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ELIZABETH NOLAN: OK, so we're going to get started.

And we're going to continue on with folding.

So we had some introduction last time

about this module and thinking about in vitro

versus in vivo studies.

And where we're going to move on today

is discussing molecular chaperones.

And effectively, there'll be three case studies

over the next two to two and a half

lectures-- so trigger factor, GroEL/GroES, and DnaK/DnaJ.

And so first is some background.

We need to talk about what are molecular chaperones.

And so effectively, these are proteins

that influence protein folding within the cell.

And they can do this by a variety of ways.

So they can help to prevent aggregation and intermolecular

interactions between polypeptides.

They can facilitate folding by limiting conformational space

and preventing side reactions.

An important point to keep in mind throughout this

is that these chaperone proteins bind to proteins transiently

here.

What are the types of processes they can assist in?

A variety are listed here.

And we see that it's quite broad.

So they can help in de novo folding, so for instance,

folding of a nascent polypeptide chain emerging

from the ribosome.

They can assist in refolding.

So for instance, if proteins have unfolded or become

aggregated because of stress, they can help here.

They can assist in the assembly of oligomeric proteins

in protein transport, and they also

assist in proteolytic degradation here.

And so we can classify these chaperones

into three main groups depending on how they act,

and those are listed here.

So we can have holdases, foldases, and unfoldases.

So something you might want to ask yourself

as you see these different chaperone systems is to ask,

what is the role?

Is it one or multiple?

So holdases help to stabilize non-native confirmations.

So effectively, the chaperone will

bind a polypeptide and a non-native confirmation

and stabilize that for some period of time.

Foldases assist in folding of a polypeptide

to its native state.

And unfoldases, as the name indicates,

can help with unfolding proteins, so for instance,

if a protein has misfolded and that needs to be undone,

or maybe a protein needs to be extracted

from some aggregate that's formed in in multiple proteins

that's a problem.

And so we're going to think about the chaperones

in the cytoplasm in two main groups for the ones

that interact with newly synthesized polypeptides.

So first, we can think about trigger factor, which

is a chaperone that's associated with the ribosome,

as we'll see.

So trigger factor is involved in co-translational folding,

meaning the polypeptide is still associated with the ribosome

and de novo folding.

And then we'll examine some downstream cytosolic

chaperones.

So these are chaperones that do not bind to ribosome--

GroEL/GroES, DnaK/DnaJ.

And just as a general overview of this molecular

chaperone concept here--

so this is taken from the required reading--

effectively, what's shown in this scheme

is a variety of different states a polypeptide

can find itself in.

So here we see a partially folded protein.

This protein may form from an unfolded protein or maybe

a native protein.

We have an aggregate.

And if we look down here, we're seeing the effect

of some generalized chaperone.

OK, so one important point to make from this

is that the chaperone's not part of this final structure.

It's just helping the polypeptide

get to its native state.

OK, and we can think about different rate constants,

whether it be for folding or aggregation,

chaperone binding Kon, chaperone dissociation Koff.

So for instance, if we look here,

we have a partially folded polypeptide.

And imagine the chaperone binds that.

Or maybe the chaperone binds an unfolded polypeptide.

OK, it's going to act as a holdase or a foldase.

And what can we see down here--

or an unfoldase-- what we see down here

is an indication of an event that's

driven by ATP hydrolysis.

And so what we'll see and what's known

is that many of these chaperones switch

between low and high affinity states

for some substrate polypeptide.

And these low and high affinity states

are somehow regulated by the ATP binding and hydrolysis.

So here, for instance, we see that step.

And imagine a Koff getting us back into this direction

here, right?

So you can begin to ask yourself questions like,

under what conditions and terms of these rate constants

is folding efficient?

When would a chaperone act as a holdase?

When would aggregation occur?

So aggregation would occur if this Kagg

is much greater than, say, for instance, Kon here

to work systematically through this scheme.

And here are just some points and words

related to that scheme and things

to think about from a broader picture.

So in terms of the systems we're going

to examine in the cytoplasm, this is the overview slide.

And where we're going to begin in this overview

is with the ribosome.

And we see that in red here we have a nascent polypeptide

chain emerging.

OK, so what does this scheme indicate?

What we see is that here is the player trigger

factor, which is involved in co-translational folding.

And we see that about 70% of nascent polypeptides

interact with trigger factor.

And these can arrive in a native conformation.

We see there's two other systems here.

So on the right, we have GroES and GroEL.

So GroEL provides post-translational folding.

We look and see about 10% to 15% of peptides in the cell

interact with GroEL/GroES.

And as we'll see, it provides this folding chamber

on a protected space.

We also see here that this system uses ATP.

OK, and here we have another two players, DnaK

and its co-chaperone DnaJ.

And we see they're binding to some sort of polypeptide

in a manner that's different than GroEL/GroES.

OK, about 5% to 18% of polypeptides

come into contact with these two players.

We also see this system as ATP-dependent,

and there's another player, GrpE,

which we'll see is the nucleotide exchange

factor needed here.

OK, so we see that maybe there's some crosstalk here.

And here we have some needed native polypeptides.

So just some things to keep in mind--

it's important to think about concentrations

and some approximate concentrations are listed here.

If we're thinking about the ribosome

DnaK/DnaJ, GroEL, and trigger factor.

Just to note that many chaperones are also

called Heat Shock Proteins, and this

is because their expression increases with increased

temperature or stress.

So Hsp70, Hsp60, that's for heat shock protein.

So where we're going to begin is with an overview

of trigger factor.

Yes?

AUDIENCE: I wanted to ask.

What do you mean by native and this protein

exist in several conformations in this slide?

ELIZABETH NOLAN: So proteins are dynamic, right?

We know that.

So native means a native fold, so a native state

of this protein as opposed to the protein being unfolded

if it's supposed to be globular or being

some undesirable oligomer aggregate.

So when this polypeptide comes off the ribosome,

that's a linear sequence of amino acids.

And it needs to adopt its appropriate conformation

to do its job, OK?

And as I said last time, we're not

discussing natively unfolded proteins

in the context of this class.

AUDIENCE: So proteins can have many different native receptors

in this slide?

ELIZABETH NOLAN: Yes, they're dynamic.

But there is going to be, like if you need a beta sheet,

a domain that has beta sheets, for instance, that

needs to fold and form there.

So we can discuss further if you have more questions about that.

But think about ubiquitin, for instance,

from recitation week one.

That had a very defined shape, right?

So it's native fold from looking at that PDB file.

AUDIENCE: OK, I think I just need to understand

in that previous slide.

When I talk about one protein here, right?

70% percent of the proteins--

ELIZABETH NOLAN: Yeah, this is thinking

about all the proteins and all the peptides in the cell.

AUDIENCE: OK, I thought it was one type of protein.

ELIZABETH NOLAN: No, this is looking

at proteins in broad terms.

And so what we'll see as we move forward

is that certain types of proteins

interact with GroEL where others don't, right?

Trigger factor interacts with many, many of them

because it's associated with the ribosome,

and the ribosome's synthesizing all polypeptide chains there.

OK, so we're going to start with trigger factor.

And the first thing just to be aware of when thinking about

trigger factor and where it acts--

so we saw it sitting on top of the exit tunnel of the 50S

in the prior slide--

is that there is a lot of things happening near the exit

tunnel of the ribosome.

OK, so here we have our 70S ribosome.

Here's the polypeptide coming out.

A few proteins are indicated.

In addition to trigger factor, just

be aware that there's other players here.

OK, one of these is Signal Recognition Protocol, which

I mentioned briefly last time.

This is involved in delivering membrane proteins

to their destination.

We also have enzymes that do work here,

whether it's an enzyme for removing

the N-terminal methionine, enzymes for deformulation

of that N-terminal methionine, et cetera.

So somehow, trigger factor needs to work

in the presence of these other constituents there.

OK, so when we think about trigger factor, what

you want to think about is a protein

of about 50-kDa that's shaped like a dragon.

OK, this is ATP-independent.

So what I said earlier about low and high affinity states

and switching between these states being driven

by ATP binding and hydrolysis, that

does not apply for trigger factor.

It's the exception in what will be presented in this course.

And it's associated with the ribosome.

And what trigger factor does is it provides a folding cavity

or cradle over the exit tunnel.

And by doing so, it gives this emerging polypeptide

a protected space to begin to fold,

so reduction of intermolecular interactions.

So if we take a look at trigger factor,

one depiction is shown here.

And as I said, think about a dragon.

And it's actually described as having a head, arms,

and a tail.

And so we can think about this also

in terms of N and C-terminus.