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Kevin Ahern: Happy Friday!
Student: Whoo!
Student: Happy Friday!
Kevin Ahern: Shall
we all celebrate?
Call off class?
Don't quote me on that.
How's everybody doing?
Everything making sense?
Student: Mostly.
Student: So far.
Kevin Ahern: Mostly?
Mostly?
Kevin Ahern: Working
through the problems?
Recitations?
Okay.
Everybody's got a
smile on their face.
That's good.
So, I'm happy to see that.
Okay.
So, as always,
if you have problems
or questions,
or there are things that
aren't working right,
let me know.
That's my job to,
obviously, hopefully,
help you to make things right.
So that's what I want to do.
What I want to do today
is talk about amino acids,
because we're going to
start turning our attention
now to protein structure, a bit,
and getting a better
understanding about
how these things we've
been talking about
with respect to pH affect charge
and how charge can affect
the structure of proteins.
As I've alluded to in
the very first lecture,
structure is
essential for function.
And so things that
affect structure
we really need to have
an understanding of.
Okay.
So that's the topic
of today's lecture
and will be the topic of actually
the next three lectures,
including today.
So as you can gather,
by the fact that,
here we go, okay.
We'll try this.
And yes, I have it on vibrate
so that you guys can't hear it,
but my vibrate seems to
make that thing vibrate.
Okay.
[buzzing noise]
Shutting down.
By virtue of the fact that
I give three lectures on this,
it says something
about the importance
of the topic of
protein structure.
It's a very, very important
thing for us to understand.
Well, structure is
a pretty remarkable thing.
We think of protein structure
as actually occurring
at four different levels,
and I'm going to
talk in some detail
about each of those four levels.
The four levels are
what we call primary,
secondary, tertiary
and quaternary.
Primary, secondary,
tertiary, quaternary.
And those four levels of
structure you can think of
as resulting from interactions
between amino acids
that are farther and
farther and farther away.
So therefore the interactions
between amino acids
that are the closest are
those of primary structure.
The next closest is
secondary structure,
the next closest is
tertiary structure,
and the farthest away,
quaternary structure.
And as I describe these,
I think you'll see
why that's the case.
So I want you to
keep those in mind.
They're not difficult concepts.
They're not difficult
concepts, at all.
But because of these four
levels of protein structure
that exist, all of
the proteins that exist
in the world can be
explained and understood.
Okay?
Structure implies function.
This is one of the best
examples I can give you
of structure
determining function.
Next term, in BB 451,
I will talk about this protein.
This protein is called PCNA.
You don't need to
know that for this.
The important thing
about this protein
is it helps DNA polymerase
to stay stuck to DNA.
Okay?
Stay stuck to DNA.
DNA polymerase copies DNA.
It's got to move along the DNA.
DNA polymerases that
bind to this protein
remain stuck to
DNA for a long time.
Why?
Well, the structure of
this protein is a ring,
and like a ring
sliding along the DNA,
it doesn't let anything go away.
DNA polymerases that
stick to this protein
stick to the DNA.
DNA polymerases that
don't stick to this protein
go along the DNA for
a little ways, they pop off,
they pop on, they
pop off, they pop on.
Okay?
Meaning, therefore, structure is,
again, implying function.
If they don't have the
ability to bind to this,
this protein itself binds
to DNA because of
its ring structure.
This ring structure is
essential for the function,
not only of this protein
but the proteins
that interact with it.
I like to show
this figure because
it's a really nice example,
when we look at proteins, of how,
what I like to describe as
nano-machines can assemble.
When we look at,
for example, a virus,
a virus is composed
of a protein coat
and a nucleic acid inside of it,
at its very simplest level.
That protein coat is
a protection for the amino acid
that's inside and that protein
coat has to be assembled.
Viruses don't have factories.
They don't have workers
that can hammer rivets
and make things stay together.
They don't have glue.
Instead, they make proteins
that do something
really remarkable.
Those proteins
can self-assemble.
Now, imagine having a puzzle
that you put together
that's got 500 pieces
and the puzzle puts
itself together.
That's what proteins
in a viral coat can do.
And understanding how
that assembly process works
has medical implications,
as we will talk about later.
And it has tremendous
implications
for the virus being able
to make copies of itself,
because if it can't
assemble its protein coat,
it can't function as a virus.
So when we think about
what I like to describe
as nano-machines, and
we have a lot of people
who are interested
in nano-technology,
it doesn't get any more
nano than this right here,
things that can put
themselves together.
Remarkable, remarkable, proteins.
Another important concept
that I'll talk about,
with respect to
structure of proteins,
relates to it indirectly,
and that is the concept
that proteins are flexible.
This is a very important concept.
You're going to hear me
talk about the flexibility
of proteins over and
over and over this term.
Flexibility is key to
understanding what proteins do.
It's key to understanding
what enzymes,
which of course are
also proteins, do.
And this example
here is a prime one.
It shows the effect of binding
of a single atom of iron
to a protein,
a single atom of iron.
When this protein binds
to this atom of iron,
right here, the shape
of this protein changes
from PacMan over here to...
I don't know...
globular nightmare, alright?
But it changes its shape.
And this change in its shape
has very drastic effects
for the function of this protein.
This protein over
here will do something
that this very same protein
over here will not do.
These are the same protein,
differing in shape by the
binding of a single atom.
Okay?
We will see later, when
I talk about enzymes,
that this flexibility
allows enzymes to do things
that chemical
catalysts cannot do.
It's the key to why enzymes
can speed reactions trillions,
quadrillions of times.
Flexibility.
So flexibility is a very,
very important concept
for us to understand,
and it's a very,
very important phenomenon
for a protein to exist.
Well, as we talk about
and think about proteins,
we have to go down to the
level of the building blocks,
and, of course, everybody
in basic biology,
I hope, knows that
the building blocks
of proteins are amino acids.
And there are, in
fact, 20 amino acids
that are most commonly
found in proteins.
There's another amino acid,
called the 21st amino acid,
that is occasionally
placed into proteins.
And of those 20 amino acids
that we always find in proteins,
some of them get chemically
modified after they get in.
So we see quite
a variety of things arising
from the structures of
amino acids in proteins.
There are some things that we see
not so variable, however.
One of the almost
invariant things
that we see in proteins
is that when we look at the
stereoisomeric configurations
that exist of proteins,
what we discover is that
there's a very strong bias
for biologically-made
amino acids.
We can synthesize amino
acids in a test tube.
If we synthesize amino
acids in a test tube,
we get a mixture.
They have two stereoisomeric
configurations.
You can see the
configurations on your screen,
D and L.
If we make them in a test tube,
no cells involved,
we make them chemically,
we get 50% D, we get 50% L.
If we examine the amino
acids that a cell makes,
if we examine the amino
acids that are in proteins,
99.999% of them will be
in the L configuration.
Okay?
There's only a very tiny
number of amino acids
and I'm going to actually
give you the exceptions
later in the term of amino acids
that appear in proteins that
are in the D configuration.
How do cells make such a bias?
Well, they make
such a bias by virtue
of the fact that the amino acids,
that the enzymes that
make the amino acids
have their own bias.
They will only make one
type, preferentially.
Why do they only make one type?
They only make one type
because they can
only use one type.
Well, how about
that cell over there?
Well, that cell
over there is eating,
using amino acids from proteins
it's gotten somewhere else.
The language of
biology says that,
if it's not L, I can't use it.
So if I'm a cell and
I've got D amino acids,
I couldn't use all the
L's that were out there.
I couldn't eat!
So by default, everything
ends up being L amino acids,
because that's what
cells universally use.
Now, this is a really
cool phenomenon because
if we are interested in
a phenomenon of what's called
"astrobiology," you know...
is there life out there,
floating around
out there in space?
There probably is.
We would like to know that.
One of the things that
people do when asteroids...
asteroids...
when meteorites fall to
Earth is that they grab those,
they try to get them before
they get any contamination,
before people get
their hands on them,
before all of Earth
can its contaminating
amino acids in it.
They bust open the meteorite
and they analyze the
amino acids that are in it.
Is, yes, astroids,
I keep saying "asteroids"
meteorites are
full of amino acids.
The question, then,
is do we see a bias?
Were those amino acids made like
they make them
like in a test tube?
Or where they made by an organism
that has its own built-in
bias for those structures?
Well, so far, we don't
have any meteorites
that show any strong bias
that we can tell that.
And we probably won't.
Floating out in space
is not a real good place
for life to be.
But, it doesn't
hurt to look, right?
So it'd be kind of
cool if we could see,
we find the meteorite that
has everything in the D,
or everything in
the L, either way.
It'd be kind of cool.
We'd know that it didn't happen
by that simple
chemical process that
we can do in a test tube.
Is there life?
Okay.
Now, I'm not going
to ask you to draw,
actually, let me go back.
I'm jumping ahead.
I'm not going to ask
you to draw an amino acid
in the D versus the L.
Okay?
You're not going
to have to do that.
You should know that L's
are the predominant form.
And you should know the
constituents of an amino acid.
That's something
I forgot to mention here.
Every amino acid has
four basic constituents
that we can point to.
Actually, five, if we
count the central carbon.
There's a central carbon
called the "alpha carbon."
Attached to that alpha carbon
are four different things,
and because there are four
different things attached to it,
that's why we have
stereoisomeric forms
of each of the amino acids,
all but one, that is.
Okay?
Four different things.
One is called the "alpha amine."
It's this blue guy, over here.
An alpha amine is attached
to an alpha carbon.
We also have something
called an "alpha carboxyl."
An alpha carboxyl is
attached to an alpha carbon.
In addition, we have a hydrogen.
And the fourth thing that
we have is an R group.
Now, if you look at this,
all the amino acids
have a hydrogen.
They all have an alpha carboxyl.
They all have an alpha amine.
The only way that
the 20 amino acids
differ from each other
in structure is in the
configuration of their R groups.
So we can think of the R
group as really defining
the chemical properties
of the amino acids.
Okay?
So if we understand the R groups,
we understand the chemistry
of each of the amino acids.
Alright.
Well, I've been talking for
a couple of days about ionization.
I want to point out
to you that ionization
is critical for amino acids,
because amino acids
have ionizable groups.
Now, so far, the ionization
that I've talked about
has been about acetic acid.
Acetic acid has
a single ionizable group.
It has a single pKa.
All of the amino acids
that we find in biology,
all of the amino acids,
have at least two
ionizable groups,
and therefore, at least
two different pKa's,
at least two ionizable groups,
at least two different pKa's.
The alpha carboxyl can ionize.
It starts out as a COOH,
which has a charge of zero.
When it loses its proton,
it has a charge of -1.
The alpha amine can also ionize.
Okay?
If it has an NH2, which
is what we see here,
and which is what we
think of as an amine,
it has a charge of zero.
But if it gains a proton,
it has a charge of +1.
NH3, +1, okay?
So two possibilities
for each one,
a charge of +1,
a charge of zero, for the amine.
A charge of zero, a charge
of -1 for the carboxyl.
The amine has its own pKa.
The carboxyl has its own pKa.
Let's examine what happens
during the loss of protons
with this amine.
Let's say we start over here
with all of the protons on.
What would it take for me
to put all the protons on?
It would take a low pH, right?
Today I'm going
to give you a rule
that I want you to understand.
It's going to simplify
things for you,
and you can actually derive
this rule mathematically
or you can memorize the rule.
This is one rule I won't
put on your exam, okay?
We are concerned, in ionization,
about protons being on or off.
Okay?
We're concerned about
them being on or off.
The rule I'm going to
give you is as follows,
If the pH of a solution
is one or more
units below the pKa,
the proton of that group is on.
If the pH of a group,
I'm sorry, the pH of a solution
is more than one unit
below the pKa of a group,
the proton is on that group.
If the pH of a solution
is more than one unit
above the pKa of a group,
the proton is off.
And you're saying,
"What if we have it
somewhere in the middle?"
Well, if we have it
somewhere in the middle,
some molecules will have it on
and some molecules
will have it off.
We can't say one
versus the other.
Okay?
Now that's what this graph
on the screen is showing you,
and I'm going to
describe it to you.
Does anybody want me
to repeat the rule?
Okay.
pH more than one unit
below the pKa, proton on.
pH one or more units
above the pKa, proton off.
One or more below,
one or more above.
Right?
Okay.
Well, here is
a plot that shows us,
it's a little
confusing of a plot,
we'll just first
look at the things
up at the top.
You told me that we had
to have a low pH to start,
if we had all the protons on.
Well, we know we've
got all the protons on,
because there's the proton
on the carboxyl group.
That carboxyl group
has a charge of zero.
And there's that extra
proton on the amine group,
and it has a charge of +1.
What would it take for me to pull
a proton off of this molecule?
An increase in pH.
How much would I have
to increase the pH
to know that I'd
got a proton off?
One or more units above the pKa.
And you're saying,
"Well, which pKa?"
Well, I haven't
given them to you.
The pKa of an alpha carboxyl
group is approximately 2.2.
I'll give you that on an exam.
Okay?
The pKa of an alpha amine
group is approximately 9.5
and I'll give you
that on an exam.
Okay?
So if I wanted to pull off
say this guy has a pKa of 2.2
and this guy has a pKa of 9.5,
which proton's going
to come off first?
The carboxyl, because the
one that's got the lowest pKa.
Which one's the stronger acid,
the carboxyl or the amine?
The carboxyl, because it's
got a lower pKa, right?
Okay.
Basic rules.
Alright.
How high would
I have to raise the pH
to be pretty sure I've got
the proton off of the carboxyl?
3.2, one or more
units above, right?
Alright.
So when I look out
here at about 3.2,
look what's happened.
We've pretty much
gotten this proton off,
and that's what this
graph is showing.
The percentage of pink is
dropping, dropping, dropping,
dropping, until we're
essentially down here.
We've got the proton off.
Instead of having this molecule,
we essentially
have this molecule.
Student: I thought
the proton was on.
Kevin Ahern: It was, until
we started pulling it off.
So we're pulling
the proton off here.
[clears throat]
Excuse me.
Pulling the proton off.
Student: [inaudible]
Kevin Ahern: I'm sorry?
Student: Because
you're above the pKa?
Kevin Ahern: Because I'm
getting above the pKa.
That's right.
So the pH is rising.
Well, what if I was at 2.2?
What if I was at 2.2?
What would I have?
I would have half
and half, right?
Half of this guy and
half of this guy, right?
Notice both these
guys have the NH3+.
Why is that?
Because at 2.2 I'm more than
one pH unit below the pKa
of the amine group.
The amine group proton stays on.
Okay?
If I said,
"Which of these two is the salt
and which of these
two is the acid,"
you would say?
The salt is?
The salt is this guy right here.
The salt will always have
one less proton than the acid.
This has the most protons.
This has lost a proton.
Salt's in the middle.
There's the acid, right?
That's going to
change, over here.
Between these two,
which one's the salt
and which one's the acid?
Salt on the right,
acid in the middle.
So whether something
is a salt or an acid
depends upon which ionization
we're talking about.
Okay?
Well, notice, we keep
adding sodium hydroxide,
we keep adding sodium hydroxide,
the pH keeps changing
and the pH keeps changing.
And all of a sudden, we
start seeing the green
form start to appear.
And where is the green form
going to start to appear?
Well, within about one pH unit,
this thing's a little bit
more exaggerated than mine
but within about one pH unit
of that pKa we start
seeing ionization happening.
By more than one pH
unit above the pKa,
it's essentially all happened.
At a pH of 9.5, you
would expect that
we would have approximately
50% this, 50% this,
and that's exactly what we have.
So this graph is showing
you, in graphic terms,
what's happening
with these molecules.
Notice, and this
always confuses people,
there are three molecules.
There are two ionizations.
pKa's refer to ionizations,
not to molecules.
Okay?
"How come there's
three molecules?
"I've only got two
pKa's in this problem!"
Well, think back
to what I just said,
pKa's refer to ionizations,
not to molecules.
They refer to the
process of this happening,
where this happens.
Okay, questions about that?
No dazed looks?
You guys read your—yeah?
Student: Could you
repeat what you said
about [inaudible].
Kevin Ahern: In this case,
the molecule is the salt,
and this molecule is the acid,
if we're talking about this one.
And, specifically, you're right.
This guy is the thing
that's lost the proton.
This is the thing
that's gained the proton.
But specifically it's
the entire molecule.
Okay?
Okay, good.
Oh, I thought I saw a hand.
Okay, yeah?
Student: So the third
can only ever be an acid?
And the first could
only ever be...
Kevin Ahern:
This can never be an acid.
Student: That could
only ever be a salt.
Kevin Ahern: This can
never give up a proton.
That's right.
Yeah.
Okay.
And this could only
ever be an acid.
That's right.
Alright.
Good.
Very good.
Let's move forward.
Now, first of all, I'm not
going to ask you to memorize
the structures of
the amino acids.
Okay?
If you're in the majors class,
I would expect you to
memorize all 20 structures,
and you would really love me.
But now, because I didn't
make you memorize that,
you really should love me, right?
I need love.
Alright.
But, but, and
there's a big "but",
I said that the R groups
determine the chemistry.
You should know
a bit about the R groups
in terms of categories, alright?
Now, your book, in this edition,
went to something that's
a little different scheme
than most books use, and
I'm going to use their
convention to keep
it simple for you
and so you won't get confused.
If you looking in the
sixth edition of the book,
you're going to see this
is going to look different.
So you might want to refer
to the figures of the seventh
when you're learning
your amino acids.
Okay?
Your book groups amino acids
into several categories.
One category you
see on the screen
are what they call "hydrophobic."
And they're called hydrophobic
because they have R groups
that will not interact
with water very well.
They don't like water very much.
So if I ask you to identify
the hydrophobic amino acids,
I would expect that
you would know this.
You should know the names
of all 20 amino acids, yes.
But you should know, when I say
"hydrophobic amino acid,"
you're going to have these
things pop into your head.
"Oh, there's alanine, there's
leucine, there's proline."
There's also other ones,
and these include
these guys here.
These are all in the
category of hydrophobic.
They have side chains
that really don't interact
very well with water.
Some of these have
big side chains.
Look at tryptophan.
That's the biggest side
chain right there, okay?
That's a big honking molecule
and it doesn't like water.
Now, this hydrophobic
nature of these side chains
of these amino acids have
very important implications
for the location of
amino acids in proteins.
I'll talk about that later
when I talk about tertiary
structure, but I want you
to keep that in mind, okay?
The chemical nature of
the R group will determine
a lot about where these
things are found in proteins.
Okay.
Another group that
your book refers
to are what are called
"polar amino acids."
Polar amino acids
have side chains
that interact with
water very well.
They're either ionic
or they have something
that can hydrogen bond.
Okay?
Cysteine, for example,
can ionize its sulfur, its
sulfhydryl, reasonably easily.
It will interact with
water very favorably.
Threonine, hydroxyl side
chain, hydroxyl group.
When we think "hydroxyl group,"
we think "hydrogen bond,"
therefore, likes water.
So these are polar amino acids.
They tend to be
hydrophilic, liking water.
Student: You said they
have a hydrogen bond
or ionic bond?
Kevin Ahern: They either
hydrogen bond or ionize.
We'll see there's a separate
category that ionize,
and I'd describe
those to you here.
But these guys,
here, if they ionize,
it's usually not
to a large extent,
with the possible
exception of cysteine.
Cysteine actually
ionizes reasonably easily.
Now, here's a group
that I call the positive
what happened there?
call the "positive R groups."
Oh, I've got the
wrong figure linked.
Okay.
Oh, blast it.
Okay.
I'll have to fix that.
This category includes
lysine, arginine and histidine.
What you see here on the
screen is only the ionization
for histidine, unfortunately.
I thought I had all three
of them up there for you,
so I'll fix that.
But these guys all
have side chains
that have amine groups.
They all have side chains
that have amine groups
and therefore, that means
if they have a proton on them
they will be positively charged.
So these guys can have
an R group that definitely
is positively charged.
Now, the R groups
of these guys vary,
but if I said to
you that the R groups
of these amino acid side
chains are on the order of 10
or 11, what would you
say about their charge
at physiological pH?
Are they charged?
Are they uncharged?
Are they positive?
Are they negative?
What are they?
pKa of, let's say, 10.
Physiological pH of 7.
The rule tells you
what about the proton?
Proton on, right?
pH more than one
unit below the pKa.
We're talking about
an amine group.
Proton on, an amine group.
Charge?
+1, right?
So this is the kind of
thing you should be able
to go through in your
head just like that,
just like I'm doing here.
It's not hard.
But when you get the basic rules,
you'll understand these
components of charge, okay?
Now, histidine actually
is an exception.
It has a rather odd pKa,
but I won't talk about
that, at the moment.
There are two amino acids
that ionize very readily,
okay, in their R groups
at physiological pH.
These are the negatively
charged R groups.
They are what we
oftentimes refer to as the
"acidic R groups."
By the way, the last
group, your book calls
"basic R groups."
I tend not to like that term,
but if you want to call
it that, that's fine.
I like it the "positively
charged R groups."
That's the way
I like to think of them.
Alright.
These guys have carboxyls
in their R group,
and they have a pKa
typically of about 4.4.
Again, I'll give
you that on an exam.
You won't need to know that.
And at physiological
pH, if the pH is 7,
and these guys have a pKa of 4.4,
you should run through your head,
"Well, the pH is more than
one unit above the pKa,
"the proton will be off.
"Proton off the carboxyl,
negatively charged."
So these guys are
usually negatively charged
when they're found
in proteins in cells,
or when they're found in
cells alone, either way.
Okay.
Now, you do not need to memorize
the three-letter abbreviation.
You do not need to memorize
the single-letter abbreviation.
You do need to
memorize the names.
Student: Do we have to
spell them correctly?
Kevin Ahern: Do I have
to spell them correctly?
Well, since something
like aspartate really isn't
a very difficult word,
I would say, in general, yes.
I'm not inflexible, except
for graduate students.
Graduate students have to
spell everything precisely.
But we will not have
a very wide latitude
for something that's a simple
name, I'll tell you that.
Aspartate is aspartate.
It's not asperilmarilbartlebate.
Literally, I've had
students on an exam,
and they're like,
"Well, it was close,"
but, you know, no.
You need to know the name.
But, again, it's not
absolute for undergrads,
but it has to be pretty close.
Here's a pKa table
showing you some
of what I've just
described to you.
And though I think their
numbers in some cases
are a little odd,
I'll show it to you.
Here's a terminal
alpha carboxyl, okay?
Approximately 3.1.
Most of them are
actually below that.
There's acidic side
chains, about 4.1.
I told you histidine's
a little odd.
Histidine's about 6.
And, again, you don't need
to memorize these at all.
I will give you
any relevant pKa's
that you need on an exam.
But I just show you these to
show you the various groups.
One that's of
interest is cysteine.
I'll talk a lot about
cysteine this term.
Cysteine ionizes
reasonably readily, okay?
8.3.
There's that stupid
bouncing thing.
And not only does
it ionize readily,
but it turns out that sulfhydryl,
that SH group on the
side chain of cysteine,
is very chemically reactive.
It will readily react with
other sulfhydryls of cysteine,
and make chemical bonds.
And we'll see that this is
an important consideration
in stabilizing the
structure of many proteins.
Tyrosine has an OH that
can go to an O-minus.
It takes a fairly high
pH to get that proton off,
but it can happen.
If I had pH 12,
this tyrosine would
have a charge like this.
Lysine, of course,
the positively charged
polar side chain there.
Arginine is even higher.
Arginine, by the way,
has a resonant structure
and we will just treat it
as if it's a single NH3.
We won't treat it as
which one is which.
It's resonant and it's
possible to go to either one.
So we'll treat it as
if it has a single NH2
that can become an NH3 out there.
Okay.
There's the abbreviations
that you don't need to know.
And I think we're there.
Alright.
Now this figure shows you
very much what I showed you
in that earlier figure.
It's actually not
even quite as nice
as that first figure that I
showed you, the ionization.
This is a simple
amino acid that has
two ionizable groups.
An example might be alanine.
Alanine only has two
groups that can ionize.
The R group of
alanine can't ionize.
If I had aspartic acid up here,
I would have three
ionizations that could occur.
Okay?
Well, this actually
comes up as important
when we think about titration.
So I showed you a titration curve
the other day for acetic acid.
Actually, it was
an acid I made up.
It had a pKa of about 2.5.
I showed it at the end
of class the other day?
And you saw that
single flattening.
And that flattening corresponded
to the buffering region.
That was the place where
that buffer was resisting
the change in pH.
I told you earlier that anything
that has a pKa indicates
it's a weak acid,
and anything that's
a weak acid can be a buffer.
And so amino acids can
be buffers, as well.
And they act as buffers.
So this figure,
it's kind of a dumb scheme,
but I'll show it to you,
this dumb scheme can show us
a little bit about what
a titration plot looks like
for the amino acids.
Okay?
If you look on the problem
set videos that I work,
I'll draw better ones than this
because these tend
to be a little odd.
But here, we can see,
here is the titration
plot for alanine.
Alanine has no R
group that can ionize.
But it does have
an alpha carboxyl
and it has an alpha amine.
Student: Is it a hydrophobic
or [inaudible]?
Kevin Ahern: Alanine
is a hydrophobic,
because it has nothing that
can interact with the water
and the R group has no
side chain that can do that.
The important thing are
the ionizable groups,
the alpha carboxyl
and the alpha amine.
We see the pH rising as we add
and here we're adding,
by the way, NaOH, alright?
We're adding NaOH
to this solution.
We're seeing the pH rise.
Okay?
The rising is going on.
And we see it flatten.
It's flattening right here.
Why is it flattening there?
Which group is being affected?
The alpha carboxyl,
because, again,
we're at a pKa of about 2.2.
Alright?
Within one pH unit of that,
it's going to act like a buffer.
We get out of that
buffering region,
and look what happens.
The pH goes, boing!
The pH is rising rapidly,
even though we've
added very tiny amounts
of sodium hydroxide.
But then we get up
to another region
where there's buffering,
and look what happens.
Well, that's the alpha amine,
and that's going to happen
up around 9.5, thereabouts.
Okay?
Now, in that first
figure that I showed you,
there was a term
that was on there,
that one of you mentioned,
that I didn't mention,
but I'll mention it to you now.
It was called a zwitterion.
And I want to say a
word about a zwitterion.
A zwitterion is a molecule
whose total charge is zero,
total charge is zero.
Okay?
Now, if its total charge is zero,
that means it must have
equal numbers of positive
and negative charges, right?
And we saw in that graph
that I drew for you,
on that ionization earlier,
that we had a molecule
that had a charge
of +1, zero, and -1.
Right?
Let's go back to that, since
I'm referring to it here.
So if we look at that
ionization, here's our molecule.
Here's our amino acid.
It's got a charge of +1.
It loses a proton, it's
got a charge of zero.
It loses another proton,
it's got a charge of -1.
This guy's a zwitterion.
Every amino acid can
exist as a zwitterion.
Now, let's think
about these structures
and let's think about, what
did it take for this guy
to become a zwitterion?
What did it take?
We had to pull that
first proton off, right?
We have sort of a range over
which we have
a zwitterion, right?
But, in fact, when we look
at a pH plot, what we see...
oh, don't start that again.
Okay, blast.
I shouldn't have gone away.
What we see is there's actually,
on the titration plot,
there's a specific
place where it will exist
as a zwitterion.
That first graph gives
us an approximation.
That rule I gave you about
+1, -1 charge, from the pKa?
That is an approximation.
It's all it is.
The titration plot
will allow us to see
exactly where this is.
Now, let's think about this.
Here's a molecule down here.
Let's say we're at pH zero.
What's the status of the
protons on this molecule?
They're all on, right?
We're just like that very
first molecule we had before.
We have a charge of?
Well, zero from the carboxyl
and +1 from the amino,
so we have an
overall charge of +1.
Right?
We take that first proton off,
where's that going
to happen on here?
Where are we going to
get that proton off?
What's it going to take
to get that proton off,
in terms of pH?
We're actually more than
one unit above the pKa.
The where I would wager 25%
of the students on the exam
will make a mistake on
the exam is right here.
They'll say, "Oh, there's
that first proton off,
"at the pKa."
Nooo.
It's only half off there.
To get it off, we have to get
more than one pH unit above.
We actually have to be
right exactly right there.
Okay?
Student: Wait, shouldn't
it happen at 3.4, though?
Kevin Ahern: Hold on, hold on.
Just bear with me.
It happens exactly right there.
Right?
How do I know it's
exactly right there?
Remember, I said this
is an approximation.
When I say it's more
than one unit above,
the proton is off,
I said we could assume that.
It's an approximation.
Student: [inaudible]
Kevin Ahern: The place...
Please, please.
The place where a proton,
where we have a zwitterion,
is a precise place.
There's a precise pH at
which we have a zwitterion.
That's known as the pI.
The pI is the pH at which
the charge of a molecule
is exactly zero.
The pI is the pH at which
a molecule has a charge
of exactly zero.
It gets the approximation
out of that thing
that I gave you before.
Well, how do I calculate
this number right here?
Well, in this case,
it's very simple.
It's the pKa's on either side
of the place where it's zero.
Well, there's only
two pKa's here, right?
If there's only two pKa's,
then it's the sum of
this one, plus this one,
divided by two.
The average of that
will give me the pI.
If this were 2.2
and this were 9.5,
the correct answer on the
exam would be 2.2 plus 9.5,
divided by 2.
I wouldn't even make
you calculate that.
2.2 plus 9.5, divided by 2.
You would have the
pI of this amino acid.
Now, what if I
have three of these?
Do I just average all three?
No, if you use the
rule I just gave you,
the two pKa's on either
side of the place where
the charge is zero.
Now, the TA's are going to
be going through with you,
in class, in the recitations,
pH plots for the amino acids.
And you're going to see how
you decide where the charge
is here, where the charge is
there, where the charge is.
So you can find this magic
place, where the pI is.
That is the two pKa's
on either side of it.
And once you identify that,
then you have the knowledge
to calculate the pI.
It's the average
of those two pKa's.
So something that
has, let's say lysine,
which has a positively charged
R group, it can, in fact,
it'll have three
places of flattening
because it'll have
three pKa values.
I've got to decide which are
the two that are relevant.
Does that make sense?
Now, we'll see later in the term,
actually in about
next week, I think,
where knowledge of pI
gives us an incredibly
powerful tool for
understanding proteins.
So this is not just an
exercise in calculation,
but it's an important concept
for understanding structure
and function of proteins.
Okay, questions about that?
Student: Would the
pI be [inaudible].
Kevin Ahern: The pI
would be right there.
Student: I mean, would the
pI, just cross [unintelligible]
Kevin Ahern: It will, it will.
Now, if you go and
you look at these,
that's whyI don't
like these graphs.
So, I'm going to
show you, for example,
their graph for aspartic acid.
Theirs doesn't draw
this so clearly.
In fact, it should be
flat, up, flat, up, flat.
Right?
But here, because
they're close together,
it sort of runs them together.
So I'm not real fond
of their plots for this.
But when you look
at my videos online,
where I'm working these,
or what the TA's are
going to show you,
you're going to see some
more defined flattenings where
you can have no doubt that
you're in a buffering region.
Student: If you zoomed in,
would that cause you to see it?
Kevin Ahern: To some extent,
but these two pKa's are
pretty close together,
so it makes it a little
bit more complicated.
But you'll see the
TA's will show you that.
Arginine's a little better.
Let me clear that one.
Here's the arginine.
You can see the three here.
Here's one, two.
Here's three.
Okay?
Yes, sir.
Student: So with three pKa's,
which two pKa's
would you average?
Kevin Ahern: You would have
to calculate what the charge
is at each place and assign that.
I'm not going to go
through that here.
The TA's are going to show
you that in the recitations.
But you understand the concept.
You have to get the
two pKa's on either side
of the place where it's zero.
Okay?
Okay.
That's good.
Let me get through here.
So I haven't said much
about primary structure.
So I want to spend at least
a couple of minutes
talking about that,
and then we will,
I think finish with a song.
[scattered chuckling]
Just something to keep you going.
I've been doing all this
talking about ionization,
but I haven't said a word
about primary structure,
and that's how
I started the lecture.
Why did I do that?
Well, we'll see that the
charge of these amino acids
affect the secondary,
the tertiary,
the quaternary
structure of a protein.
They affect all three
of those structures.
They don't affect the
primary structure, however.
The primary structure
is essential, however,
for all of the other
structures of a protein.
Underline that.
The primary structure
of a protein is essential
for all the other.
It determines what the
secondary structure will be.
It determines what the
tertiary structure will be.
It determines what the
quaternary structure will be.
The primary structure of
proteins relates to the sequence
of amino acids, joined
one to the other.
Lysine.
Arginine.
Glutamic acid.
Valine.
That's a sequence,
one to the next.
And the sequence happens
because the amino acids
are joined together
by peptide bonds.
You see a peptide
bond being formed here.
We see there's the peptide bond.
It goes between the alpha
carboxyl of one amino acid
and the alpha amine
of the next one.
There's my R group
of the first one.
There's my R group
of the second one.
We'll say more
about this next time.
We see in this orientation
that this guy has an end.
This is known as the "amino end,"
because there's the alpha amino
and it's not bound to anything.
And there's the alpha carboxyl,
there's a carboxyl end,
because there's an alpha carboxyl
and it's not bound to anything.
However, this alpha amine
and this alpha carboxyl are
tied up in a peptide bond.
All proteins will have
one free alpha amino
and one free alpha carboxyl.
All the other alpha amines
and all the other alpha carboxyls
will be joined in peptide bonds.
Okay?
So I can always tell which is
the amino end or the protein,
and which is the
carboxyl end of a protein.
Okay, you guys have been patient.
Let me see if I can
get the audio going.
I think instead of us...
you can sing along,
but I've actually got somebody
who's going to sing for us.
And I hope this works,
so be patient for me.
He's a way better
singer than I am,
so you may like that.
And, let's hear it!
[music, "Alphabet Song" tune]
Sing along!
Lyrics: Lysine, arginine and his
basic ones you should not miss.
Ala, leu, val, ile, and
met, fill the aliphatic set.
Proline bends and cys has "s."
Glycine's "R" is the smallest.
Then there's trp and tyr and
phe structured aromatically.
Asp and glu's side chains of R
say to protons "au revoir."
Glutamine, asparagine
bear carboxamide amines.
Threonine and tiny ser have
hydroxyl groups to share.
These twen-TY amino
A's, can combine...
