Lecture 10 Protein Folding & Degradation
- In the past lectures we’ve looked at going from DNA to protein
- Now we address the question why cells don’t explode from the protein, we look at the proteome and he wants to
ask how we get rid of the proteins in the proteome
Slide 1 - In the remainder of today’s lecture, we will cover protein folding with molecular
chaperones, something we’ve heard about before and he wants to look at protein
degradation, the proteosome and ubiquitin.
- Short readings today but at the end of last lecture, the handout in the last lecture
had the accurate readings, pay attention to the handout. He made a booboo for the
lecture but the handout is accurate.
Slide 2 - He has just said wow that’s a lot of protein, why don’t his cells explode?
- Let’s consider for a moment, where that protein came from as it came off of the
- Let’s take a look as just a reminder, at translation. We see the peptide bond being
made starting with methionine in eucaryotes and the formation of a peptide bond
within the large subunit of the ribosome and the transfer that occurs from one tRNA
- There is a movie playing capturing the elongation of the peptide strand very
slowly. All of this should be looking fairly familiar, the same is true on the next
slide, the multistep process involved in making polypeptides, the process of
translation and away we go.
- This is a very simple multistep process involving the migration of the tRNAs from
multiple active sites within the ribosomal complex.
Slide 3 - We want to focus on what's happening to that protein as it comes off from the
- We know that a number of things must happen to it, we know that as it emerges
from the ribosome it enters its life within the cell, that it has to be folded, that there
may be association with cofactors there may be some form of covalent
modifications of the proteins, intra and intermolecular interactions that can occur,
assembly into multi-protein complexes etc.
- Eventually we have a 3D structure that performs a function in the cell.
Nascent polypeptide chain enters cell from ribosome Folding and cofactor
binding (non-covalent interactions) covalent modification by glycosylation,
phosphorylation, acetylation, etc binding to other protein subunits mature
Slide 4 - The way you can think of a molten globule is the way the name implies, like a
bead of wax dripping off a candle or lava from a volcano.
- It's something that is very malleable, flexible in its shape but it's not yet assumed
its final shape and that is captured in the textbook.
- You see the transition in the slide.
- The beginning of this folding of the molten globule begins right on the ribosome.
Slide 5 Co-translational folding
- As the protein rises from the ribosome during the process of translation, folding
occurs and this is called co-translational folding.
- Folding occurs as the protein emerges from the ribosome.
Slide 6 - The problem is that not all proteins are folded properly, most are, most proteins
will assume the leftmost route (going downwards).
- It is the on-pathway folding meaning it is on the route towards a properly folded
protein, it starts its life as a molten globule and eventually through
thermodynamically driven interactions between the components of the molecules,
between the AA R groups, you end up with a protein in its final properly folded
- Every now and then you get folding that occurs off of this pathway, where misfolding has occurred, and it turns out that the cell has mechanisms to recognize
these improperly folded proteins and get them back onto the properly folding
pathway route again.
- You have every now and then, proteins that misfold that can’t be refolded back to
a properly folded state.
- The bottom line is the cell has mechanisms that ensure a misfolded protein gets
back onto the pathway towards correct folding but if it is completely irrecoverable,
it gets trashed.
Heat shock protein 70
- Here is one of the mechanisms that get things back onto the properly folded
pathway or to ensure they are on the pathway to begin with and that they fold
properly, this is the role played by molecular chaperones.
- An example of a molecular protein is heat shock protein 70, HSP 70 (remember
not HSP90 which is an inhibitor).
- The heat shock protein component of this is misleading, the name came about
because in studies where people were originally looking at the proteome and this is
some of the earliest proteomic work ever done, what they did was take a look at the
differences of protein content and normal cells and those treated with heat. Those
cells treated with heat created an abundance of a lot of these proteins which were, at
that point in time named heat shock proteins because they were created in response
to heat shock.
- It turns out that these proteins are actually made every day and used every
femptosecond of every day to ensure that proteins fold properly but they get
expressed on much higher levels during heat shock because it is more likely for a
protein to be misfolded when higher heat is present.
- The thermodynamics changes so to protect the cells from the heat, the cell ensures
that these molecular chaperones are there to make sure the folding process goes
correctly, but they’re still there every minute of every day to ensure that the proteins
in your cells are folded properly, this HSP 70.
- We met HSP 90 earlier which is a chaperone which ensures that proteins stay
outside in the cytoplasm and we saw that with the glucocorticoid receptor, yet
another example of the chaperones that ensure proteins do what they do.
- HSP 70 it binds to short stretches of hydrophobic amino acids in this instance and
as he already said, it was named based on its appearance after heat shock. It turns
out that different organelles have different HSP 70s but they all fulfill a similar
role, recognizing hydrophobic patches and in an energy dependent fashion involving
the hydrolysis of ATP, ensures that the protein folds into a proper shape on the basis
of thermodynamic properties, it ensures that the protein is properly folded.
Slide 8 Barrel - like
- Another kind of heat shock protein which is a molecular chaperone is heat shock
protein 60 (HSP60) sometimes called TCP – 1 or GroEL.
- Again it is named due to its abundant expression after heat shock but again this is
also functioning every moment of every day.
- In an energy dependent fashion, it folds misfolded proteins properly.
Through hydrophobic interaction
- Here is how HSP 60 functions. You have a misfolded protein called a client
protein, if this client protein has hydrophobic patches exposed, it's wrong in the
aqueous environment within the cell. If you have a hydrophobic patch on the outside
of this protein (trust me it's hydrophobic even though he says aqueous), it indicates
that it is misfolded.
- Sometimes the misfolded protein is fed in by heat shock proteins.
- In a reaction that involves physical movement of the barrel structure itself causes
the thermodynamic properties within the barrel to change and causes the protein to
refolded and hopefully cause the protein to refold into a properly folded state. - What you’ve done is that you’ve taken what you knew was misfolded and there is
a good chance that it's going to get properly folded.
We will now see a movie - This movie captures what we’ve talked about in the last couple of slides, starting
with HSP70 and moving on to HSP60.
- There is a properly folded protein where we see hydrophobic amino acids are
properly found in the interior of the structure. We see the two types of chaperones,
starting with HSP70 bound to ATP, then we have proper co-translational folding
where as hydrophobic amino acids emerge from the ribosome, they are protected by
HSP70 which by now are involving the hydrolysis of ATP to ADP. An exchange
with ATP, the chaperone will let loose and the protein will fold into its properly
folded configuration, that’s example number 1.
- Let’s take a look at HSP60, there we see barrel-like structure, the misfolded
protein is being fed in and we see the hydrophobic patches interacting with the
surface of the barrel, the barrel changes shape and out comes a nicely folded protein.
- What he wants to do in the next lecture is talk about what happens to proteins when they aren’t properly folded,
how do we get rid of the trash. Let’s see what happens to incompletely folded forms and digested in the
**Lecture 10 Ends Here**
**Lecture 11 Starts Here**
- Last lecture: we’ve taken a look at the elaboration of the central dogma & we’ve got ourselves as far as protein.
We’ve talked about the proteome, how one analyzes individual proteins & determines their function within the
proteome & what we started talking about in the last lecture was how proteins fold to give rise to appropriate
structures & we started to consider what happens when we focus on that proteome, what happens when things
don’t fold properly, when they don’t function properly, how do we get rid of that protein?
Slide 10 - These are the pathways that proteins may assume as they are post-transcriptionally
folded offstf the ribosome & find their way to their function in an eucaryotic cell.
- So the 1 part of the pathway is that proteins are correctly folded without help.
- The 2 part of the pathway involves the role of the molecular chaperone HSP60 &
HSP70 in the folding of proteins.
- The final pathway is what happens to proteins that are incompletely folded or
completely off the folding pathway & they are described as irretrievable
errors/mistakes – these are digested in the proteasome, they are put in the
equivalence of the cellular garbage can.
- Think of this as a nice quality control mechanism to ensure that misfolded proteins
don’t find their way inside the cell to cause havoc.
Slide 11 - Has 2 major components: 1) the 20 S core proteasome that contains proteases & 2)
the cap: structure that effectively feeds proteins into that core catalytic activity.
- How do proteins get marked/designated to get put into the trash?
Slide 12 - One of the most effective ways that proteins are targeted for the trash, and of
course eventually there is going to be turn-over of proteins that even are folded
properly, the most effective way of marking them for the trash is to put a tag on
them that says that this particular protein needs to go into the proteasome. That tag
is what is known as ubiquitin – small protein that is added covalently to proteins to
modify where they end up in the cell.
- Not all instances of addition of ubiquitin (also known as ubiquitination &
ubiquitylation) – this particular tag that gets added to the protein in some instances
functions to ensure that that protein gets targeted for the proteasome. How does that