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ELIZABETH NOLAN: Welcome to the class.
We're going to discuss the themes that
are going to basically permeate every topic
and module we'll talk about here.
And one of the central themes of this class
is that we're interested in studying
the cellular processes of life at a molecular level, right?
So as biochemists and chemists, we're
interested in this level of understanding.
And what we see here is a cartoon depiction of the cell.
And we see that there's many types of biomolecules
in this environment.
So what are our core themes for this year?
First, we believe that life must be studied on a molecular level
to truly understand it.
And so we need to think about the cellular environment,
both on a macroscopic scale, and on the molecular level.
And this environment is complex, and it always
needs to be considered, right?
So as experimentalists in biochemistry, often
we're doing experiments in aqueous buffer
with proteins or some other biomolecule.
How does that relate to a context like this one
here where the environment is very different and much more
complex?
Something we'll see, especially in the first half,
the first four modules of this course,
is that in cells, complex processes
are carried out by macromolecular
machines and elaborate systems.
And these systems are fascinating.
You'll see that we know a lot, but as we learn more,
there's more and more questions that come up,
and more questions we need to address with that.
In addition to these macromolecular machines,
some additional themes for this course
involve homeostasis and signaling.
And these will be especially emphasized
in the second half of the course when Professor Stubbe takes
over there.
So how do we think about homeostasis and signaling
in these contexts?
Something that will come up again and again
is how, basically, understanding cellular processes
at a molecular level, or the molecular features,
can help explain mechanisms of human disease,
as well as therapeutics.
So an example we'll see in the early part of this lecture
involves the ribosome.
So many antibiotics target the ribosome.
And by understanding ribosome structure and function,
we can understand how these small molecule therapeutics
work.
Another example involves the proteasome
which we'll hear about in the second half of the course.
So there's therapeutics that target the proteasome,
for instance, for cancer.
And cholesterol biosynthesis will come up,
and how does our understanding of cholesterol biosynthesis
lead to ways to treat coronary disease?
Something that JoAnne and I really
like to think about day-to-day and convey
to you in this course is the importance
of experimental design, and choice of methods.
So as scientists and experimentalists,
how do we think about designing an experiment,
because that design is really critical to the outcome,
and what we can make of the data?
And so throughout lectures and recitations,
things to keep in mind, and that we'll reiterate,
are that all techniques have inherent strengths
and limitations.
And so it's something we all need
to keep in mind when we analyze data
and think about how an experiment was done.
And these systems we're going to look at in 5.08
are very complex.
And what that means is that many different types
of experimental method are needed
in order to answer complex--
and sometimes not so complex--
questions.
So one method alone just often isn't enough.
We need insights from many different techniques and types
of expertise.
And so we look forward to informing you about different
types of methods-- whether they be established and quite old
or new--
that are important today.
And as I alluded to before, something
we have to keep in mind when doing biochemistry in the lab
is that the test tube is very different from the cell.
These environments are vastly different,
and so we always need to think about how to relate data back
to a cellular or physiological context.
If you measure a dissociation constant of one micromolar,
what does that mean in a cell versus one picomolar,
for instance.
Another point to make is that the hypothesis
is a moving target.
So we have the hypothesis, experiments
are designed to test this hypothesis,
and there's some outcome.
Maybe that supports the hypothesis, maybe not.
Or maybe there's some new insight
from a related field that really changes
how we think about something.
So in many cases we're integrating
data and insights that are quite new, and Professor Stubbe
and I won't have all of the answers.
And so that type of uncertainty is something
that we aim for you all to gain some level of comfort with.
So there's many complexities in primary data, often
uncertainties.
And that's just an aspect of this course.
And scientists, it's something we grapple
with every day in our own work.
So we're introducing that to you here.
And along those lines, just keep in mind, we know so much.
And I think it's amazing, and-- if I step back and think
about this for some of the systems
we'll see-- actually overwhelming.
And it's really due to dedicated efforts of many, many people
over many, many years.
But with that said, there are so many remaining unanswered
questions, and we hope that you'll find inspiration
in some of these questions as looking forward
within this field.
There.
OK, so what about the cell and macromolecular crowding?
Just to emphasize this point a bit more,
here we have an E. coli.
OK, so E. coli are laboratory workhorses for biochemists.
They're fascinating, I love E. coli.
But I just show you this simple E. coli cartoon
and this depiction here to emphasize how crowded
the cellular environment is.
So we have an equal E. coli of about two microns long,
and maybe half a micron wide, a volume of about a femtolitre.
And if we think about the E. coli genome for a minute,
it encodes about 4,000 proteins.
That's a lot of proteins.
And if we think about one E. coli cell of this small size,
can just ask a simple question, how many ribosomes are there?
So we all know the ribosomes are needed
for polypeptide biosynthesis.
How many ribosomes are packaged in one E. coli?
Any guess?
So, 10, 100, a million.
AUDIENCE: Order of 1,000?
ELIZABETH NOLAN: Pardon?
AUDIENCE: Order of like, 1,000?
ELIZABETH NOLAN: Yeah, let's say 1,000 times 15 or 20.
So there's about 15,000 to 20,000 ribosomes
in one E. coli cell.
And as we'll see in Friday's lecture,
the ribosome is very large.
How did they all fit?
And there's not only the ribosomes,
but there's many, many other players,
just as noted here in this cartoon.
So you can think about what does that mean
in terms of concentrations.
We'll bring up concentrations of biomolecules in the cell
throughout this course, and what does it
mean having them packaged together so much here?
So, very different than the test tube.
Our goals, some of which I think have
been communicated by me so far.
But just to emphasize, we're interested
in these macromolecular machines and chemical
processes responsible for life.
We hope by the end of this course,
everyone gains an appreciation for the complexity of life,
and our current understanding of the topics
we present to you this spring.
There's close links between basic fundamental research
and medicine, and technology development as well.
Understanding the experimental basis
for understanding, methods and hypotheses.
And what we think is something that we hope to achieve,
and that you can bring to other places
after this course is really to be able to knowledgeably
and critically evaluate methods and results, especially
primary data.
And we also hope that we convince you
that biological chemistry is really
thought provoking and fun, and hope
you all think that right now as well.
So what are the actual topics we're going to cover?
We organized this course into modules,
and these modules are listed here.
And different modules will have different numbers
of lectures dedicated to them.
But where we'll go between now and spring break--
I'll present to you during these weeks--
is that we're going to focus on the lifecycle of a protein
for the first three modules.
And many of you are familiar with aspects of this.
We're going to present these topics,
I think, a bit differently than what you've seen before.
Again, very much from the standpoint
of experimental methods and hypothesis testing.
So we'll cover protein synthesis,
doing a careful case study of the ribosome.
We'll continue with protein folding.
So asking the question, after the ribosome
synthesizes a polypeptide chain, how does that polypeptide
assemble into its native form?
What happens when proteins are misfolded?
And then we'll move into protein degradation,
and we'll look at proteases and machines that are involved
in proteolytic degradation.
And where we'll close the first half
is with module four, which is on synthases,
or often called assembly-line enzymology.
And this is a different type of template-driven polymerization
that's involved in the synthesis of natural products.
And then after spring break, Professor Stubbe
will take over, and the focus will
be on cellular processes that involve homeostasis,
metabolism, and signaling.
And so these topics will involve cholesterol biosynthesis,
and a type of molecule called terpene.
And so a third way to make a carbon-carbon bond
will be introduced in this section.
So you've heard about Claisen and Aldol condensations
in prior biochemistry courses, this will be another route.
And then, we both love metals and biology,
so there's a whole field of bioinorganic chemistry,
and it will be introduced to you here with iron homeostasis
as a case study.
And moving from here, and something quite related,
involves reactive oxygen species.
So I'm sure you've all heard about these somewhere, maybe
in the news, maybe from your lab work.
What are these reactive oxygen species?
Are they all reactive?
What kind of chemistry do they do in a cell?
How do we study that here?
And then, of course, we'll close with a section,
a module on nucleotide and deoxynucleotide metabolism--
excuse me-- as well as regulation.
And then an integration of course concepts.
So we have a lot of exciting topics and exciting things
to tell you about.
In terms of level of understanding
for this course, as I said, many of these systems are complex.
We're going to look at huge macromolecular machines,
and multi-step processes.
This is a biochemistry course, and we
are interested in molecular level, in addition
to this big picture.
And so things to keep in mind when thinking about structure.
You need to think about the amino acids,
and please review these if you're a bit rusty.
So to know the side chains, PKAs, et cetera, that's
all important to have in mind.
What are the protein folds?
What are the arrangements of these macromolecular
assemblies, and how do we study that?
In terms of reactivity, we'll see
bond-breaking and bond-forming reactions.
So again, we need to think about things like PKAs, nucleophiles,
and electrophiles.
If you need to brush up, organic chemistry textbook
or biochemistry textbook is a good place to go.
And then something to keep in mind is dynamics.
So the macromolecular structures and enzymes and proteins
we'll look at are dynamic.
Often we only have a static picture or some number
of static pictures.
But there's conformational change,
transient binding occurs, and we always
need to think about kinetics.
So these are things just to keep in mind when you're
reading and questioning to yourself about any given
system here.
So what about experimental methods?
This is just another topic to go over in this course overview.
So there's many methods that come up in 5.08.
And we don't expect that you have
knowledge of any or all of these at the stage of starting
the course.
The difficulty that comes up is that we can't introduce
all of these methods to you at once
in a level of detail that's needed for everything we do.
OK, so what will happen is that if methods come up
in problem sets that haven't yet been addressed,
we'll give you enough background information
in the problems that material, such that you
can think about the questions and answer them.
And we'll let you know when a method comes up.
You know, you'll hear this in recitation x,
or we'll talk about it more in class.
So right now, what I'd like to do
is just go over a few of the methods
that you're going to see multiple times.
And the thing to keep in mind is that the context in which
these methods are being used may differ,
but the underlying principles are the same.
And we choose methods that are being used today,
and are important.
Some of these were developed decades ago, some of these
are very, very new, and hot off the press.
So if it's an older paper, please don't brush it off as,
like, oh, this is old.
And so, you know, it's not new.
We're all really excited by technology and everything here,
but many of these older methods are robust,
and used all the time here.
So what are some methods and tools
that we'll have under our belt?
The first to point out are methods involved
in macromolecular structure.
So we care a lot about structure,
because we need structural understanding
to be able to comprehend how these systems work.
And so one method we'll see a lot--
and you'll discuss in recitation this week--
is x-ray crystallography.
And in addition, a method that will come up quite a bit--
and we'll see both of these in the initial discussions
of the ribosome--
is electron microscopy.
And another method to be aware of-- and if you're curious,
talk to your TA, Shiva--
is NMR.
OK, so NMR has a lot of applications
here within biological chemistry,
but we won't discuss that.
What can go along with methods is bioinformatics.
So how many of you have used BLAST?
How many of you know what BLAST stands for?
AUDIENCE: Basic Local Alignment Search Tool.
ELIZABETH NOLAN: Yeah, Basic Local Alignment Search Tool.
So what does this let you do?
It lets you find regions of similarity between sequences,
whether that's amino acid sequence,
a nucleotide sequence.
And you can use that information to make hypotheses and design
experiments there.
So that will come up.
I have additional methods and possibilities.
What about fluorescence?
So how many of you have done an experiment that
involves fluorescence, either in lab, or in your research?
How many, did that involve a fluorescent protein?
What about a small molecule?
Yeah.
That's fluorescent.
So have you thought about why the protein was
used, versus maybe why a small molecule, and what
are inherent strengths and limitations or one
or the other, depending what you want to do?
So fluorescence is used in many, many different contexts.
We can think about proteins like green fluorescent protein,
we can think about using small molecules.
And we like fluorescence because it allows us to see.
We can get visual information.
And so, where fluorescence will first come up in this class
is with the ribosome.
And in recitation week two, there'll
be some discussion about using small molecule fluorophores
to label tRNAs, and using fluorescence
as a readout of steps in the translation process.
And there's a lot of considerations and caveats
to that.
Do we have a pizza delivery?
Thank goodness no.
Often in this class, we get pizza deliveries for someone
else.
I didn't know if that's already starting.
Yeah, yeah.
We'll also see GFP being used in the proteasome section
for degradation.
So a folded protein has fluorescence,
a degraded protein does not.
What about kinetics?
So what different types of kinetic studies can be done?
So what do we all hear about in introductory biochemistry
class?
Pardon?
AUDIENCE: [INAUDIBLE].
ELIZABETH NOLAN: Yeah, steady state kinetics, right?
Turnover.
So we have steady state, which I encourage you to review
Michaelis-Mentin Kinetics here.
And you'll also be introduced in the first weeks
of this course, and especially recitation three--
so recitation two is going to build up to this--
pre-steady state kinetics.
So here, you've heard about this in 5.07
or another course, introductory course.
And we're looking at multiple turnover of an enzyme.
And these experiments are set up with an excess of substrate,
right, in order to afford conditions
that allow multiple turnover.
So there's formation of an enzyme substrate complex,
and then there's product formation.
So review as needed.
So what about pre-steady state kinetics?
How many of you are familiar with this method?
Not so much.
So what does the name suggest?
Pardon?
JOANNE STUBBE: So I'm deaf, you have to speak louder.
ELIZABETH NOLAN: Yeah, we're both deaf.
JOANNE STUBBE: I'm really deaf.
So if you want to say something, so I can hear it.
Speak up.
AUDIENCE: Yeah, maybe observing single molecule
by some spectroscopy.
ELIZABETH NOLAN: Yeah, a single turnover, maybe, I
think is what.
If we're having multiple turnovers here
in the steady state, right?
If we're before the steady state,
what does that mean, right?
It means we're in the initial, really
initial part of this reaction, where we're looking
at a single turnover here.
And how would you do this?
Basically, you look with subs having limiting substrate
rather than excess substrate.
And this is just to give a little prelude
in terms of thinking about experimental design.
So here, look at the first moments of a reaction.
So what type of time scale is that?
AUDIENCE: Small.
ELIZABETH NOLAN: Yeah, small.
Maybe a millisecond time scale, compared to a timescale
of seconds or minutes.
So what does that mean?
It means you need some different experimental setup.
You can't do pre-steady state kinetics
in the way we've done steady state kinetics, say in a lab
class for instance.
So you need a special apparatus.
And what does it let you see?
Here you're looking at multiple turnover, products forming.
You know, here in the early stages, what can you see?
Maybe intermediate formation.
And why might that be important for thinking about mechanism?
So those will come up in the first weeks of recitation.
Another topic that will come up, and is something
that you always need to think about, and relates
to integrity of materials, is that of purification.
So how are proteins purified.
For studying the ribosome, how do we
get ribosomes that are pure and are correct?
Or what if you'd like to use a mutant ribosome?
How does that get generated?
So here, you can talk about ribosome or protein
purification.
And so, I'll present to you on ribosomes and mutant ribosomes
in week four of recitation.
And this topic more generally of proteins
will come up in passing again and again.
So how many of you have purified a protein?
Many.
How many of you used an affinity tag?
So are they the answer to all problems?
No.
They can be a huge help, but they can also be problematic
in one way or another, right?
So with the ribosome we'll look at a case
where there was really some elegant work done
using an affinity tag approach to allow researchers
to obtain new ribosomes.
We'll also, though, talk about the limitations
of that type of methodology, and the things
you need to think about if you're doing protein
biochemistry, and how a tag may affect your experiments
and data there.
In addition, to think about is assay development,
and analytical methods.
And so there will be many different types of assays that
are presented in this course.
And something just to think about--
how do you develop the right assay, and what
are all the considerations?
How do you know your assay is a good one for the question you
want to address there?
This is actually really complicated.
And so there'll be some case studies
that come up in the course, but just more broadly
to think about.
So often in lab classes, you may have an assay,
but you might not be aware of all of the considerations that
went into actually developing that assay such that it works.
And then there's the analytical methods
that are used, either for analyzing
assay data or other data.
And again, these have strengths and limitations.
Just some that will come up, to present western blots
and immunoprecipitation.
So these methods involve antibodies,
and so we need to think about the antibodies themselves here.
Radioactivity.
OK, how does this work?
Why do biochemists like to use radioactivity and assay
development?
And how to think about this productively and correctly.
So should you be afraid of iron-55, yes or no?
How does that exposure compare to being in an airplane,
for instance.
Seriously, because there's a lot of fear associated
with radioactivity that may or may not be well-founded,
depending on what you're doing.
And so this gives us a lot of sensitivity.
And JoAnne will talk in week two of recitation
about radioactivity, and designing experiments
that use this as a read out.
What else?
So affinity measurements.
OK, so dissociation constants, or affinity constants,
how are these measured?
When reading the literature, is the value a good one,
or a not-so-good one, and how can you make that distinction?
Mass spec and proteomics.
So these will be in the later half of the class--
I believe recitations 11 and 12--
and many others.
And we're introducing CRISPR this year,
in the context of the cholesterol unit as well.
So as I said, we can't take care of all of these methods
immediately.
We'll let you know when they're coming up,
when you need to know more details about them as we
go through the course here for that.
So we can get started.
And in the last few minutes, what
I'll do is just give you a brief overview of the macromolecular
machines we'll look at through modules one through three.
And basically, what is the big picture?
And then we're going to break that down
into looking at individual components.
So if we think about the lifecycle of a protein,
basically, we'll fast forward to having mRNA from transcription
of the genetic code.
And then we have the macromolecular machine,
the ribosome that allows for translation of this method
message to give us a polypeptide chain.
So some linear sequence of amino acids.
And then what happens?
We need to get from a polypeptide chain
to some functional unit.
And so there's a whole number of interesting players
that are involved in protein folding.
So we have folding, which is enabled by chaperones,
is what we call these proteins that facilitate folding.
And that's going to give us some structure that
has some function here.
And this protein has some lifetime in the cell.
So at some time, for some reason,
it will be time for this protein to get degraded.
In which case, we need machinery that
will facilitate the process to break down this folded protein
into smaller fragments-- whether that
be individual amino acids, or short polypeptide chains
of seven to eight amino acids.
So from here, we have degradation
to give us small fragments.
And the players here are proteases and chambers of doom,
one of which is the proteasome.
And actually, I forgot to mention
there will be a second guest lecturer in recitation
this year, Reuben Saunders, who is a senior,
and does research in the Sauer Lab
on one of these chambers of doom, called ClpXP.
And so he'll present on single molecule methods,
and fluorescence methods to study how this degradation
chamber works.
So that will be really exciting.
He was a student in our course two years ago.
So let's just take a look.
We have the ribosome here.
What are the structural features of this macromolecular machine,
and how does it do its job?
We'll look at a number of seminal studies that were done.
And it is truly fascinating and incredible.
What about protein folding?
So look at this macromolecular machine here,
GroEL, GroES, look at how big this is.
So how does this chaperone allow some nascent polypeptide
that's unfolded or partially folded
to obtain its native structure?
And there is many details in this depiction here
that probably aren't apparent yet.
But by the time we're done with module two, it will be there.
Protein degradation.
So here is just a cartoon-type depiction of a chamber of doom
and its accessory protein from E. coli, ClpZ, ClpP.
So look, we have a folded protein here, it's
a beta barrel, our friend GFP that emits green light.
And somehow, this protein gets threaded through ClpX,
enters this chamber--
which has multiple protease active sites--
and that protein gets all degraded.
So how does this work?
How did ClpX and P work together to allow degradation
of this condemned protein?
And then finally, where I'll close is on something
I think a little bit different for most everyone,
and it's a type of template-driven polymerization
involved in the synthesis of small molecules
like penicillins and erythromycins.
So these are antibiotics.
So how do we get at molecules like these
from simple amino acid precursors,
or precursors like those you've seen in fatty acid biosynthesis
here?
And often, these are described as assembly lines.
And something we'll just need to keep in mind in this unit
is, are these proteins really acting like an assembly line,
or is this just a way to help us think about the templates
and what's going on here?
So that's where we'll close.
OK, so with that I'll finish up, and on Friday
we'll begin with looking at the structure
of the prokaryotic ribosome.