at Oregon State University.

Kevin Ahern: So we're moving along nicely with the schedule.

And though we don't have to stay on it exactly,

we've been on it pretty good.

I've been pleased with the interactions

and also pleased with the questions I'm getting,

both in class and out of class.

So you guys seem to be engaging in this material

and that's a very good indicator of success.

So if you have questions, please feel free.

Come see me. Come see the TA's.

And we're here to help in any way that we can.

I only have one tiny little thing to say today

regarding the last of protein structure.

It's actually sort of an anecdote more than anything else.

And then I want to talk about techniques

for characterizing and/or purifying proteins.

One of the things that biochemists spend

a tremendous amount of time doing is just that: isolating,

characterizing, understanding proteins, enzymes, etc.

And so what you've learned so far about structure of proteins,

you will discover will be useful as tools

for learning how to isolate them.

And so I'll spend some time talking about that today

and also on Monday.

The anecdotal thing I wanted to mention to you

is the very last item on the protein structure page,

and it's actually this right here.

I've mentioned hydroxyproline to you already

and I want to reiterate something here.

Now, if you recall, I said that there are 20 amino acids

that we find commonly in proteins,

but we find modified amino acids in proteins.

And the point that I want to emphasize is that those

modified amino acids that we see happen post-translationally,

meaning that the modifications occur

after the amino acid is built into the protein.

So, in the case of hydroxyproline, for example, I gave you,

I showed you or described to you how Vitamin C

was involved in that reaction that modified the proline.

That happened after the proline

had been built into the protein.

The same is true of all the other things there are here.

Carboxyglutamate is an important modification,

as we will see,

that occurs as an important consideration in blood clotting.

Carbohydrate-asparagine adduct,

where we see, in this case,

addition of a carbohydrate to an asparagine residue,

this is really imporant in the production synthesis

of glycoproteins that we'll talk a little bit about later.

Phosphoserine, Phosphorylation is something

that you're going to hear a lot about later in the term

because phosphorylation is a means of controlling

or signaling through proteins.

And it's a very, very important mechanism for us to understand.

It's specifically phosphorylation

that I want to address briefly at the moment.

And that is that phosphorylation of amino acids

has to occur on side chains and side chains

that have hydroxyl groups.

So the three amino acid side chains that have hydroxyl groups,

of course, are the tyrosine, serine and threonine.

These are the three amino acids that get phosphorylated

or can be phosphorylated.

And we'll see a bit of a pattern

to how that phosphorylation occurs.

Not surprisingly, you might think,

well, why do these have such big effects?

You saw a big effect with hydroxyproline

because it was a part of that important structural

consideration for making a strong collagen.

In the case of phosphorylation,

what we're doing is we're converting,

excuse me, we're converting a side chain

from being hydrophilic to actually being ionic.

And so, in essence, what we've done is we've changed it from,

say, a partial charge to a fully negative charge.

In this case, we see two minus groups there.

Now, based on what I told you so far about protein structure,

you might imagine that changing the charge

of a specific location of a protein

might have structural considerations for that protein.

Imagine that previously we had a negative charge,

let's say a glutamic acid residue,

that was close to this proline

before we put the phosphate on there.

When we put the phosphate on there,

here's this negative charge before

that didn't really have that much interaction with the OH,

but now there's two minus charges over here.

What's going to happen?

Well, of course they're going to repel,

and when they repel,

that's going to change the configuration.

It's going to change the shape of that protein slightly.

And, as we will see,

and I've mentioned previously,

changes in the shape of proteins

can have some dramatic effects on the action of those proteins,

and we're going to talk more about those

as we get further along.

So those are some things that are other modifications

that can happen to proteins.

But I want you to be aware

that virtually any time you see a modified amino acid

in a protein it is because it has happened

after the amino acid has been put into the protein.

Okay, so that's the last of what I want to say

about general considerations of protein structure.

Now I'd like to turn our attention to characterizing proteins.

The first part of the characterization

I'll talk about is actually purification.

And purification isn't a spiritual purification,

but it's actually a physical purification.

I'll tell you a brief story.

When I was working in my very first lab after I had graduated,

I worked in a laboratory where we did HPLC,

and we had to have very pure solvents.

And I was very impressed by this notion

of purification that happens in there,

the need for purity in all biochemical materials.

And so I was very, very impressed

with these solvents that we used,

and we got them from this company that had purified stuff.

So I remember writing a letter to the companyótongue in cheek,

of courseósaying that, you know,

we found that not only were their solvents very pure,

but we had to do a spiritual purification

of these solvents before we used them, as well.

Of course, I wrote this as if it were completely serious,

sent it to the president of the company,

and, to my delight, I got this letter back

from the president of the company

congratulating me on describing for him

a new way of purifying his solvents that he could use for HPLC.

It was a good exchange.

So purification really had a big impact on me

as a very young biochemist.

Purification is important.

When we want to characterize,

let's say, a protein or an enzyme,

we need to have it isolated away from everything else.

When we try to understand an enzymatic reaction,

for example, we say, "Okay, well, ìI'm interested in this enzyme.

ìI'm interested in the reaction

that this enzyme catalyzes."

If I only have the soup of the cell, that is,

the cytoplasm of the cell that contains this,

I not only have that one enzyme that I'm interested in,

but I have several thousand other enzymes in there.

So it's important for me to understand what

this enzyme does that I be able to purify this

enzyme away from all those other proteins.

And so understanding how to purify one

protein apart from others is a very,

very important consideration in biochemistry.

Well, there are several techniques that

we use in order to do this,

and I'm going to go through and sort of describe

a few of the basic ones to you and then show you some

of the applications of these technologies.

You can't walk into a biochemistry

lab without finding a centrifuge.

It's almost impossible to do that and

that's because the use of centrifugal force as a means

of separating molecules on the basis of their size is a very,

very valuable tool.

Not surprisingly,

different things can be spun down.

We talk about "spinning them down."

That is, will they precipitate out of solution or

will they move to the bottom of the tube?

The function by which that,

by which they occur,

is a function of their size and the speed

with which we spin things.

So the largest things,

of course, as you might imagine,

spin most easily to the bottom.

So if I take and I'm interested in studying an enzyme in

E. coli cells, I can take a batch of E. coli cells

and I could use a fairly light

centrifugation and spin,

and those cells would come to the bottom of that tube.

Let's say I took that pellet which we get out of that,

and I'm interested in, not just the cells,

obviously, because I'm interested in

the enzymes that's inside,

I can use some techniques to bust 'em open.

I might use sonic waves to do that.

I might use enzymes to do that.

I might use mechanical agitation to do that.

It doesn't really matter the means I use.

But when I do that,

I basically open up the contents

of the cell and the insides spill out.

Those insides are going to have some things in them.

And, in addition,

I'm going to have some cell walls that are sitting there,

that now are empty of their contents.

I could spin those down again.

And if I did that,

I would basically have done my first separation.

I would have, on the one hand, the pellet,

which would contain the very big things,

like those cell walls,

and I would have the liquid component,

which would be the cytoplasmic material

that I was interested in.

I could take that cytoplasmic material.

I could do various centrifugations on it,

if I chose to.

And I could separate them on the basis

of the size of those complexes that are in there.

Now, we don't need to memorize numbers

or anything like that,

but I do want you to understand that centrifugation

allows us to do a sort of a rough separation based on size,

a very rough separation.

The stronger the centrifugal force,

the more things I'm going to pellet,

I'm going to drive to the bottom of the tube.

And there's a lot of different techniques

involved in centrifugation that allows me to purify things.

Now, centrifugation alone will notóunderline

"not"ógive me pure material.

So it's used mainly as a means of what

I would describe as fractionating.

When we fractionate things,

we break them into smaller pieces and

then we work with those pieces to do things of interest to us.

Let's imagine, for a moment, we've got two possibilities.

I took these E. coli cells

that I was describing to you,

and I'm interested in understanding a particular protein.

The first question I would ask is,

"Well, where's this protein?"

Is this protein in the cytoplasm?

Or is this protein embedded in the cell membrane?

Because both of those are possible.

The beauty of this is,

if I've fractionated it in this way,

I've got one fraction that has only things

in the cell membrane and I have another fraction

that has only things that's in the cytoplasm.

Then I can subdivide those further,

and that's some of the other things