5. Kevin Ahern’s Biochemistry – Protein Structure II

Captioning provided by
Disability Access Services at Oregon State University. Professor Kevin Ahern:
Middle of the week, hump day! How are you guys doing? Student: Better. Professor Kevin Ahern:
Better, better than what? Student: Better than yesterday. Professor Kevin Ahern:
Better than yesterday. Biochemistry makes things better? No? [laughing] I hear some dissent
from that, I don’t know! I hope it’s making
things better for you. We’re going to continue
today and hopefully finish up with our considerations of the
elements of protein structure and some things specific to that. And then on Friday I plan to
start talking more specifically about some of the
applications of that knowledge. That will be something
that will hopefully resonate a little bit with you after you’ve learned the things
that we’ve talked about here. Last time I talked about, mostly, primary, secondary,
and tertiary structure. Today I’m going
to finish that off, talk a little bit about
quaternary structure, and you’ll see that
quaternary structure is probably one of the
easiest ones to understand. Then we’ll talk about some
of the other ramifications of that structure, and talk about a very, very
important process for proteins, and that’s the phenomenon
known as folding. Folding is a really
amazing process, as I hope to convince you today. The first thing I
want to talk about is something I’ve alluded
to on a couple of occasions, but I haven’t explicitly
shown it to you, and I think it’s a pretty critical
component of protein stucture, and they’re known
as disulfide bonds. Disulfide bonds
are, you may recall, covalent bonds that form
between the side chains of cysteine residues. Cysteines, you should
remember, have a sulfhydryl, that is, an SH as a part of
their R group in the amino acid, and two SHs, if you put
them very close together, will basically oxidize
and form a disulfide bond. Meaning an SS bond splitting
out the hydrogens in the process. That covalent bond turns
out to be something that is a very, very strong structural
component of some proteins. You’re going to see this protein
I’ve got on the screen today on two or three occasions, so I’ll get you warmed
up to it a little bit. It’s called ribonuclease. Ribonuclease is an enzyme that is ubiquitous
among living organisms. It is an enzyme whose
catalytic activity is aimed at degrading RNA. You might think, “why do
we want to degrade RNA?” We’ll talk about that
a little bit next term, but most importantly
we degrade RNA because RNA can
code for proteins, and we don’t always want to make
all the proteins all at once. So being able to degrade
it is one of the ways we can stop a protein
from being produced. RNases are very important
players in that process. By the way, ribonuclease
is called RNase, R-N-A-S-E. RNases are, in many cases, really interesting
and important proteins. These enzymes can, in fact, in addition to catalyzing
the breakdown of RNA, they are very stable. Now what I haven’t told you
too much about so far is that the protein structures
that we’ve talked about, hydrogen bonds for example, are the forces that
are holding together many of the protein
structures that we have seen, and those hydrogen bonds
are relatively weak. They can easily be
overcome with heat. Today I’m going to
talk a little about other forces that help
stabilize proteins, and one of those
is disulfide bonds. So disulfide is the
strongest of the bonds that are stabilizing
tertiary structure. So tertiary structure
has a variety of forces that help give it stability. Hydrogen bonds are one form,
and disulfide bonds are another, and the disulfide bonds,
as I said, are very strong. I like to think of them,
if you go into a building and you see support beams, you can see the
support beams in here- There’s a support beam,
there’s a support beam, there’s a support beam. They’re critical for
the overall structure because of their strength in
holding up this room, hopefully. These support beams
here in this protein are also very important
for their strength. In contrast to
these support beams, which are pretty critical
in holding up the room, and might survive an
earthquake fairly well, these guys, these disulfide
bonds, in some proteins really help to hold a protein
together under very difficult conditions, and other times,
most proteins they don’t. I’m not going to go into
that in too much detail, but suffice it to say, that they are nonetheless
important components. First I’m going to talk
about these disulfide bonds, then I’m going to tell
you about some other forces that stabilize the tertiary
structure of proteins. So we see this ribonuclease
that has these disulfide bonds, and these disulfide bonds arise because the structure
of the protein has folded to a point
where the SH of one cysteine is brought into close proximity
of an SH of another cysteine, and from what I’ve
told you so far, getting them in close proximity is sufficient for them
to react with each other. So they’re brought into
close proximity, they react, and they form that structure. When you see the term “native”
it means active, normal, whatever you want to call it. It’s the way that the
enzyme is made and folded, and this enzyme, if I took it
and I mixed it with some RNA, it would, in fact, degrade
the RNA very readily. Very, very readily. If I take ribonuclease
and I boil it, ribonuclease has an
interesting property. In contrast to most enzymes that lose their activity
when you boil them, ribonuclease does
not lose its activity. When it cools back down it’s just as active as
it was before you started. One of the reasons that happens
is that the disulfide bonds in ribonuclease
help to stabilize it even under high temperatures. Most other enzymes, even though they almost
all have disulfide bonds, most other enzymes
don’t have that property. Ribonuclease does
have that property. So, we think it’s because these
bonds are in very critical places that really help to hold
everything else into place, yes? Student: Does it work
at the high temperature? Professor Kevin
Ahern: Good question. Does it work at the
high temperature, or does it work only at
the lower temperature? Ribonucleases can work in
relatively elevated temperatures, they can work up to say
60 degrees centigrade where most enzymes
wouldn’t make it to that. They’re not active
at 100 degrees, but when you cool
them back down to 60, they’re active again. So that tells us that there
are other considerations of structure that help give
rise to function of proteins. We’ll talk a little bit
about those in just a bit. Now what this figure
is showing you is, is it possible to
break disulfide bonds, and if it is, what happens
if I break the bonds? There are chemical
reagents that I can use to break disulfide bonds. One of the chemical reagents is
what you see on the screen there, it’s called beta mercaptoethanol, and what it does it
converts an SS bond into two SH bonds,
so they come apart. Well if they come apart, it would be kind of like taking
the beams in this building out and then putting an earthquake
and seeing what happens. If we did that this
building, or this room, wouldn’t be here for very long, and similarly if I take these
beams out and I boil this guy or I use something that
destroys hydrogen bonds, urea is one of those things, either of those will cause this
protein to completely come apart and not be active. When you do that you
have done what we call denaturing the protein. We have denatured it. ‘Denatured it’ you can
think of as unfolding, and when you talk about
a denatured protein, a denatured protein is inactive. This protein on the right, the
denatured reduced ribonuclease, will not, in fact, catalyze
the breakdown of RNA. I’m going to say more about this
protein in just a little bit, but I wanted to give you
some background about this because it’s going to come up
later in the lecture again today. Yes, question? Student: [inaudible] Professor Kevin Ahern: Okay,
she’s got a very good question. This is what I love. You put this up here and
people get thinking about it. Seriously, she’s thinking! Well they started out with an SH, and if I convert them to SH
what keeps them from going back into their original SH form? So she’s actually getting
ahead of me in the lecture. There are two things, but I will answer your
question since you’ve asked. One of the things
that happens with this is the fact that we’re boiling
it and we’re driving them apart. The other is, if we
want to keep them apart, we need to keep a
sufficient supply of the beta
mercaptoethanol in there so we keep them in
the reduced form. If we don’t have sufficient
amounts of this in there, we’re going to see that this
guy can come back together, and it’s an interesting problem,
of it coming back together. Good question. Every body got that? Yes, question back there? Student: What about low pH? Is that the same effect as heat? Professor Kevin Ahern: Oh
boy, very good question! What about conditions
of very low pH? What do you think? Student: [inaudible] Professor Kevin Ahern:
Probably same effect. For most enzymes, and again this is not
an absolute rule book, for most enzymes, change in the pH drastically
will also unfold the protein because in that case we’re
disrupting ionic interactions. When we change the pH
we’re adding a proton or we’re taking a proton off, depending if we’re making
a low pH or a high pH. Imagine if you will that
I’ve got a protein structure that is folded up, and I’m thinking of the
forces that hold it together. So here is, let’s say, one
strand over here in my left hand, and here’s one strand
over here in my right hand, and these two fingers up
here, this guy on the left is a glutamic acid that
has a side chain of COO-, and this guy over
here on my right hand has a lysine with a
charge of +1 from an NH3. They’re going to be
attracted to each other. That attractive force
is going to help hold and stabilize this
protein, right? If I change the pH drastically and I add protons
to that solution such that the COO
– becomes a COOH, it now has a charge of zero. The NH3 is still
is a charge of +1, but a zero is not going
to be attracted to a +1. I have destabilized
the force holding that, it may come apart. That make sense? So, very good question. So what I have just described
to you is another force that is stabilizing the
tertiary structure of proteins. Ionic interactions. Ionic interactions are forces
that help to stabilize a protein. We’ve seen disulfide bonds. We’ve seen hydrogen bonds. We’ve seen ionic forces. There’s another one that’s very
common, it’s known as metallic. I don’t talk much about metallic, but suffice it to say, some proteins are
stabilized by a metal getting in the middle of them
and helping to hold them together. Student: You’re
talking about unfolding. What if you break
down the amino acids? Let’s say under certain
pH they break down. If you remove it from the pH, would the sequence
go back to the same? Professor Kevin Ahern: Oh
okay, very good question! You’re saying break it down to
individual amino acids, right? The question is if I were to
hydrolyze a polypeptide chain, a protein, from its
constituent amino acids- so let’s say I’ve got a
polymer that’s 110 amino acids that are there, and I break it up so that
it’s 110 individual amino acids and I go back to the original
pH, will it come back together? The answer is no. Because that original
structure came about because of the genetic code and they were assembled
sequentially in the ribosome. So there is no assembly
mechanism, number one, and there is no ordering
mechanism, number two, that’s there. We’ll talk about that next term. Good, other questions? Okay, so the last force
that I want to talk about that helps to stabilize the
tertiary structure of proteins is one that’s a little
hard to describe, but you sort of know it. It’s what we call
hydrophobic interactions. Hydrophobic interactions. Hydrophobic interactions we’ve already sort of
talked about last lecture. Last lecture, what I talked about was the fact that those nonpolar
side chains of amino acids tended to be where in a protein
that was dissolved in water? They tend to be on the inside because they’re avoiding water. Well it turns out that when
they’re interacting with each other, that’s a force. That avoidance of
water is a force. Maybe some body set
you up for a blind date, and you really didn’t
want to go on a blind date, and you heard something
about this blind date, and you locked
yourself in the bathroom because you didn’t want to
go out with that blind date- that’s a force. The hydrophobic amino
acids are locking themselves in the bathroom by folding
and being in the middle. That is a force. It turns out that
that hydrophobic force is actually a
reasonably strong force. It helps to hold
the protein together. If I want to break the
bonds of a hydrophobic force I have to basically
insert something into there that is going to disrupt
those hydrophobic interactions. Do you know what we use for that? Soap. The reason we wash our hands
is that we’re interrupting those hydrophobic interactions because the long
nonpolar tail of the soap interferes with those. It destabilizes the hydrophobic
interactions in proteins and allows them to unfold. When they unfold,
they are denatured. So again, the reason
we wash our hands before we eat, and get rid of the
germs and so forth, or kill germs and so forth
that are on our hands, is because we’re denaturing
the proteins in them. Just for the same reason
that we cook our food- We’re killing the proteins
in a different way, we’re killing them with heat. So heat versus soap or detergent. Now you know something
about the forces that help to stabilize tertiary structure. None-the-less, you’ve
heard many forces here most proteins you’ve got
to be pretty careful with. If you’ve ever tried to
isolate proteins in a laboratory some proteins are exquisitely
sensitive to temperature. Some of them are exquisitely
sensitive to the buffer in which they are located. If you’ve been trying to isolate
a protein and keep it pure, and keep it active, it’s really, really
hard to do in some cases. Others like ribonuclease
you can boil them. One of the ways you can purify
ribonuclease is you can boil cells and take the stuff left
over that still works. That’s kind of good. Alright, protein structure. Let’s see… That finishes pretty
much what I want to say about tertiary structure. I’ll talk about helix-turn-helix
later in another lecture, I’m not going to
talk about that here. I want to turn our
attention briefly to quarternary structure. I will remind you last
time that I said that, as we get higher and
higher levels of structure, we start talking
about interactions that are further
and farther away. Primary sequence had interactions
between adjacent amino acids because they were peptide bonds. Secondary involved regular
repeating structures, like an alpha helix
or a beta strand, that arose from interactions between amino acids less
than 10 amino acids away. Tertiary structure interactions are happening between amino acids that are farther than
10 amino acids away because if I have a very
large and folded protein, like I have with myoglobin, I could imagine that this
might be amino acid number six right here, and this might be amino
acid number 60 over here and that’s more than
ten amino acids apart, yet they’re close
enough to interact, and that interaction
helps to stabilize them. So tertiary
structure interactions are happening between amino acids that are not close
in primary sequence. Well then you might wonder how do quarternary
interactions arise? They must be not close
in primary sequence, and it turns out that
quarternary interactions happen between
individual sub units. That is, they’re
separate proteins. Most proteins that
we see in cells are actually
multi-subunit components. Hemoglobin is probably
the best example we’ll talk about in the term. Hemoglobin is comprised
of four subunits, that is four separate
polypeptide chains. Two of the chains are identical,
and ther’re called alpha. The other two chains are
identical, and they’re called beta. You see two alphas
and you see two betas. Alphas and betas are very,
very similar in structure as we will see, and very
very similar in function, essentially we will treat
them identical in function. Nonetheless, there are
four separate units. When we start thinking about well what is going to
stabilize quarternary structure? And it turns out that
all of the things we saw that stabilize tertiary structure also stabilize
quarternary structure. Hydrogen bonds, hydrophobic
interactions, metals, ions, disulfide bonds, all of those can help to stabilize
quarternary structure. And so just as adding a detergent to a protein might denature it, so too might that detergent
separate and take apart the individual polypeptides of a protein that has
quarternary structure. Notice all proteins don’t
have quarternary structure. Not all proteins
are multi subunits. Myoglobin, that I
showed you earlier, is not a protein that
has quarternary structure because it only has one subunit. It takes at least
two to interact. Hemoglobin which is related
to myoglobin has four subunits. We’ll talk more about
both of those later. So that’s quarternary structure. Here is a nice depiction
of quarternary structure. It’s actually the viral
coat for a rhinovirus. The thing that has
caused many of you to have a cold in the past week. You can see individual subunits. You can see there is
many greens, many blues, there’s many reds. All the greens are the same. All the reds are the same. All the blues are the same. This is an example of something that has assembled
itself very, very nicely to encase within
it a nucleic acid. We have quarternary interactions that are between these
individual subunits helping to stabilize
this structure. Yes sir. Student: What is
considered a subunit? Professor Kevin Ahern: What
is considered a subunit? A subunit is a polypeptide chain. So I use the term polypeptide
chain as identical to protein. So we can say something that
has quarternary structure really has four
proteins within it. The nomenclature is
a little confusing we talk about it being a protein, and then it has four proteins. Which is why sometimes
people say polypeptide units as the subunits. Make sense? Let’s go back and talk
about ribonuclease. Ribonuclease is of
interest in several levels and it’s also because it gives us some information about
structure of proteins. Here it is again. You can see the disulfide bonds that I described to you earlier. Ribonuclease is of
historical interest because it was the first protein that we determined
the sequence of. That is the sequence
of amino acids. The first protein we
discovered the sequence of amino acids of
was ribonuclease. I talked about
reducing disulfide bonds and I described it to you but I didn’t show it to you. So let me show you at the chemical
level what’s happening when we reduce a disulfide bond. Here is a protein that
has a disulfide bond. This is mercaptoethanol. Mercaptoethanol you see
starts out with an SH and you see our protein
starts with a disulfide bond. Basically what happens in this
reaction is they swap places. This guy which has an SH
becomes a disulfide bond. And this guy which was a
disulfide bond becomes two SH’s. That’s possible because
this guy is a reducing agent. Meaning that it’s donating
electrons and protons to make possible the formation
of the disulfide bonds. Mercaptoethanol is one
of several compounds that we use in the laboratory to reduce disulfide
bonds in proteins. Another reagent that
we commonly us is DTT. Not DDT but DTT. It stands for dithiothreitol. It has a very similar
chemistry to mercaptoethanol in terms of what it’s doing
with donating electrons. The end result is the same though when we treat a protein that
has disulfide bonds with DTT we end up with sulfhydryls
instead of disulfide bonds. I showed you earlier
that there was urea and I said that urea had
the same effect as boiling because what urea does is it
breaks down hydrogen bonds. It interferes with
hydrogen bonds. If I add urea to a protein there is a very good
chance I will denture it because it’s going to
disrupt its hydrogen bonds and those hydrogen bonds
are going to be necessary for secondary structure, they are going to be necessary
for tertiary structure, and if there’s
quarternary structure they may be necessary
for quarternary structure. So all three of
those may be critical and may be affected by
the addition of urea. Quanidinium chloride
is another reagent that interferes
with hydrogen bonds. Also interferes
with hydrogen bonds. Also will act to help
denature proteins. Ribonuclease is a pretty
interesting protein as I’ve said earlier because ribonuclease teaches us and reminds us about the
importance of primary structure. Primary sequence of amino acids. So this is related to couple
of questions I had earlier so let me step you through this. Let’s say I’ve got a protein. I’ve got a ribonuclease. And I do like I said before. I take the ribonuclease and I treat it with
beta mercaptoethanol and I either boil it or treat
it with urea, either one works. What I hope you remember
from what I showed you earlier is that the ribonuclease
will not be active. It will unfold and be
in the denatured form. It’s interesting that if I
take away the urea very slowly and I take away the
beta mercaptoethanol what will happen is over time I will see a little bit of the ribonuclease
activity begin to return. What does that mean? That tells us as
convincingly as anything that the sequence of
amino acids can determine the folding of a protein. Because in order for that
protein to become active again it has to refold. I haven’t put anything else
in there to help it to refold. It’s refolding on its own,
using its sequence as a guide. I’m going to repeat that now. So I take this
unfolded ribonuclease I remove the urea I remove
the beta mercaptoethanol and this guy will start to refold and I can see it
refolding by the fact that I start to get
ribonuclease activity back. Yes? Student: Is that the
shape of the sequence? Professor Kevin Ahern:
So his question is, is that based on the
shape of the sequence? And the answer is the
sequence determines the shape. So in essence the answer is yes, but it’s the sequence
that’s doing it. The sequence because it has
shapes in it are determining that. That’s really interesting. That tells us like I said
that the sequence drives it. That’s not true
for most proteins. You say “well is
the rule not good?” No, it turns out that folding is an
extraordinarily complex process. Once you’ve taken things apart and unfolded them
for most proteins they don’t have the inherent
ability to come back together because their folding was
a function of the sequential addition of amino acids
as they were being made. That is they started folding
as they were being made and if you completely
take it apart and you put them back together there is going to be some
misfoldings that might happen that would not allow
it to come together in the same way as it would
if it were being folded sequentially while
it was being made. Ribonuclease doesn’t
have that limitation. Ribonuclease doesn’t need
to sequentially start to fold as its being made. It can come together just from
the sequence of amino acids. Most proteins, as I
said, won’t do that. But none the less
this tells us that that sequence there is important. I’ll tell you something that
will confuse the heck out of you. I said when I did what
I just described to you, which is that if I take away urea and I take away the
beta mercaptoethanol I get some of the activity back. I don’t get all of
the activity back. That’s a little confusing. And I’m going to add to
your confusion but maybe for some of you give you an
understanding of what’s going on. If instead of taking away
all the mercaptoethanol I take away most of the
mercaptoethanol but not all of it, I see that I can get close
to 100% activity back. Now, there’s a puzzle for you. There is a puzzle for you. How come it is when I take
away all the mercaptoethanol that I see some of
the activity back but when I take
away most but not all I see almost 100% of
the activity return. What’s going on? Any thoughts? Yes? Student: The disulfide bonds
form between opposite ones. If you still have the
beta mercaptoethanol it will continue doing it. Professor Kevin Ahern:
She get’s an A for the day. Her answer if you didn’t
hear it I will repeat it, her answer is that the formation,
if we have random mixture and random unfolded protein, the way in which it
comes back together because of that sequential
thing I talked about, doesn’t necessarily
lead to everything forming in the right order. Sometimes that will happen and some of those
you get together. What if you have the situation
like you see on the screen where you have a red with a green instead of a red with a red? Once a red forms with a green, this guy is never
going to be active. If I have a little
mercaptoethanol in there guess what happens? It allows that to come apart and gives it another
chance to fold properly. And that is the
answer to the question. That’s what happens, basically. That’s very cool,
very, very cool. Yes? Student: [inaudible] Professor Kevin
Ahern: Good question. Is this energetically
favored to be in native form? In general yes, but when looking at the
energies associated with folding, there is energy all over
the wazoo for each step. So we have peaks, valleys,
peaks, valleys, peaks, valleys, peaks, valleys, happening
over the folding, so we can imagine if we
started to misfold something we’re not going to
have enough energy to get over the next
peak in the process. So yes, this is
energetically favored, but to get to this
point is a long path that might have
misdirections along it. And that’s why most
proteins in fact don’t have this property. They get in those
misfolded directions and they don’t ever
get to this point. Make sense? Other questions? If you understand that,
that’s really cool. That really tells you an awful
lot about protein structure. No questions? You’re a quiet group today. Everybody’s ready for Friday
and it’s only Wednesday. Student: [inaudible] Professor Kevin
Ahern: Say it again. Student: [inaudible] Professor Kevin Ahern: His
question is, why does it, once we get it in
the native form, why doesn’t it unfold again? The answer is actually
these guys right here. Just like these stabilizers, these guys are stabilizers. For many proteins they
don’t have enough of these in the critical places that
they keep it from unfolding. This guy has enough and in the right places that it pretty much
stops that from happening. This enzyme is a real bear if
you work in the laboratory on RNA. I see people smiling about that. Do you work with RNA? Anybody work with RNA in here? Yeah, you know what
I’m talking about. RNases are murder because if you’re working with RNA, you have something
on your fingertips, something on everything
that is chewing up the RNA. You have to be
really, really careful in how you work with those. Okay, that’s a cool
lesson on ribonuclease. This is another thing
I wanted to talk about. Last time I had some people
ask me questions about what happens with the tendency
for a given amino acid to be in a given structure. So I promised you a table, and
this is the table that I have that tells you the
relative likelihood that a given amino acid will
be in any particular structure. Here is glutamic
acid, for example. The higher the value, the more likely it will
be in that structure. So there’s glutamic acid 1.59. Very likely to be in alpha helix, much less likely it’s
gonna be in beta sheet. And reasonably likely it’ll
be involved in a reverse turn. We can’t look at one amino acid- You can see from this table
we can’t look at one amino acid in a sequence and say
“What’s it gonna be in?” We have to look at
a sequence of them and say “Oh, well here’s
a stretch of sequence “that has a glutamic
acid, it has a leucine, “it has a methionine,
it has an alanine.” And we see that their
all lining up nicely with an alpha helix,
we recognize that that is very likely
to be in alpha helix. On the other hand if we
saw something that had a leucine followed by
methionine, followed by a valine, followed by an isoleucine we would say there
is a good likelihood it is going to be
in a beta strand. As I said we can have a
computer program go through and analyze a given
protein sequence and say “Hey what’s
the likelihood “that this stretch
is in an alpha helix?” “What’s the likelihood this
stretch is in a beta strand?” And a computer
program can tell you that answer pretty accurately. We have pretty good ideas
of the secondary structures of proteins from
computer analysis. Tables like this are
really invaluable for that. They tell us where the turns are. They tell us where
these sequences are. I’m sorry where
these structures are. And we can predict
with reasonable accuracy the location of those
secondary structures within a given protein structure. What we can’t do,
and this is amazing, what we can’t do
is we cannot predict what the overall tertiary
structure of a protein is from its amino acid sequence. People would love to do this. They would love to do this. It’s not possible
for the most part. There are some minor
exceptions there. It’s not possible for
the most part to do this because there are so many
possible variations in sequence. I’m sorry I mean in structure. Think about yesterday I
talked about the turns. You have turns that can happen. And you say “Okay well here’s a turn.” Does the turn have
a 60 degree angle? Does it have a 51 degree angle? Is it in three dimensions so that it’s rotated
slightly this way? There are all kinds
of possibilities. That even slight
structures in one turn have big effects down the line as you try and predict
where that structure is. So if you miss the first one, then everything else
is going to be off. If you get the first one, but miss the second one then everything else
is going to be off. When people first started working with the sequence of
proteins they said, “Oh well this is
a simple problem. “We know the chemical structures. “We know the dimensions. “We know some of the
angles and so forth. “So we can predict the
overall structure of a protein “if we get just a
powerful enough computer.” There were a lot of
people spent a lot of time trying to do just that. There are people today
still working on it in a different way. They quickly came to realize was that no matter
how hard they tried they weren’t getting anywhere. They weren’t able to make
very accurate predictions of tertiary structure. Secondary structure yes,
tertiary structure no. Their predictions were way off. They know they are way off because we can actually
determine what the structure is if we do what’s called
X-ray crystallography. Where we shoot X-rays
through a protein. We can tell where
those atoms are. It would be nice
if we could predict where we didn’t have to shoot X-rays through it and do all that. So people work on it very hard. What I’m getting ready
to describe to you is what’s called the
protein folding problem. It’s also called
Levinthal’s paradox. Levinthal’s paradox. This is gonna blow your mind. It blows my mind. Imagine we’ve got
a simple protein. It’s 50 amino acids long. That is relatively
short as proteins go. 50 amino acids long. It’s got 50 peptide bonds in it. 49 peptide bonds in it. We start thinking about all
the different possible angles and all the different
possible interactions that each of those
guys could have. Levanthol did this and came to the conclusion that if he looked at
the possible angles, and if protein folding was
occurring by a random process where it went through
every possible configuration before finally ending
up with the right one. If it went through that process, Levanthol looked at it on a
computer and says “Oh my God.” The possibilities blow my mind. “It can’t be a random process.” I’ll tell you why. What he discovered was considering all those
possible different structures, that if it were on a random basis and they were to
go iterably through and analyze every one. If you took the most
powerful computer on earth, that’s available today, and you had it
analyzing a structure every nanosecond or so. So you have all of these
different possible structures that you could analyze on a
very rapidly working computer, this is a very simple protein, how long would it take to go through all those
possible structures and determine the
ultimate correct structure? Well it turned out that
computer couldn’t do it. Then he said, “Well, what if you took “all the computing power
on the face of the earth “and you applied it to the
problem, could you do it?” The answer was
you couldn’t do it. What he discovered was if you took every computer
on the face of the earth and they were all the
most powerful computers on the face of the earth and they worked on
this problem forever it would take a
million times longer than the age of the
universe to do it. It’s probably not going to
be solved on a random basis. That tells us a couple of things. One we don’t want to
use random methods, for trying to identify structure. We know that. Number two it tells
us that protein folding does not occur by a
random basis either. We may not understand
all of the things that are happening
in protein folding. And yes we are making
good progress in learning a lot about how those
things are in fact happening. But they’re not occurring
by a random process because a protein
inside of your cells can properly fold in
a matter of seconds. In a matter of seconds. And fold the same way every time or almost every time. Pretty cool stuff. We also know that folding
is a very critical process in our cells. What you’ve seen is that
it doesn’t take much energy, and in some cases it doesn’t
really take any significant energy, for something
to not fold properly. You saw that with
ribonuclease right? Even ribonuclease
didn’t come back together 100% of the time unless
I added mercaptoethanol. I had to give it more chances
to come back together properly. Some of those chances
didn’t work out right. In the case of
ribonuclease, no consequence. No consequence because it means that ribonuclease
simply isn’t active. There are proteins in your brain for which there’s
enormous consequence if they don’t fold properly. Enormous. Everybody in here has
heard of mad cow disease. Mad cow disease is not
caused by bacterium, it’s not caused by a virus, it’s caused by a protein that’s found in every
one of your brains. My brain, cow brain,
they’re all there. Everyone of us has this protein. And when this protein
that we have in our brain doesn’t fold properly
we’re in deep doo-doo. We see it in mad cow. We also see it in human diseases
known as Creutzfeldt-Jakob, and if you ask me later
I’ll spell it for you but I won’t spell it here. Creutzfeldt-Jakob syndrome is
almost identical to mad cow. It’s a neurological problem and people who
develop this disease last a few months before they,
basically their brain is gone. Dementia out the wazoo. It’s happening
because this protein that was in their brain,
one of them, mis-folded. Didn’t fold properly. It didn’t fold properly. As a result of it
not folding properly, this is the bad
part of it, you say “Well ribonuclease didn’t
fold properly no biggie right?” Well this protein is a
biggie because this protein, once one of them
doesn’t fold properly, it causes others
of the same nature to also not fold properly. This protein is called PrP. Here’s the normal PrP,
it’s a normal brain protein, you have it in your brain,
and here’s its proper folding. Here’s one that started
out not folding properly. What one misfolded can do
is cause others to misfold, and they form large,
large, amyloid plaques that destroy nerve
cells in our brain and they destroy
them very rapidly, that’s why Creutzfeldt-Jakob
works very rapidly and causes dementia very quickly. It’s not completely
known why it misfolds, it’s not completely known, for example, if it
can be transmitted or how easily it
can be transmitted, it does not appear to be
easily transmitted in humans, but there is some
evidence, that it can be. People who work for example
with the brains of dead people who suffered from this disease, sometimes have an increased
incidence of developing it. There was an outbreak of mad
cow disease in the late 1980’s in Great Britain, that was
well known, well documented, and at that time they
didn’t inspect British beef, and so it made it
into the food supply, and about ten years later they saw a pretty good
bump in the occurrence of an odd form of
Creutzfeldt-Jakob in humans. The concern being, can it be
transmitted in the the food? It’s a controversial issue, some people say yes,
some people say no, but here’s the really bad news, cooking it doesn’t protect you. This protein is
extraordinary stable. If you want to
completely denature it so that it can’t cause affects, you got to heat it up to
about 700 degrees Fahrenheit. I don’t know of any recipes
that call for cooking at that temperature. So, that’s a concern. A real concern. Questions, comments about that? Student: [Inaudible] Professor Kevin Ahern:
Yeah, once someone has it can you do anything about it? Can you unfold the unfolding? Is basically what you are asking. I know of no single incident,
no single occurrence, where a person has been cured
of Creutzfeldt-Jakob, no. It’s a disease that is prevalent throughout
the animal kingdom. Probably every animal has it. I mean there are
animals who have it. For example, beef is one, but there is a disease
in sheep called scrapie that is caused by the same thing. It’s known to be in deer, it’s known to be in
a variety of things, it probably is in
virtually every animal that’s out there, and there’s evidence
that it may in fact, be a problem throughout
the kingdom of life. There are mis-folded proteins that cause some unusual
problems inside of yeast. Yeah, that’s kind
of a scary thing. It is. Now, the last thing I’ll tell you and then I got a
fun thing at the end, the last thing I will tell you is that folding of proteins
is really critical. I hope I convinced you with this, but our cells know
they are very critical, and our cells actually
have things in them to help proteins
to fold properly. They are called
chaperones or chaperonins. You can call them either one. Chaperones are structures
that proteins get put into to allow them to fold properly. You might think what in
the heck does that mean? Well, they’re basically
a little chamber. Let’s think about a
protein being made. A protein is being made
one amino acid at a time, and if I think about
a typical protein that’s dissolved
in water, I think well, there are hydrophobic
amino acids there, right? And if I have hydrophobic
amino acids there and they want to fold
away from everything else, what happens before they fold? Do we have problems? We might. They don’t like water. They want to get away from water, and they will
interact with anything that allows them to
get away from water, so imagine I’ve got this
protein being made over here, and I’ve got a few hydrophobic
amino acids come off and I’ve got the same protein, another copy being
made over here, it’s little bit further along. They’re close to each other, the hydrophobic amino
acids of this protein might interact with the
hydrophobic amino acids of this protein, and thus
prevent proper folding. What chaperones do is they allow
the proteins to be isolated, and fold on their own, so they don’t have that
interaction with each other, and misfold. That turns out to be
really, really useful. Okay, I promised
you something fun, and lets finish
with something fun. It relates to the fact that I’m tired of
all this sunshine. I’d like you to think about that. [music] [David Simmons singing “Let it Rain”] Lyrics: Oh the
Oregon weather’s dowdy cuz the sky is mostly cloudy, you can’t stop it if you complain so let it rain, let
it rain, let it rain! It doesn’t show signs of slowing and it’s rarely right for snowing though it’s driving
some folks insane, let it rain, let
it rain, let it rain! When it finally turns out dry we’ll be putting
away our rain gear. It will probably be July but I’ll surely
miss the rain dear! Cuz the sound of the falling rain pitter pattering down the drain makes music inside my brain so let it rain, let
it rain, let it rain! Professor Kevin Ahern:
Alright, see you Friday. [students clapping] [END]

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4 thoughts on “5. Kevin Ahern’s Biochemistry – Protein Structure II

  1. Thank you SO MUCH!! I am a prospective biochemistry student but I cant afford the cost of college especially with out-of-state tuition. I love your videos 🙂

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