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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.
