#05 Biochemistry Protein Tertiary/Quaternary Structure Lecture for Kevin Ahern's BB 450/550

Uploaded by oharow on 05.10.2011

Kevin Kevin Ahern: Maybe not.
Hold on.
Ah, now let's get started.
How's that?
Too many cords.
I vote for cordless.
How's everybody doing today?
"Good, professor, that's very good."
Male Student: Well caffeinated.
Kevin Ahern: Well caffeinated!
Then learning shall happen, right?
Okay, so, today I'm going to finish talking about
protein structure.
I've talked about primary and secondary so far,
today I'll finish talking about tertiary and quaternary.
Then we'll probably spend most of our time down here
talking about sequence and structure
and there's several things in there
that are kind of really interesting things
that start to bring together, I think,
the whole perspective of why protein structure's important.
Of course we'll be talking about that
over the next week are two actually.
Protein structure relates to every property of a protein
as I've been saying almost endlessly.
I started talking about tertiary structure.
Before I talk about tertiary structure,
I want to give you a definition for it.
Tertiary structure is...
So first of all I'll give you secondary structure
I said resulted from interactions between amino acids
that were close in primary sequence.
Tertiary structure arises because of interactions between
amino acids that are not close in primary sequence.
Well what's close versus not close?
Close, roughly ten or fewer amino acids.
Interactions between ten or fewer
are what we would categorize as secondary structure.
Something that's more than ten apart
is something we would refer to
as interactions giving rise to tertiary structure.
Well tertiary structure has a very different appearance
than secondary structure does, okay?
This is a schematic example on the left
and a space filling example on the right
of the protein known as myoglobin.
Myoglobin is a very important protein that's found in our body,
primarily in our muscles, and in our muscles,
it serves the function of storing oxygen.
And we'll see later the significance of that storage of oxygen.
Myoglobin is closely related to hemoglobin
which is the protein in our blood
that carries oxygen and myoglobin's function
is better described as storing oxygen.
It's kind of what I like to think of as an oxygen battery.
Now if you we look at the structure of myoglobin
and this is the 3D image of the structure of myoglobin,
what we see is that it has secondary structure in it.
It has these alpha helixes that we've seen before
and it also has those turns.
And it's because of those turns that portions of the protein
that wouldn't otherwise be close together
are brought into close proximity.
So we could think of tertiary structure
as a rising between interactions
between one part of the protein here
and say another point of the protein here,
but they're not close in primary sequence.
This might be amino acid number amino acid #41,
this might be amino acid #200 for example, alright?
And the only way those interactions those happen
is because they've been brought into close proximity
and we'll see a little bit later how that actually occurs.
It occurs in a process we refer to as folding.
And folding is as close to a magical a process
as you will find in biochemistry.
Folding is an absolutely phenomenal process.
Now, tertiary structure is simply that.
Tertiary structure gives rise to something
that we call globular proteins.
And the structure go of globular proteins is not random.
It looks like it's fairly random but it's not.
Myoglobin when it's given the proper chance
to fold will always fold in the same way.
It will always have that same structure.
That tells us that there's something
that's driving that specific structure
and as I said in the very first lecture on protein structure,
the thing that drives all the properties
of a protein is the amino acid sequence.
The amino acid sequence determines
what this folded structure is going to look like.
The vast majority of proteins that we find
in the real world are not fibrous.
I talked about fibrous proteins last time.
The vast majority of proteins in the real world,
no, okay, let's see.
Is that ringing, are people hearing a ringing?
Okay, and it's not a phone, it's me.
The vast majority of proteins in the world
are in the globular form, okay.
Fibrous portions are not nearly so common
as globular proteins are, okay.
If I change the amino acid sequence,
I will change that folded form.
The amount that I change it will determine
how much actually varies.
If I change one amino acid in here,
I probably won't change it much.
If I change 50 amino acids,
I will change it a lot, alright?
So we start to think now that every protein
has its own specific amino acid sequence.
And if those specific amino acid sequences
give rise to specific structures,
then we will have a different structure for every protein,
bear [inaudible] amino acid sequence,
and the answer is that's exactly correct, okay?
So again structures function.
When I mutate, if I mutate and I change an amino acid,
I could change a structure very slightly
and sometimes very slight changes have enormous effects.
Sometimes they have virtually no effect.
You can't product necessarily that a mutation
is going to be what we think of as bad.
Some mutations are what we think of as good
because they actually make the enzyme more efficient, alright.
But in any event, when we change an amino acid,
we're going to change something about the structure of a protein.
We're going to spend a lot of time
talking about enzymes in structures and I think you'll see
how very, very tiny changes really can have enormous effects
on the properties of a protein.
Okay, this shows the amino acid...
Forgot my code here.
So this shows the amino acid distribution
of the amino acids in a protein and they've been color coated.
So it's color coated is the amino acids that are the most
or charged are in blue and those that are the most hydrophobic
are shown in yellow and those that are sort of
in between are shown in white, alright?
Now what we see and it may not be real obvious at first,
but what I will tell you is the case is
there is an uneven distribution of amino acids
in this folded protein.
The hydrophilics are generally on the outside,
and that makes sense because myoglobin is found
in the dissolved portion of the cells
found in aqueous solution, the cytoplasm, okay?
Now hydrophilics like water,
they associate with water very well,
and because of that,
this protein is soluble in the environment in which it's found.
That's not totally surprising.
What about the hydrophobics?
The hydrophobics don't like water,
so just like oil doesn't like water,
and oil forms that layer that stays away from water,
it stays with itself,
so to do the hydrophobic amino acids associate with themselves.
We find an uneven distribution,
we primarily find for proteins that dissolve
in the aqueous solution of the cell,
we find that the hydrophilics are on the outside of the protein,
and the hydrophobics are on the inside.
Now this isn't just a random phenomenon.
This tendency of hydrophobic amino acids to like to associate
with each other provides a driving force,
one of several driving forces that cause a protein to fold.
Hydrophobics like to associate with each other.
This really helps get them they need
so we can think of this as being a hydrophobic glob
coated with hydrophilics on the outside.
That enables this protein to be soluble.
Now we'll see later in the term when I talk about things like LDLs,
and HDLs which are complexes in our blood that carry fat,
that they're multi protein complexes
that arrange themselves in exactly the same way.
They put that most hydrophilic portion
of themselves on the outside,
and the most hydrophobic portion on the inside.
When we examine proteins that are desolved
in the aqueous solution of the cell.
As I said, we almost universally see this arrangement.
It tells us that structure,
and function are important.
If I try to put too many things hydrophobic on the outside,
I'm going to have problems.
Why do I have problems?
Well if I have a lot of hydrophobics on the outside,
and hydrophobics like to associate with hydrophobics,
what do you think is going to happen
when one full of hydrophobics on the outside
encounters another full of hydrophobics on the outside?
They're going to glob together.
Now I'll show you later an example
where that actually has important human health implications
when proteins don't fold properly.
By the way, this little thing in the middle, this little,
you're probably wondering what that is,
that's a group called heme,
it's the group that gives hemoglobin its name.
It's actually the portion of the protein that carries the oxygen,
and myoglobin has a heme just like hemoglobin has a heme.
We'll talk a lot more about that later.
Now, I described to you a situation
where proteins that are dissolved in the aqueous environment
of the cell have hydrophilics on the outside,
hydrophobics on the inside.
Are there violations of that rule?
For every rule that is made,
there are violations,
and one of the violations that we see
are in proteins that are found in membranes of the cell.
Not all proteins are dissolved
in the aqueous environment of the cell.
Many proteins are embedded in the membranes of a cell,
and that's important because cells use those proteins
in the membrane to perform very important functions,
and we'll talk about some of those again later.
One of the proteins that I want to show you in this regard
is a protein called porin.
Porin is found in the membrane of the cell,
and when we examine the distribution
of amino acids protein,
what we see is what people describe as an inside-out protein.
The hydrophobics are on the outside,
and the hydrophilics are on the inside for the most part.
Why is that the case?
That's the case because membranes have long,
fatty acid chains in them that are hydrophobic.
So the outside of this protein is interacting
not with an aqueous environment,
but instead with the hydrophobic side chains of fatty acids.
Structure, function go hand in hand.
If I'd try to put hydrophilics out there,
associating with it it doesn't work.
Well why do I even have hydrophilics in this protein at all?
The answer is the function of this protein.
This protein is called porin,
and porin has the function for the cells
that have it of letting in water.
It's channeled to let water in.
Well water of course is water,
and water likes hydrophilic molecules,
and look where the water goes.
It goes right there where the hydrophilic molecules are.
So yet another example of structure matching function,
and teaching us something about protein structure in the process.
So that's a very important consideration.
Okay, so I'll stop there and take questions.
Any questions of what I've said so far?
You guys are all asleep today, huh?
Male Student: [inaudible]...
Kevin Ahern: There are beta barrels,
and beta barrels are like the structure I showed yesterday.
Beta barrels can have several functions
besides doing things inside of membranes,
but one function can be actually what you described, yeah.
Male Student: Now with that being,
are they only one layer thick, and if so,
are they neutral or are they predominately polar, or...
Kevin Ahern: Okay, so the question is are they only one layer thick,
and the answer is most proteins in the membrane you find
are not really one layer thick.
No, they're sort of surrounded by things kinda like what we see
I'll show you an interesting example later
of a an interesting protein in that respect.
Alright, so that's the sort of general things
I want to say about these.
I do want to spend a few minutes talking about
tertiary structure in terms of stabilizing it.
Tertiary structure is actually a fairly fragile thing.
It's fairly fragile.
If I look at an alpha helix, alright,
I see an alpha helix coiling, coiling, coiling, coiling,
and that coiling might go on for 40 amino acids.
And every 4 or 5 amino acids,
I've got a hydrogen bond that's helping to hold
that alpha helix together.
That structure I showed you for myoglobin
has an alpha helix over here
interacting with an alpha helix over here,
but there might only be a couple of hydrogen bonds
stabilizing that arrangement, alright?
It means that there's not as much force
or not as much energy holding that tertiary structure together
as there is the secondary structure,
and that means that tertiary structure
is relatively unstable by itself.
I'm going to show you some things that help to stabilize it,
but it's important that we consider all the forces
that help to stabilize the tertiary structure of a protein.
If you've ever tried to purify a protein in a laboratory,
some people find that they pull their hair out
more than I've already had go off on the top of my head
trying to get a protein purified
because what they discover is that the protein literally
falls apart or stops working half way through
the purification because the tertiary structure doesn't,
isn't very stable.
There's not much energy holding it together.
Not all proteins are that way,
gazuntite, not all proteins are that way.
So let's talk about some of the forces then
that stabilize tertiary structure.
One of these is actually a very,
very strong stabilizing force.
It's actually a covalent bond,
and that covalent bond is called a disulfite bond.
It's a bond between two sulfurs,
and it arises as a result of two cysteine side chains
being brought into close proximity.
Cysteine, I hope you remember,
in the amino acids is the one that has an SH side group.
If you didn't get that before,
you should know that.
Cysteine has an SH side group.
If I put one SH side group next to another,
an oxidation will occur that will join the two sulfurs,
getting rid of the hydrogens.
And that forms a covalent bond.
That disulfite bond is very strong.
Remember that covalent bonds are much stronger than hydrogen
and that can be a very important force
in helping to stabilize a protein.
So we see that here, here's a folded protein.
It has disulfite bonds, disulfite bonds, disulfite bonds,
here's the same protein unfolded without those disulfite bonds.
I'm going to talk more about this protein
in just a little bit, okay?
But disulfite bonds are the most important stabilizing form
for the tertiary structure of proteins.
What other forces do we have that help to stabilize them?
Well, we obviously have hydrophobic forces.
Those hydrophobic amino acids associating with each other
on the inside do help to stabilize that protein, okay?
We've got disulfites, we've got hydrophobics, we've got ionic.
Imagine I've got a plus amino acid up here,
and a minus amino acid down here,
they're going to be attracted to each other,
those are also forces that help to stabilize tertiary structure.
We can think of hydrophilic in sort of a loose sense
of that happening because it's associating with water,
and that may help contribute to the structure
though I don't think it contributes
a tremendous amount to the stability.
But the hydrophilic interactions can play somewhat of a role.
Hydrogen bonds, we saw hydrogen bonds help to stabilize
the alpha helix, the beta strands,
hydrogen bonds also help to stabilize
tertiary structure of a protein.
The last force that helps to stabilize a protein
I'll just mention it here,
and I won't mention it again are known as metallic bonds.
There are some metal carbon bonds
that help in fact to stabilize protein structure.
We don't really deal with them too much in this class.
Now I'm going to come back to this figure in just a bit
after I talk about quaternary structures so bear with me on that.
Okay, that's the last of what I want to say about tertiary
And I want to move our consideration now
to quaternary structure so you've seen what we've seen
in each case are forces between amino acids
that are getting further, and further away,
and so quaternary structure arises as a result of interactions
between amino acids that are actually on separate protein units.
Separate protein units so when I think of an enzyme
or I think of certain proteins,
they may have multiple polypeptide chains that hold them
Prime example is hemoglobin, okay.
Hemoglobin actually has four separate polypeptide chains
that comprise it.
It has two units called alpha that are identical,
and it has two other units called beta that are also identical.
And alpha, and beta are fairly closely related to each other,
and they're also closely related to myoglobin.
This is hemoglobin, yeah.
So hemoglobin has four polypeptide chains
that are held together by, they actually,
hemoglobin has quaternary structure, alright?
So quaternary structure arises
when we have completely separate polypeptide chains
that are interacting with each other.
This is a very common phenomenon in biochemistry.
It's not at all unusual.
Many enzymes, many proteins have multiple subunits,
they're called subunits that come together, okay?
Now, the interactions between the subunits
that stabilize quaternary structure are exactly
the same ones that stabilize tertiary structure.
Hydrogen bonds, ionic bonds, metallic bonds,
hydrophobic bonds, disulfite bonds.
All of those can also stabilize quaternary structure.
Now quaternary structure gives rise
to some really interesting things so first of all,
you have to have multiple sub units to have quaternary structure.
Myoglobin because it has only a single subunit
doesn't have quaternary structure.
But hemoglobin which is very closely related
with multiple subunits therefore has quaternary structure
because those sub units have to interact in some way.
Hemoglobin will be a lecture I will give,
I think it's next week,
that is one of the most interesting proteins
we'll talk about in the entire term.
The amount of functionality that's built
into this protein is nothing short of astonishing,
and some of the most subtle things you can imagine
give rise to properties like being able to be an animal,
being able to move, okay?
Being able to adjust your oxygen
as a result of exercise, alright.
These things all arise because of the properties
built into hemoglobin,
and that just scratches the surface.
So that will be coming up in a lecture very soon.
Okay, and here is a case of a super quaternary structure.
This is the coat, I'm sorry the protein coat,
of a virus that has infected several people
in here I hear people coughing, and hacking.
this is a picture of the cold virus.
And we can see that there are multiple proteins,
each protein has its own distinctive color on here,
and these proteins are interacting to form the coat of this virus.
When I talked about the ability of viruses to make their coats,
and have the proteins self assemble very much
like the pieces of a puzzle putting themselves together,
that's actually what's happening here,
and what you're forming in the process
of that are quaternary interactions,
different proteins interacting with each other.
So this is a great big example of quaternary structure.
Okay, questions on this before I dive into some other stuff?
Yes, sir?
Male Student: [Inaudible]
Kevin Ahern: So your talking quaternary structure, Kev?
Is that right?
So Kev's question is if we look at the quaternary structure,
is there only one force or one bond that stabilizes that,
and the answer is no.
Just like we don't have one force or one bond
stabilizing tertiary structure,
so too can we have many bonds,
and many forces stabilizing quaternary structure.
Yes sir?
Male Student: So with that virus,
are there stabilizing forces [inaudible]
sheer number of quaternary interactions?
Kevin Ahern: Yeah, there's definitely,
when we see quaternary structure,
we have stabilizing forces.
Proteins will not associate with each other
without stabilizing forces.
Absolutely, you will see that.
And you might imagine a case with a viral coat
that the stronger those stabilizing forces are,
probably the better off the virus is
because the virus has part of its life cycle
in the nice cozy environment of the cell,
but for those of you who are sneezing,
and hacking, what you're doing
is you're expelling cold virus into the atmosphere.
That's a pretty harsh environment for a coat to have to survive.
So a strong coat is important,
and having those stabilizing forces is important.
I saw a hand over here, yeah?
Female Student: So do the subunits,
are they considered proteins or only once they [inaudible]
Kevin Ahern: So her question is are individual subunits proteins or
And so the answer is individual subunits are.
And I call the individual subunits,
actually that's, I said we, I, use protein,
and polypeptide chains together interchangeably,
technically, the subunits are polypeptides,
and proteins are the complexes that arise from that.
That makes it a little bit confusing,
but I'm not going to get you one way or another on the exam
so don't sweat that.
Yes, sir?
Male Student: So how does a virus acquire the energy
to get the structure?
Kevin Ahern: How does a virus acquire the energy to get its structure?
Well, there's not a simple answer to that.
Virus has all the things that it does from cellular energy
in the first place,
so making the proteins, making the RNA, making the DNA,
they use cellular machinery to do that
so the cell's energy contribute to that.
The self assembly of these doesn't require a giant amount of
So that's a pretty cool trick they have on a nanoscale.
Yeah, that was the one I saw earlier, yeah?
Male Student: Do subunits have to be tertiary structure?
Kevin Ahern: Do subunits have to be tertiary structure?
You mean could I have a fibrous subunit,
and then interact them?
I can't think of any good examples.
But I guess in biology, you never say never, right?
So I can't think of any good examples.
The vast majority of proteins have tertiary structure, okay?
99.9% of proteins out there have tertiary structure.
The examples I gave: hair, or nails, or silk,
spider silk, something like that.
Those are relatively rare examples of fibrous proteins.
Collagen was another rare example,
but collagen is pretty much it.
Yes sir?
Male Student: Is it more common for a protein
to have a functional stand alone tertiary structure
or are most proteins amalgamated subunits quaternary structures?
Kevin Ahern: So are most proteins loners or are most proteins social?
That's the question, right?
I haven't done a number count,
but I would say in my just off the top of my head,
most proteins are multisubunit.
Most proteins are multisubunit.
So you don't don't see nearly as many single subunit
as you see multi subunit.
And a multi subunit protein has tertiary structure as well.
It has all 4 levels, okay?
Well, good questions, good thinking about this stuff.
What I want to do is talk a little bit now about that structure,
and it's relationship to sequence.
So I first showed you this guy here, ribonuclease.
Ribonuclease is a protein that sort of violates
that rule I said earlier.
I told you that most proteins have a tertiary structure
that is relatively unstable so we have to be careful
when handling them, okay?
We can disrupt most proteins' tertiary structure
by treating them with detergent.
Because detergent interacts with hydrophobic things,
and it gets in the middle of that protein,
and it literally peels it apart.
We wash our hands to kill bacteria
because we're denaturing their protein.
And by the way, when we unfold a protein,
we describe it as denaturing it.
It no longer has it's original shape,
it no longer has it's original function.
Most proteins come apart fairly readily if I add detergent.
Most proteins come apart fairly readily if I heat them.
Because heat provides enough energy to break hydrogen bonds.
I can break hydrogen bonds with heat quite readily.
So by putting a protein at a high temperature,
I break hydrogen bonds.
That's how I'm killing bacteria when I cook food, okay?
Now most proteins don't have good stability to,
for example, heat.
Heat's going to be the example I give you
for the most part, alright?
Ribonuclease is an exception to that.
Ribonuclease is a protein that breaks down RNA.
If you ever work with RNA in a lab,
you will hate this protein.
You'll hate this protein because it's everywhere.
It's in your skin.
You touch your skin to any piece of glass in the laboratory,
and it's automatically full of RNAs.
If you try to work with RNA,
and you've just contaminated with an enzyme that breaks down RNA,
that's a real problem.
If you want to kill this protein,
you can't kill it by boiling.
Most protein's gone, if you boil them, they denature,
they've got no activity left.
You boil ribonuclease, and it's just happy, okay?
It's an exception to that rule.
It's very happy.
Well, why is it stable, and most enzymes aren't?
Most enzymes actually have disulfite bonds.
The answer isn't that this has disulfite bonds,
and others don't.
The answer is that this guy has two interesting properties
I'm going to describe to you.
One is it has a way of arranging itself
so that it readily forms disulfite bonds.
Even after you've taken it apart.
I'll show you that example in a second, okay?
It has the ability to rearrange itself
so that it reforms the disulfite bonds if it's gone.
To illustrate to that to you,
I have to give you an example.
Let's imagine I've got some ribonuclease,
and I take, and I do what's shown here on this treatment.
I treat this with two chemicals.
Mercaptoethanol is very simple molecule,
and it's property is it will reduce disulfite bonds,
and make them back into sulfhydryls.
Remember I said that we put together two sulfhydryls,
and they oxidize, and form a disulfite bond that's a SS bond?
What mercaptoethanol will do is it will break that SS bond,
it will put hydrogens on there,
and they won't be bound together anymore, alright?
Now, if I were to take this room,
and I look at these major supports on the side,
and I were to chop all of those,
we could imagine that this room
would probably be somewhat destabilized.
We might not want to stand down here in the front, right?
It might hold up, but then again it might not hold up.
We've just destabilized the structure of this room.
Structurally, it's not the same as it was
if I get rid of the support beams, right?
Right, we can think of the disulfite bonds as being support beams.
If I denature this protein by first of all
treating it with mercaptoethanol,
what I discover by treating it with mercaptoethanol
doesn't destroy it.
It still stays functional.
So just like this might stay sort of hang around here,
you might not want to be in it,
we still have the basic structure,
the enzyme still functions, but guess what?
The enzyme is not nearly as stable as it was.
I can make it come apart.
Before I try to treat it with mercaptoethanol,
I boiled it, it was still active.
But now I've taken out the support beams,
the disulfite bonds, and I try to denature it,
I can denature this protein, and it won't work, okay?
This tells me that these support beams are very important.
Very important.
So I get rid of the support beams,
I can denature.
So the other thing that's here is urea.
Urea breaks hydrogen bonds,
and this guy comes apart.
Yes, sir?
Male student: [inaudible]
Kevin Ahern: The protein itself wouldn't work, that's correct.
Yeah, okay?
So the combination of breaking the support beams,
and breaking the hydrogen bonds causes this protein to unfold,
that's denaturation, and now this protein doesn't work anymore.
This protein's an enzyme,
it doesn't catalyze its reaction anymore.
It loses its shape.
You see its shape on the side.
I could use instead of urea, I could use heat.
Same thing.
Heat will break hydrogen bonds.
I can heat that guy, and everything's fine.
Now most proteins, once I denature them,
they will not come back to their native form.
They will not come back to the way they were.
This room, if I cut the beams,
and I take a slight bulldozer to it,
it's probably after I've let it fall down,
not going to reassemble itself back
if I magically wave my wand at it.
Well ribonuclease has the property,
but if I'm careful, I can wave that wand,
and it'll go back, and redo itself.
Now that's really cool, okay?
That's really cool.
How does that happen?
Well let's imagine in this experiment.
I start, I use, this is a very concentrated solution of urea,
by the way, let's say I take that urea,
and start very slowly removing the urea, okay?
I very slowly start taking the urea out of there,
and each time I measure,
does this thing that remains have the ability to break down RNA?
That's the question because this guy's an enzyme
that breaks down RNA.
Before I take the urea out,
none of it will break down RNA.
But as I start taking the urea out,
what I discover is all of a sudden something in the solution
starts being able to break down RNA.
Now that simple experiment tells you
a very important thing, okay?
It tells you that the information necessary
to make this structure is the sequence of amino acids.
Because that's the only thing that's there to make this happen.
There's no other proteins,
there's nothing in the cell,
there's only the sequence of amino acids that's there,
and the sequence of amino acids are telling this thing
this is the shape that you want to have.
That's pretty cool.
Now, I'm going to take this one step further.
If I let, if I take all the urea out of the cell,
and I look, and I say,
"yep I've got a heck of a lot more of this activity
than I had over here where I had none,
but do I have as much activity as I had
before I took it apart?"
The answer is no, I don't.
Now my question to you is why?
If the information that's necessary to fold this protein
is there in this thing over here,
why doesn't it all go back to this?
It clearly does not because I don't get everything,
I don't get as much activity as I started with.
Now, I'll give you a hint,
and the hint will surprise you, okay?
One of the ways in which I can increase the amount of activity
when I'm taking out the urea
is to put a little bit of mercaptoethanol in there.
Now, what does that tell you?
Female Student: [Inaudible]
Kevin Ahern: What's that?
Female Student: Some of the sulfurs don't bond
where they're supposed to?
Kevin Ahern: Okay, so sometimes the sulfurs bounce into each other,
and not into the places where they do.
Because all it takes is bouncing into each other,
and once you've got two malformed ones,
there's no way it's going to form properly.
But if you put a little bit of mercaptoethanol in there,
you allow it to come back apart,
and give it another chance to fold.
It's like giving a person a second chance.
If you've ever taken a class,
and you got a poor grade in the class, right?
And you said I want to improve my grade point
so I take the class a second time,
you get that second chance.
Mercaptoethanol is that second chance.
I see a bunch of people looking at each other,
I hope that's not a common phenomenon.
Okay, does that make sense?
Male Student: Is that a repeatable process?
Kevin Ahern: That is a repeatable process.
Male Student: So you can add a little bit more after that
[inaudible] back up to a certain point?
Kevin Ahern: Yeah, you could ultimately optimize it, you could.
Yeah, very interesting insight.
Yes, shannon?
Female Student: Are you ever going to be able
to get back to full activity?
Kevin Ahern: That's what he was asking,
and the answer is if you are very careful, you could, yes.
Yes, back there?
Male Student: Is it a random event, then?
Kevin Ahern: That's a good question.
Is it a random event?
The answer is folding itself is not a random event.
We don't fully understand it,
and I'm going to give you some statistics on that in a little bit.
If not, in the lecture next time,
I'll try to get to that today.
Question back here also?
Male Student: I was just going to ask if folding
is built in to all the proteins,
how is this the only protein that does that?
Kevin Ahern: This is not the only protein the does it,
but it's a rare protein.
If folding is built into all proteins,
how come all proteins don't do that?
Because we can imagine once we take them apart,
they all can make disulfite bonds,
maybe they make the disulfite bonds the wrong way.
And this one has the unique ability not to make those,
and come back, and fold itself.
It's a complicated answer to your question.
Your question is very complicated,
so I don't have a simple answer to that,
but that's one way.
What we discover is that when we take proteins apart,
and we misfold them,
we can drive the folding in the wrong direction.
That's also something that happens.
And when we do that,
then there's no way of getting back over that hump.
But I'll say more about that in just a minute.
Yes sir?
Male Student: What's stopping one ribonuclease
from bonding with a neighbor?
Kevin Ahern: Nothing's stopping it.
That's why the mercaptoethanol is important
in helping improve that folding later.
Okay, good questions.
You're thinking about this.
Happy to see that.
Okay, I talked about reduction.
Here's the actual reduction that happens,
it's kind of hard to invision,
some people say they like to see that on the screen.
When I talk about reducing disulfite bonds,
this is what's going on right here, okay?
This is mercaptoethanol.
That's what it looks like.
You don't need to draw this structure.
But I'm showing you that you're going
from this structure over to this structure.
We've broken that support beam,
and made this over here.
So when I add mercaptoethanol,
and there's another reagent that we can add
that does the same thing it's called dithiothreitol
or you can call it DTT.
Not DVT, but DTT, okay?
DTT will do the same thing, okay.
I've mentioned things that can disrupt structure,
and now you've seen two of them.
Mercaptoethanol, there's urea,
urea's the stuff in your pee that stinks, okay.
And guanidium chloride is another reagent
that can disrupt hydrogen bonds.
So this guy will disrupt hydrogen bonds,
this guy will disrupt hydrogen bonds,
this guy will disrupt disulfite bonds.
What did I say would disrupt hydrophobic bonds?
Detergent, right?
Detergent will disrupt hydrophobic bonds.
All these are important reagents.
Okay, let's see, how am I doing on time?
Since I had a question on folding,
let me just say a couple things about folding.
So first of all, folding is,
his question was if folding is built into all proteins,
how come I can't refold this protein properly.
I said it's a complicated question we can't completely answer,
but we can imagine there's some misfoldings,
and so forth that happen.
Some proteins have a better ability to fold than other proteins do.
Because of that, our cells have special structures in them
that help proteins to fold properly.
And as we will see,
folding proteins properly is important.
Let's imagine that I have a protein that I'm making out of here,
and this protein for whatever reason
is full of hydrophobic amino acids.
A lot of hydrophobic amino acids, okay?
Now the mature protein is going to fold
it's going to put those hydrophobic amino acids on the inside,
but in the process of being made,
there's all this long string of hydrophobic amino acids
that's floating out here in the cell as it's being made.
One amino acid at a time.
Could that pose a problem?
The answer is it could.
Because if I have an identical protein being made right next to it,
we can imagine the hydrophobic amino acids
of that protein might interact with this one,
and prevent it from folding properly
because the hydrophobics like each other,
and they'll start associating with each other
before the folding can really get going properly.
We can make a great big conglomeration of proteins
that would be of no use.
So cells have a structure called chaperones.
Molecular chaperones
proteins called chaperonins,
and you can call them either as far as I'm concerns,
concerned that take proteins,
and allow them to fold without interacting with other proteins.
How does this work?
Okay, well a chaperone basically is a barrel like chamber,
and that barrel like chamber when you have a protein
that needs to be folded properly,
its synthesis goes into that chamber.
It doesn't give a chance to interact
with hydrophobics of other proteins.
The inside of this chamber doesn't allow it
to interact with the chamber.
This protein is left to its own devices.
This protein is left to fold on its own.
So the chaperone allows this protein
to go through its own folding,
and not interacting with other things.
That's a very important consideration for some proteins.
Makes sense what that's doing then?
What happens if we allow misfolding to happen?
If we allow misfolding to happen,
in some cases we have disaster.
We've heard of mad cow disease.
There's a related human disease called
Creutzfeldt-Jakob syndrome that's caused
by the very same problem that causes mad cow disease.
Mad cow disease is caused not by a virus,
but by something we call an infectious protein.
It's called a prion.
And people were very puzzled
when they first started studying prions
because no matter how hard they looked,
they could take an infected animal,
it's also found in a disease in sheep called scrapie.
They're both neurological diseases
where the brain basically ceases to function.
Same things happen to humans that get Creutzfeldt-Jakob.
They can take these infected samples,
and they can transmit it from one organism to another,
but when they analyzed what was there,
they couldn't find it in nucleic acid.
There's no RNA, there's no DNA no matter how hard they looked.
At finally a man named Stanley Pruisner said,
"Well the problem is that we don't have any RNA or DNA."
What we have is an infectious protein.
And people said, how can you have an
infectious protein?
What are you, some kind of an idiot?
And what happened?
What he discovered was that this protein was a misfolded protein.
Now misfolded protein isn't infectious
by itself you wouldn't think,
but it turns out this misfolded protein
has a very bizarre property.
It induces other identical proteins to fold in the same way.
The protein that causes mad cow disease,
the protein that causes Creutzfeldt-Jakob syndrome
is in every one of your brains.
If you get a single misfolded protein in those cells,
it can induce those proteins to start misfolding
which can in turn cause others to misfold,
which cause others to start misfold.
That's a scary phenomenon.
That's how an infectious protein propagates itself.
It's a normal, it's not a mutant protein,
it's a normal protein found in your brain.
Yes, sir?
Male Student: So are prions ever recognized by the immune system?
Kevin Ahern: Are prions ever recognized by the immune system?
I would never say never, but as far as I know, no.
There's some effort right now to make antibodies against it
to see if they can treat it,
and there's been some limited success with that,
but naturally, no.
Female Student: So the reason people get, you know,
like, mad cow disease is because they're eating infected meat?
Kevin Ahern: Will eating meat give you mad cow disease?
That's debated.
Okay, there was an increase in mad cow disease
in England in the late 1980s,
and it followed thereafter an unusual form
of human Creutzfeldt-Jakob syndrome that arose from that,
and the thinking was that maybe that was related.
That is argued.
I will tell you the thing that scares you though.
You talk about stable proteins,
the prion protein is stabler than ribonuclease.
If want to denature the prion nature by cooking your meat,
you gotta take up to 700 degrees.
And I haven't seen any recipes that say,
you know, baste 700 degrees for three hours.
That's not considered a good move, okay?
Now, whether it can be transmitted through your food,
as I said, that's argued,
I won't say that it can or it cannot.
I'll make some big enemies if I do that.
But that's an important consideration.
So prions are really scary things.
They induce other proteins to do what they've already done.
Here's a normal, it's called PrP,
here's the normal protein.
Here is a bad one, and what do you suppose is happening?
Well we've got some hydrophobics
that started associating with each other.
And associated, it doesn't fold properly,
it folds improperly, and it makes this abhorrent structure,
what we call amyloid plaques.
And when we analyze the brains of animals
or people that have these,
they have these big, humongous,
ugly structures that are just polymers of this protein
that look like this.
Okay, that's a bad piece of news.
I thought we would finish on a good piece of news.
We'll finish with a song.
You guys ready for a song?
Let's do, please join me.
[professor starts to sing]
"Oh little protein molecule..."
Join me!
"You're lovely, and serene
"with twenty zwitterions like cysteine and alamine.
"Your secondary structure has pitches and repeats
"arranged in alpha helixes and beta pleated sheets.
"The Ramachandran plots are predictions made to try."
Can't hear you!
"To tell the structures you can have,
"four angles phi and psi
"and tertiary structure gives polypeptides zing
"because of magic that occurs in protein folding.
"A folded enzyme's active and starts to catalyze
"when activators bind into its allosteric sites.
"Some other mechanisms control the enzyme rates
"by regulating synthesis and placement of phosphates.
"And all the regulation that's found inside of cells
"reminds the students learning it of pathways straight from hell
"So here's how to remember the phosphate strategies..."
We'll talk about this.
"...they turn the GPb's to a's and GSa's to be's."
Alright guys, see you Friday.