#03 Biochemistry Amino Acids Lecture for Kevin Ahern's BB 450/550

Uploaded by oharow on 01.10.2011

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at Oregon State University.
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?
Kevin Ahern: Working through the problems?
Everybody's got a smile on their face.
That's good.
So, I'm happy to see that.
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.
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.
[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.
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.
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.
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.
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.
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.
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.
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...
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?
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.
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.
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.
So if we understand the R groups,
we understand the chemistry of each of the amino acids.
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.
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.
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.
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?
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.
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.
The pKa of an alpha amine group is approximately 9.5
and I'll give you that on an exam.
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?
Basic rules.
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?
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.
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.
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.
"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, 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.
And this could only ever be an acid.
That's right.
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.
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.
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.
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.
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.
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.
Oh, blast it.
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.
+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.
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.
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?
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.
There's the abbreviations that you don't need to know.
And I think we're there.
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.
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.
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.
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.
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.
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.
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.
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.
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."
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.
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.
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.
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.
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.
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.
Glutamic acid.
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.
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...