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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.

  • OK, and depending on which atoms they are,

  • they have different stabilities.

  • And they decay spontaneously into some new configuration.

  • They have a nuclear decay spontaneously into a new state.

  • And during this process, during this decay,

  • they emit ionizing radiation.

  • They emit energy.

  • So during this process--

  • so this is the whole thing