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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