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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%