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JOANNE STUBBE: We talked last time

about kinetics, steady-state kinetics,

pre-steady-state kinetics, how you design the experiments,

what kinds of information you can get out

of each experimental design.

And we introduced all of that material.

And today, what I want to do is come back to the model.

You saw it at the very beginning,

and you've seen it in a lecture.

And specifically, where did this model come from?

That's what we're going to focus on.

OK, and so in order to be able to understand this model,

you have to design assays.

And you're going to see over and over again

over the course of the semester figuring out

how to design an assay, in this case, isn't so hard,

but in many cases is really tough.

And that's the key to being able to get kinetic information

is designing the assay.

So if you look here, today, we're

going to be looking at GTP is hydrolyzed.

So you need to think about, as a chemist, how

you could study that reaction.

How would you look at starting material?

How would you look at product as a function

of time, which is what we were talking about last time?

And we're going to talk about that first.

We're going to talk about use of radioisotopes first.

And we've already been talking about radioisotopes in class

the last couple of lectures.

So we decided to focus most of our energy

now on radioisotopes.

And then the second kind of probe you're going to see

is a fluorescent probe.

We're going to use fluorescent probes over and over again.

And the details of the fluorescent probes

and how they work isn't going to come

in until the last recitation, which is recitation 13.

So from the point of view of thinking

about Rodnina's paper, what you need

to think about is, if you have a probe,

and you stick it in a different environment, it changes.

And you can watch it change, OK, without looking at the details.

But that's something you do need to think about,

but we're not going to talk about that.

OK, so we have a way of monitoring potentially GTPase.

And we'll talk about that today.

What other reaction can we monitor in here?

We can monitor formation of the polypeptide chain.

And so that's the other thing.

And both of these chemical transformations

use radioactivity.

OK, so that's where we're going to focus on it initially.

And then hopefully-- how many of you

went back and reread the paper for this week from last week?

Did any of you go back and reread it?

OK, so I think it's good.

I just think, you know, every time I read a paper--

I read a paper.

Sometimes, I've read it 10, 15 times over the course

of my career.

And as I learn more and think about things differently,

I keep seeing new things.

And this paper is just packed full of information.

So I could say you could read it another 10 times,

and you'd still keep learning stuff out of reading it.

And in the very beginning, that's

what we're trying to teach you to do.

What do you look at in the paper to learn

how to critically evaluate what's

being presented in the model, which

is maybe what you're going to build your research program on?

Somebody else's data, is it correct?

Is it not correct?

OK, so we're going to use radioactivity.

I'm going to start there.

And then to look at these first few steps,

which are binding steps, that's where

we're going to look at the fluorescent probe.

And there were three different kinds

of experiments that were described in this paper--

looking at the rates of the reactions as a function

of the concentration of the ribosome--

you need to think about why they looked at a concentration

dependence--

measuring fluorescence changes, and then they

used non-hydrolyzable GTP analog.

Why did they use that?

Do you remember what the non-hydrolyzable GDP

analog was?

So where's the n?

AUDIENCE: It's between beta and gamma.

JOANNE STUBBE: So it's non-hydrolyzable.

It is hydrolyzable, but not under

the experimental conditions.

So what does it do?

Why would you want to use something

like that to get information about the first few steps?

AUDIENCE: It's along the reaction continuum.

JOANNE STUBBE: Yeah, so you don't

let the reaction continue.

So what that does, if it's working correctly,

is it puts a block here.

And then you can potentially monitor what's going on here.

And from the data that you looked at,

it's not really so clear what was going on there

unless you went back and read the preceding paper.

So there had been a decade worth of experiments on this system

before this paper came out summarizing

the conclusions about what they are thinking about fidelity.

OK, so what we're going to do is talk about radioactivity.

And our objective is simply-- and we'll come back to this

at the very end--

is to use all this experimental data, the concentration

dependence, the radioactive isotope experiments,

the stop flow fluorescence experiments,

and try to come up with a model that

can explain all of the data.

OK, so you make some measurement.

What you're measuring is some k apparent.

And that's usually a first-order rate constant

because it's happening on the enzyme.

OK, so you measure these numbers.

Well, what do they mean?

You don't know what they mean.

And why don't you know what they mean?

Because the kinetic mechanism is so complicated.

You saw that with the steady-state analysis

of km and kcat last time.

So in the end, though, if you come up with a model,

and it can explain all the data because you've

done many, many experiments, it can

be quite informative about the question we're

focused on is specificity.

How do you distinguish between phenylalanine and leucine

and proofreading?

How do you decide whether you're going

to form the right peptide bond or the incorrectly charged tRNA

is going to dissociate?

OK, so that's what you want to come out with.

You want to look at the ratio of these rate constants

and the ratio k3 to k minus 2.

And when you look at the experimental data, which we'll

look at the end today, it should make sense to you

in terms of this model.

OK, but let's put it this way.

In most cases, you don't come out with a unique model.

It's a working hypothesis that people for the next 15 years,

if it's an interesting problem, will take pot shots at

to try to understand in more detail what's really going on.

OK, so what I want to do is talk about two methods,

but the focus probably won't get very far

in terms of the second one.

But today, we're going to look at radioisotopes and how

you use that to do the assay for GDP hydrolysis

and peptide bond formation.

OK, so what is an isotope?

OK, so how many of you guys have actually

worked with radioisotopes?

Any of you?

No, OK, so you know, maybe they don't use this anymore.

Biochemists for the decades have used isotopes.

Every paper I read has isotopes in it.

But you know, I'm old school.

So maybe people don't use it.

But I think the power of it is its sensitivity.

I'm going to show you that today.

And the other power of it is that you

have no perturbation of your system

and there are almost no probes like that.

You're sticking on green fluorescent protein.

Well, what does it do to the whole rest of the protein?

You have to perturb to see, but radioisotopes

have minimal perturbation.

So it's still a very important probe,

but it probably depends on what kinds of questions

you're focused on.

So what is an isotope?

So an isotope is atoms with the same number of protons

and a different number of neutrons.

That's called the mass number.

So what you have here for carbon,

which is one of the common isotopes you guys will be using

if you do any kind of biochemistry,

we have C-12, C-13, and C-14.

OK, and so this is the atomic number, which

is the number of protons.

OK, so the only difference between these guys

is a neutron or two neutrons.

OK, so there's minimal difference.

And so what are the isotopes that you see used in biology?

So we've already seen many of these in this paper,

but we've also talked about some of them in class today

and in the preceding class.

So we're going to be using over the course of the semester

isotopes of hydrogen. Why?

Because if you look at your metabolic pathways,

you're always cleaving carbon-hydrogen bonds.

OK, so this isotope becomes incredibly important.

C-12, C-13, anybody know where you use C-13?

AUDIENCE: In NMR.

JOANNE STUBBE: NMR, so if you're working for Mei Hong,

you might be doing isotopic labeling using C-13.

If you're doing any kind of metabolic label chasing,

you're going to see the radioisotope is, which is what

we're talking about, is C-14.

Working

So you see often, all the time, you see nitrogen and oxygen.

And oxygen has three isotopes.

Nitrogen has two.

None of them are radioactive.

OK, so you're never going to be using the methods

we're describing today.

But frequently, in NMR again, you

might replace N-14 with an N-15.

And today, we will see that we're

using isotopes of phosphorus.

What about phosphorus-31?

Where do you see that?

Have you thought about this?

Maybe you have, and maybe you haven't.

Phosphorus-31 versus phosphorus-32,

what's the normal abundance isotope of phosphorus?

31, so phosphorus-31 has a nuclear spin of a 1/2.

So you frequently use that as well in NMR.

And P-32 is used--

it's radioactive and is used in today's experiments.

OK, so this is something that in the back of your mind

you should think about.

What are stable versus unstable isotopes?

And what we're talking about today is unstable isotopes.

So what I want to do is we're not

going to go into this in a lot of detail,

but I want to describe the things I think

you need to think about if you're ever going

to use radioactivity and how you make measurements,

quantitative measurements.

And so we're going to be looking at a radioisotope.

And what do we know about radioisotopes?

They're unstable.