at Oregon State University.

Ahern: We are moving rapidly through stuff

and I finally sat down and looked at when the exam is

and the exam is this Friday,

so I guess...

[classroom chatter]

Ahern: It's not?

The exam is a week from Monday.

I'm guessing you guys wouldn't be ready for it this Friday?

No?

There will be fewer things on it.

Think about that.

A smaller nice, little tidy exam.

Student: And the next will destroy us.

Ahern: And the next will kill you.

[laughs]

Or you will kill me, I don't know.

The second exam is not cumulative.

The final is cumulative.

I just have a couple fairly minor things

I want to say about characterizing proteins

and I do that in recognition of the fact

this is not a biophysics class.

The things that are there are really more biophysical in nature

than they are biochemical in nature.

I want you to be aware of what we can do

with certain techniques.

One of the techniques,

last time I talked about MALDI-TOF and MALDI-TOF

is a fantastic method as I say that I realize

I didn't post that figure for it.

I will get that figure posted showing you the set up

for a MALDI-TOF instrument

and you can hopefully see it better than my words can describe.

I will get that posted for you.

What I want to talk about today

are a couple other very powerful techniques

that come to us from biophysics.

I'm not going to go in depth

but you should know the basics of them

and you should be able to understand why they're useful for us.

The two techniques I want to talk about

are X-Ray crystallography and nuclear magnetic resonance.

You've probably had some exposure

at least with nuclear magnetic resonance,

I'm guessing in your organic chemistry class.

Has anyone had exposure to X-Ray crystallography before?

Little bit, okay.

I'll start with X-Ray crystallography.

X-Ray crystallography,

both these techniques, by the way,

are extraordinarily useful for helping us to understand

relative positions of nuclei in space.

I always tell the story when I give a tour of our facilities

in ALS in Biochemstry and biophysics to students

that using the tools of X-Ray crystallography

and nuclear magnetic resonance,

biophysicists can determine the position

in three dimensional space of every atom

that might exist in an enzyme that has 10,000 or 50,000 atoms.

That's a really remarkable thing

because knowledge about structure

leads to understanding about function.

We've heard structure function before.

When we think about how drugs get designed,

the design of drugs is happening increasingly

as a result of the molecular knowledge of the structure

of the proteins that they're targeting.

If I know the position or I know the structure

of the active site of the enzyme,

the place where the reaction is catalyzed,

I know the dimensions of the molecule

I need to design to plug up that enzyme.

That knowledge of structure is really valuable for us

to have for whatever purpose.

And there are purposes aside from designing drugs as well.

X-Ray crystallography arises from the fact

that X-rays get diffracted

which means they get bent when they encounter electron clouds.

That diffraction process is depicted here.

To do X-Ray crystallography,

one has to have first of all a crystal

and a crystal is a as you guys see crystals

you don't think at a molecular level what a crystal is,

but a crystal is a perfectly packed homogenous molecule

that has a regular repeat to it.

That is all the molecules in there are the same composition

and they're organized in a regular fashion.

That regular repeat is what gives rise to the crystal itself.

The process of making a crystal for a lot of X-ray crystal analyses

is actually the thing that takes the longest.

A lot of different, there's no one formula for making a crystal.

Different proteins crystallize in different to way.

Suffice to say that making the crystal,

which is already shown right here,

can be a very and time consuming step

and a frustrating step in the process.

Once one has a crystal,

one can take they crystal

and put in the path of an X-ray beam.

That X-ray beam will have its rays diffracted again

according to the electron clouds that it encounters.

The importance of the regularity of the molecules

in the crystal are very important

because those really add up

and give us the diffraction patterns that we see.

Now interpreting the diffraction patterns

obviously isn't a thing we're going to do here,

but sufficed to say that diffraction gives,

this is what a diffraction pattern

of a given crystal might look like and we say wow,

there are some spots.

These are the spots correspond places

where X-rays got diffracted to.

So a biophysicist can take information

from a diffraction pattern and work backwards,

ultimately to determine where the individual electron clouds

where it caused the diffraction pattern to exist.

So X-Ray crystallography is extraordinarily powerful

because it does give us three dimensional information about

the position and orientation of electron clouds

and a working map might look something like what you see right here.

Here are patterns of electron clouds and then within there

we decide what are the individual atoms that correspond to there,

you see different carbons and hydrogues and oxygens

and so fourth scattered through there.

And interpreting these patterns can again be a very time

consuming process it's very computationally intensive.

The result is at the end of this

that one has that structural information that's very useful.

Again, that's just a very cursory

description of X-Ray crystallography.

X-Ray crystallography has its advantages

and it has its disadvantages.

The advantage of X-Ray crystallography is if you get crystals,

then you can determine these positions very nicely.

Sometimes you can't always get crystals and that's one limitation.

And the other is crystals may or may not correspond to the natural,

whatever that means, shape of the solution.

So we think about the enzymes we have in our cells,

most of them are dissolved the in water of the cytoplasm.

So when I'm making a crystal,

I'm basically taking it out of the solution

so one thought is well is this reflecting

the actual structure it has when it's in the solution?

So to partly address some of those concerns,

this additional technology of nuclear magnetic resonance

is very useful because nuclear magnetic resonance analysis

allows one to determine molecular structures in aqueous solution.

They work in different ways and nuclear magnetic resonance

relies on the fact that certain nuclei have spins

that are characteristic of them and those spins

can be altered in the presence of an electromagnetic field.

So understanding the energies it takes to alter those spins

of given nuclei for example protons.

Protons are very commonly used in analysis

to understand the changes in those spin gives us

some knowledge about the structure.

I'll just show you a very brief example here.

Here's a nucleus that has a characteristic spin.

There are two possible spins that can exist.

One has a slightly higher energy than the other

and the difference between that energy

is what is excited using the electromagnetic radiation.

It turns out the different nuclei have different spins

corresponding to the electronic environment

in which they find themselves.

This depicts the nuclear magnetic resonance signal

of a very simple molecule.

This is ethanol.

We can see that ethanol has three different kinds of protons in it.

It has methyl protons that are farthest off in the end.

It has methylene protons in the middle here

and it has hydroxyl on the end.

These give rise to characteristic signals.

These signals have known positions,

we know where hydroxyl protons arise,

we know where methyl protons arise, etc.

And so we can examine the spectrum that comes from this.

The spectrum is called the chemical shift which I won't go in to.

This molecule has some hydroxyl,

it has methylene, it has methyl protons.

As you can imagine for a molecule like a protein,

it's not not nearly as this.

We might get very complicated spectra

and in fact we do get complicated spectra.

So this is a little bit more challenging to interpret.

We're not going to do that obviously here.

But sufficed to say that analysis of nuclear magnetic spectra

does allow, ultimately a scientist,

to allow which signal corresponds

to which groups inside the protein.

Now understanding the different kinds in this case of protons

that exist in a protein is useful information

but one of the things we're interested

in as biochemists is how do proteins fold.

Because remember that folding really gives the protein

its characteristic shapes so we would like to get more information

because the knowledge of different protons we have

in the protein really isn't sufficient enough to tell us structure.

One of the techniques done with that is an enhancement

as it were of nuclear magnetic analysis.

It's called the nuclear overhouser effect

and it arises from the fact that,

this clip doesn't work,

it arises from the fact that nuclei,

when they interact with each other,

also have effects.

In this case, here are two protons that,

as a result of folding,

have been brought into close proximity.

And if they're brought into close enough proximity

they actually do affect the signal of the other one.

This requires a very sophisticated analysis called 2D

and that's obviously a lot more complicated

than 2D gel electrophoresis I'm not going to go into that.

But sufficed to say that with this type of an analysis,

one generates some even more interesting spectra

but this information that we see here now

tells us not only what kinds of protons that we have

but how close those protons are to each other.

That's very, very useful when one goes to trying to determine

the overall structure of a protein molecule.

So that can be very, very useful.

Commonly these two techniques are used in combination

with each other to help elucidate molecular structure.

There is a biophysics course in 5 minutes.

How's that?

Questions or comments about that?

Let's get away from biophysics and talk about,

this is the lecture I'm going to give today

is most of the most popular lectures

I give throughout the entire term.

It's the lecture on hemoglobin.

And hemoglobin is, I hope to convince you

by the end of the lecture on Friday,

one of the most magical molecules in our body.

It is absolutely incredible the abilities

that are built in to the structure of this protein.

I start talking not about hemoglobin

but about a related protein called myoglobin

and I introduced myoglobin to you before

as a protein related to hemoglobin.

It's found in our muscle cells primarily

and there it serves the function of storing oxygen.

It's a very good way to store oxygen.

Hemoglobin is very good at delivering,

that is picking up and dropping off oxygen.