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.

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