here?

  • Why is there this extended alpha helix?

  • And I'll just note there is a study just in the past year

  • where interactions between DNA K and GrpE

  • were studied in some more detail.

  • So they used some biochemical experiments, some cryo electron

  • microscopy.

  • And what they learned is that the interactions between GrpE

  • and DNA K are more complex than what's seen here,

  • and what they observe in their cryo

  • EM is evidence for this N terminal region interacting

  • with the substrate or polypeptide binding domain

  • of DNA K.

  • So there's some dynamics and flexibility

  • that we can't appreciate from this crystal structure.

  • And so that begs into question, how else is GrpE facilitating

  • this cycle and modulating confirmation

  • and function of DNA K?

  • So you're not responsible for these details,

  • but if it's something you're curious about

  • I've included the reference.

  • So effectively GrpE accelerates the release of ADP,

  • and that in turn promotes binding of ATP.

  • So what is the functional cycle?

  • And we'll look at this depiction here.

  • There's another depiction in the notes from the reading.

  • This is the current model.

  • And in this model, we're going to start here.

  • So what do we see?

  • We have DNA K in the ATP bound form.

  • So we have the two domains--

  • the nucleotide binding domain, and here the polypeptide

  • substrate binding domain.

  • And in this cartoon, we see that alpha helical latch is open,

  • so no polypeptides bound.

  • And what we also see is that the ATPase activity here

  • is very, very low.

  • So DNA K is not hydrolyzing its ATP.

  • So then what happens?

  • DNA K-- sorry, DNA J, the co-chaperone,

  • has found some polypeptide substrate--

  • indicated by this S--

  • that needs the help of DNA K. So J

  • binds the polypeptide substrate, and it

  • delivers that polypeptide to DNA K.

  • So what does this cartoon tell us?

  • It tells us that J is interacting with K,

  • and here we see the polypeptide substrate being delivered.

  • So when DNA K is in the ATP bound form,

  • it binds peptides with relatively low affinity

  • and in a reversible manner.

  • So there's fast exchange, that polypeptide's

  • going to come on and off.

  • And when DNA J binds and delivers the polypeptide,

  • it activates the ATPase activity of DNA K.

  • So that's indicated here.

  • So the ATPase activity is enhanced substantially

  • so you can compare the values for some quantitative insight.

  • There's ATP hydrolysis.

  • ATP hydrolysis results in release of DNA J and PI.

  • So now what do we have?

  • ATP's hydrolyzed, and now we have ADP

  • bound in the nucleotide binding domain.

  • And what do we see?

  • The latch has closed--

  • open, closed.

  • So like what we saw in the structures

  • with those model polypeptides mound,

  • we have the substrate clamped in this latch.

  • So here we have a form of DNA K that

  • binds the polypeptide with high affinity and slow exchange.

  • So this state is considered to be long lived,

  • on the order of 10 to 15 seconds.

  • So the question is, if this is binding the polypeptide

  • with high affinity and slow exchange, how do we release it?

  • And that's where this nucleotide exchange factor

  • GrpE comes into play.

  • So here comes along GrpE.

  • GrpE binds.

  • GrpE binding results in release of ADP

  • from the nucleotide binding domain.

  • So GrpE is inserting its beta sheet into that clef,

  • and it looks like something else is

  • happening with that long alpha helix to facilitate this.

  • But this was drawn before that 2015 study,

  • so we just see it interacting here.

  • But imagine that this region here

  • is maybe interacting down here and doing something

  • to facilitate peptide release.

  • So now what?

  • No nucleotides bound according to this model.

  • Since the ADP is released, ATP binding

  • is facilitated so ATP can bind.

  • And what do we see?

  • There's release of the peptide, release of GrpE,

  • and this cycle can start over again.

  • So effectively, the release of ADP

  • is accelerated about 5,000 fold from the action of GrpE.

  • And so GrpE is called a thermo sensor

  • and can begin to think about why that might be.

  • If, say, there's condition of heat shock or stress,

  • maybe the cell wants DNA K to be able to hold on

  • to this polypeptide rather than release it.

  • So GrpE won't be doing its job under those conditions.

  • So another example of ATP binding and hydrolysis

  • modulating activity of these chaperones.

  • So we need to think about what are the substrates for DNA K J,

  • and what is the chaperone system doing?

  • So we define possibilities as foldases--

  • like what we saw with groEL--

  • holdases, unfoldases, what's happening here?

  • And so in thinking about the in vitro substrates, what

  • are the experiments we're going to do?

  • Or sorry, in vivo substrates.

  • So can we take the method used for groEL, groES,

  • and adapt it to this system?

  • Are you convinced that method was useful,

  • or are you down on that method?

  • AUDIENCE: It can probably be adapted.

  • ELIZABETH NOLAN: Yeah, right, it can be adapted.

  • So can imagine again going to do a pulse chase here,

  • and can imagine the same experiments

  • where we have our E coli with no methionine to deplete.

  • We can pulse with radio labeled methionine--

  • again, 15 seconds, 30 degrees Celsius--

  • to let us see newly synthesized polypeptides.

  • And this gives us a way to ask what

  • newly synthesized polypeptides did DNA K and J act on.

  • Then we can chase with excess unlabeled methionine

  • for 10 minutes.

  • And again, can take samples at varying times.

  • Do rapid lysis.

  • And in this case, rather than using EDTA to quench,

  • what they did is do rapid ATP removal

  • by adding an ATPase here.

  • So just to realize that there's theme and variations in terms

  • of how you can quench these.

  • So what do they find?

  • And we'll go over the data in more detail starting on Monday.

  • And what do they need to do to find that?

  • So in this case, they need an antibody to DNA K

  • if there's going to be an immuno precipitation, right?

  • So in these experiments, effectively we're

  • to this point.

  • They immunoprecipitated with their DNA K antibody.

  • Of course, the specificity of this antibody

  • needed to be studied, and then they

  • used SDS-PAGE to analyze the immunoprecipitates.

  • And so what we'll see when we discuss the data next time,

  • the experiments were analogous to what

  • was done with groEL, groES, but a few differences.

  • They were less sophisticated in terms of the approach.

  • So they use just standard 1D STS page rather than 2D,

  • and it didn't go through the process

  • of doing trypsin [? digestion ?] mass spec

  • to identify the polypeptide.

  • So it's more of a qualitative look.

  • But we're going to ask starting on Monday

  • what did they learn from analyzing

  • these gels about the substrate scope of DNA K J?

  • And then we have to ask the question,

  • how does that help our understanding

  • in terms of the type of chaperone activity

  • that's occurring?

  • So with that, I'll close.

  • We'll end the chaperone unit with those experiments

  • on Monday, and then we'll transition

  • into module 3, the proteasome and degradation chambers.

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B1 中級

11.蛋白質摺疊4 (11. Protein Folding 4)

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    林宜悉 發佈於 2021 年 01 月 14 日
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