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

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


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. Okay? 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 hydrophilic 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. Alright? 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 here. 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 bonds, 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 structure. 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 together. 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. Okay? 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 what? 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 energy. Okay? 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? 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. Why? 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. Why? 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. Alright? 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? Yes? 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? [Inaudible] 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? [Inaudible] 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? Chaperones? 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. P-R-I-O-N. 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? Okay. 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. [END]