[ Silence ]
>> Today, we're going to
take up on protein structure
and we're going to
talking about one
of my old time favorite
topics, which is how do you go
from a sequence of amino acids
to this truly beautiful
structure right here.
There's actually a
good friend of mine
that I spent five years writing
my PhD about and studying
and doing experiments with.
So, this is truly one of
my old time favorite topics
in the 10 weeks that
we talked together.
OK. So, that's our goal,
understand how proteins work,
understand what their
composition is.
And then we'll get into
conformational analysis,
amino acid conformations,
and then finally looking
at protein structure.
So, this week is all
protein structure,
next week will be all protein
function in our scheme.
OK, any questions
about what we're going?
OK, couple of quick
announcements.
Read Chapter 5, and
work odd problems.
As usual, there'd be
a couple of topics
that I'll have you scheme,
the synthesis of peptides
for example, we're just
going to scheme through,
we just don't have
enough time to talk
about it in great detail.
But it's the book if you
want to read more about it.
Book reports-- sorry,
this should be journal
article reports,
are due this Thursday,
Valentines Day.
A week after that the abstracts
are due for your proposals, OK?
So, you should be
probably be thinking
about your proposal topics now
and start to do some research
on them and coming
up with an abstract.
On Thursday, I'll give you the
format for this abstract, OK?
But for now don't
panic about this.
This is a single
paragraph that you're going
to write describing
your proposal idea, OK?
And this will be worth 10
percent of your proposal grade.
So it's actually a
really important part.
This gives me a chance
to give you feedback.
And I'll give you a format.
It's basically a formula
that you could follow,
five or six sentences,
pretty straightforward.
But in order to do it you
do need to have your topic
and it's almost impossible
to think
of a good topic the
night before it's due,
so you should be thinking
about the topic now, OK,
coming up with a
proposal topic this week.
And again, this journal
article report is designed
to help guide you towards coming
up with your own idea as well.
OK. And I'm also
going to ask you today
to learn the 20 amino acids,
their structures, 1-letter
and 3-letter codes, but I'm
going to say that shortly.
OK, office hours.
OK, office hours are
going to be today.
And-- let's see.
Oh, office hours
today by Kritika,
and then office hours
tomorrow by me,
and then office hours
Thursday by me again,
and then office hour
Friday by Mariam, OK?
Any questions about
office hours,
office hour stuff,
OK, things like that?
OK. So, one question that I get
asked a lot during office hours
actually came up last week
but it comes every year,
is something along the lines of
I have a big idea that I want
to something in medicine but I'm
not sure exactly what that is.
And so, I want to take just a
moment to sort of talk to you
about careers in chemical
biology and careers
in the health profession,
OK, professions broadly, OK,
because I think this
is something
that we don't spend enough
time talking about when it's
where 99 percent of you are
going to go after you graduate..
OK. So, all of us
I think everyone
in this room has one goal,
which is to treat people.
OK, at the end of the day
I think all of us want
to make the universe
a better place,
we want to make people
healthier and happier,
and so our overarching goal
I would say if everyone
in this room, I suspect
is something like this.
So, in the end we want to
have people who are happy.
OK, that's our ultimate
goal, right?
And, I suppose if anyone
is really interested
in making money as
a way of getting
to this then you're
probably in the wrong class,
you should be taking class of
real state or, I don't know,
copyright law or
something like that.
But there're other classes
on campus that can get you
to the dollar signs, OK?
So I want to talk to you today
about all the different ways
that you can contribute
towards treating patients, OK,
which is I think the goal
of most of us in this room.
OK. So, the most obvious
way is to treat patients
by being a medical
doctor, an MD,
and I think this is what many
of you have on your minds.
Of course, and these
come up with--
will prescribe pharmaceuticals
which are then given
to the patients by pharmacists.
And again, I think that this
is probably the goals of--
so these are all
contributing in here,
and then of course there's
optometrist, there's DOs,
which are also participating,
there's--
you know, there's just other
types of-- there's dentists,
all of these are sort of the
hands-on ways that we think
of when we think about
treating patients over here, OK?
But, what I want to talk to you
about are all the other ways
that you can contribute
to this ultimate goal
that don't involve say going to
medical school, don't involve,
you know, becoming a dentist
or something like that.
I think you're all
aware of what it means
to become a dentist and,
you know, what it means
to become an optometrist, OK?
But ultimately someone has
to come up with the drugs,
the pharmaceuticals,
the therapeutics
that the pharmacists
are going to put in--
pharmacists that are
going to put in bottles
and the MDs are going
to prescribe.
And so, before you can get
to this stage over here,
there's a whole series
of clinical trials
that take place, OK.
So, the clinical trials are
staged into four stages,
where the first stage
is looking at toxicity
and the fourth stage
is looking at dosage,
and every stage is
looking at efficacy
which is to say effectiveness.
OK. So, someone has to
do those clinical trials.
And the people doing these
clinical trials are--
these clinical trials
include scientist, so PhDs,
but also MDs are
running these as well.
MDs are thinking about
what dosage to use,
and pharmacologists
play a role as well
in designing these trials
and looking at side effects
and interpreting data.
OK. Now, on the PhD side
of things, there's PhDs
who are analyzing data who are
thinking about side effects,
who are thinking about all
kinds of aspects of the trial.
They're thinking about the data
that-- say DNA sequencing data,
if it's a trial that
involves a particular mutation
of a cancer associated protein,
someone has to be thinking
about the underlying biology,
the underlying chemistry
involved.
And so, this is one
role that students
from my laboratory play.
So I have students
from my laboratory
who design clinical trials,
you know, graduated from my lab
who have PhDs in
Chemical Biology
who are now designing
clinical trials
and interpreting the data.
OK. So now, in order
to get the drug
to the clinical trials it
turns out it's a long process.
It takes about 10 years
or so to get a drug
into a clinical trial.
And so, kind of at the
beginning of all of this,
a series of experiments are
done over here by biologist
and chemical biologist.
OK, and the goal here is to
identify potential targets
that would be useful in
organisms, in humans, OK?
And they might not start with
humans, it might start with say,
I don't know, zebrafish,
where you maybe identify
a particular mutation
that causes heart muscle
to grow back in zebrafish,
and zebrafish are
this clear organism
that you can actually look
in and see their hearts.
OK. So, dude, if you, you
know, find something else,
find somewhere else to go, OK.
OK. So, someone has to do that,
someone has to get the foot
in the door and identify what
the ultimate target is going
to be, that this cancer
thing is going to target.
OK. So, this again
is done by biologists
and chemical biologists.
And, these are at all levels.
So, you tomorrow can go
out and when you graduate
from UCI you can participate
in this at your level
of a bachelor science, you can
have a masters, you have a PhD,
all of these give you a way
of identifying targets, OK,
so we'll call this ID targets.
OK. Now, once the target
is on hand then someone has
to chemically synthesize
the drugs, right,
someone has to synthesize
the initial leads
that are then going
to eventually become
the therapeutics, OK?
And so, this is typically
done by medicinal chemist, OK.
So, medicinal chemist,
but also this is done
by chemical biologists
who device assays,
looking at how effective
the compounds are,
computational chemist play
a key role so someone has
to model whether or not the
pharmaceutical are actually
targeting the drugs as expected,
and in doing this they can start
to decide whether or
not they're getting more
effective compounds.
They can use this
to design compounds
that bind more effectively the
target and avoid side effects.
So, this again is done by people
who have BS degrees,
MS degrees, and PhDs.
OK, so let me just talk
very briefly about this.
So, if you went out tomorrow
and got a job at say Genentech,
as a BS chemist, let's just
say BS chemical biologist
and you worked in Department
of Protein Engineering
at Genentech, what you would
be doing is you'd be working
as part of a team to
synthesize compounds,
perhaps if you're working
in medicinal chemistry team
or you're in say protein
engineering you'd be
synthesizing proteins,
you'd be testing proteins,
you'd be trying gauge the
efficacy of those things
and trying to decide whether
or not things are working, OK?
And, your role on this
team is you're kind
of like the foot soldiers,
OK, you're the muscle
that makes it possible
to do the science.
OK. Now, the thing is if you're
working on a really good company
and you're doing
amazing science,
eventually you get promoted
up to the ranks to be
at the same level
as a PhD scientist.
OK. So, when I was at
Genentech I had a friend
who never got a PhD but
he published seven papers
in science when he was there and
they took that as the equivalent
of a PhD and he was promoted
to levels of scientists
which are usually reserved
for people who have PhDs.
The PhDs are running the teams.
They're typically running
teams of about four people
and the people on those
teams include a couple
of BS level people,
maybe one masters,
the masters people is kind
of like a super technician
who's kind
of corralling [phonetic] the
resources, directing things,
helping things along,
pushing things more quickly,
that type of thing.
Sometimes they're called
research associates,
but it's the same sort of idea.
OK. So, in here-- so there're
lots of opportunities in here,
and then eventually
someone has to do things
like test the compounds
in animal models
and then also synthesize the
compounds before you can get
to clinical trials.
And so actually I'm missing
an important step over here
and I think what I'll
do is I'll have this.
So, we'll call this scale up.
OK, someone has to
scale up the compounds.
And the scale up is typically
done by process chemist.
OK. So, if you're that person
who loves organic chemistry
and loves mechanisms,
this is for you.
OK, you're going to be basically
really understanding the details
of a particular reaction and
trying to improve its yield
so that you can get 10
kilograms of the stuff,
enough to run a thousand
person [phonetic] trial
or something like that.
OK. So, someone has to do this.
And again, this includes
some BS level people,
also MS, and also PhD.
I haven't talked very much
about the MS. MS is a good thing
if you don't plan
to go on to sort
of a leadership role
on the team.
If you're totally happy at
kind of an intermediate level,
a masters of science
gives you a higher salary.
And over the course of a 30-year
career, a difference of 10,
000 dollars a year comes
out to a lot of money, OK,
especially compounding.
OK. And so, this gives you
just a little bit more freedom
but not a lot of freedom, really
the most sort of creativity
and freedom is at the level of
PhD until you earn it if you're
at this BS, MS level through
just being successful.
OK. So anyway, in the end this
leads then to the compounds
which then can be tested
in the clinical trials,
which ultimately leads over
here to the happy patients.
OK. So, a couple of
thoughts about this.
So, I have a diversity of
students in this classroom.
I have one guy over
there who text messages
through every class, it drives
me crazy, doesn't even seem
to listen to me at all.
But I also have some people
who have 4.0 GPAs, OK,
so I have a wide range of
students in a class like this.
And I have a message
for everyone,
no matter what your GPA is,
no matter how well you're
doing here at UC Irvine,
no matter if you're
text messaging
through every class here at UC
Irvine, there's a place for you
in this majorly challenging
enterprise.
To beat a disease like
cancer we need everyone, OK?
It's not true that just the
4.0s get to go on and go
on eventually to
win Nobel prizes.
There is a role for everybody.
And I've seen people with 2.0,
3.0 GPAs coming out of college
who have gone on to
win presidential awards
for their quality
of their science.
OK. And that's one of the
cool things about science,
it's a total merit-driven
enterprise.
So, if you are really good
and you work really hard,
and you bring a lot of heart,
and you put away your cellphone
at key moments, you too
have a role for this, OK?
And so, let me talk to you
very briefly if you want to be
over here and your GPA
doesn't let you do that.
OK. So, first, if
you're applying
to medical school you should
be thinking about having
like a 3.6 or higher GPA.
I've had trouble getting
students medical schools
with three A's.
It's that unpredictable.
OK, MD, PhD should be
having like a 3.95 GPA, OK?
And, like PhD graduate school,
you should probably have
somewhere in the order
of 3.3 to 3.5 minimum, OK?
Now, don't panic, OK,
if your GPA is below
that there is a place for
you, and I want to talk
to you very briefly
about how to get there.
OK, so number one,
is you can start
at this BS levels positions
in a company, and as years go
by your GPA from your
undergraduate becomes
increasingly less important.
If you're at this company and
you're doing really good science
and you're impressing
people then, you know,
that calculus class that you
got a C- and that's been bugging
down your career ever
since suddenly becomes
a lot less important.
OK. So, every year that goes by
your GPA gets less important.
OK, and then at that point
then it's all about heart
and your ability to
rise the occasion,
OK, which is good news.
Now, the other thing
is if you want to get--
go straight towards the PhD and
you don't have say, you know,
3.6 or 3.8 GPA, don't
panic either,
what you could do is can go
off and get a masters degree
at a Cal State and then come
back to get a PhD at one
of the top universities
in the country.
I would suggest that
you don't go
down to a low quality
institution
for getting a PhD, OK?
This is not elitism
[phonetic], OK?
You can look at my record
obviously I have a lot
of vested [phonetic] in
high quality universities.
But the point is, is that
we actually have something
like 200 chemistry PhD grant
in universities in
the United States.
Of those most of the employment
for the kind of top level jobs
in both industry and
academia are coming
out of probably 50
to 60 of those.
OK. So, if you drop down to
say, you know, third-tier
or fourth-tier PhD, you can
get a PhD but it's going
to be hard then for you to
come back, so a better way
to do it would get
a masters degree.
Cal State Fullerton, Cal State
LA are outstanding institutions,
you get your masters from there
and you show that despite,
you know, that grade in
calculus you still are someone
that we should take a bet
on and then you come back
and get a PhD at, I don't
know, UC Berkeley or UC LA,
or Harvard, or Johns Hopkins,
or any of the great institutions
here in the United States,
or maybe you go abroad and get
PhD from Cambridge or, you know,
one of the good schools in
Australia or so on so forth, OK?
So, there is a role for you
at whatever level you want
to participate in this major
enterprise of treating patients.
And the real question that
I want to challenge you
with is what do you want to do?
Are you the kind of person who
likes to think about molecules
at the atoms and bonds level?
In which case then you probably
want to be somewhere over here.
If you're the kind of person
who likes customer service
and likes, you know, having
patients coughing on you and,
you know, you don't
mind blood and stuff
like that you probably
want to be over here.
OK, so you basically get
to dial in whatever level
of interest is ultimately
yours, OK?
And no matter what your current
GPA is there is some room
for you, OK?
Any questions about that?
OK. So again, this comes
up every year during
my office hours,
and this year I thought
I make it more systematic
and just tell everyone.
OK. No questions?
OK. Oh, you know,
one last thought,
none of these is set in stone.
You know, the great thing
about United States is
that you can reinvent yourself.
So, if you end up going
to dentistry school
and you don't like it, change.
OK. I've had friends, actually
I have a friend who got masters
in Computer Science and was
a programmer and he hated it,
he absolutely hated it.
So he went back and got
a degree of dentistry
and he's now a really
happy dentist.
OK. So, no matter what you're
doing you don't necessarily have
to get locked into one thing.
There's considerable room to
reinvent yourself and then use
that earlier experience
to be more successful
at your next career, OK?
So, that's one of the
great things about this,
and that's one of the things I
really prized about our system.
OK, any questions?
OK. Well, let's stop here.
I want to move and I want
to talk about proteins next.
Specifically, I want
to talk to you today
about protein structure.
Before I do I need to review
what we talked about last time.
So, last time I was
telling you about RNA
and how RNA had a diversity
of different structures.
What we saw last time was
mechanisms of RNA polymerase,
we saw this last week.
But our message on Thursday
that was after the synthesis
of the mRNA, heavy
modification takes place.
We saw that both ends of the
mRNA are cut, they're altered.
There's splicing a vents,
there's cutting and pasting
that takes place after the
messenger RNA is synthesized.
For that matter,
after a synthesis
of RNA there are other
covalent modifications
that are taking place on the
RNA, and we saw an example
of that being a transfer RNA,
tRNAs that are synthesized--
that have modifications to
their basis, those modifications
as we saw have important
functional consequences in terms
of determining the specificity
of the amino acid
that's appended
to the three prime
end of the tRNA.
OK. We talked a little
bit about that
on how aminoacyl-tRNA
synthetase reads those types
of modifications out, it
reads out the anticodon
and in the end the correct
amino acid is appended
to the correct tRNA.
RNA as we saw is also a very
versatile biopolymer beyond its
ability to be both an
mRNA and also a tRNA,
for example we saw catalysis
by RNA and this was catalysis
by a class of RNA base catalyst
that we now call ribozymes.
And it turns out this
actually works very, very well.
We also talked a little
bit about binding activity
from RNAs by aptamers.
And so, all these things make
RNA this really versatile
molecule that is
really ripe ground
for all kinds of discoveries.
Whenever I pick up science
or nature there's something
new about RNA in there.
And, you know, the chapter
that I had you read has
large sections of it
that are changing, that are
being transformed overnight
by discoveries that are being
made in laboratories here
at UC Irvine and here at other
universities across the world.
So, this is a really
exciting area to investigate.
It's an area that I really
encourage you to think more
about because RNA
is so versatile
that it continually
surprises us.
OK. Let's talk about proteins.
So, we're continuing our journey
down the central dogma
of molecular biology.
We talked about DNA,
we talked about RNA,
we're now at proteins.
And, proteins are named proteins
because they were
thought originally to be
of central importance to
the cell, they still are
of central importance despite
everything I've told you
about of RNA and DNA,
and I'm showing one
of my own little biosis.
But, here's a great picture
of Max Perutz on the left
and John Kendrew on the right,
who shared the Nobel Prize
in 1962 for determining
the first three-dimensional
structures of proteins.
And back in the early days
of the structural biology they
would build little wire frame
models of proteins, this is
before computational modeling
was available.
And so, there was actually
tremendous craft involved
with building protein structure,
and there's almost
this tactile feel
as you have these
things in your hands.
We still have a little bit
of that structural biology,
nowadays we use 3D printing
but it's the same idea.
And so we like to think about
things with three dimensions.
And one of the things
that's always entrance me
about this topic is that we're
going to be taking things off
of the flatland and talking
about them in three dimensions.
OK. So, the first thing
I'd like to ask you
to do is memorize the
structures of the 20 amino acids
and their abbreviations,
both the 3-letter
and the 1-letter codes.
Here, I'm depicting just
the 3-letter codes--
just the 1-letter codes.
And, the amino acids can be
roughly grouped according
to the functionalities
of their side chains.
So, at the very top,
here are some side chains
that are hydrophilic including
acidic functionalities
like aspartic acid which
has an abbreviation D,
glutamic acid abbreviation E,
but also the hydrogen
bond donor tyrosine.
Here are some aromatic amino
acids with aromatic side chains
and then some basic side chains.
Please do this as
soon as possible.
Starting on Thursday,
I'm just going to assume
that I could talk about these
without kind of referring back
to histidine has this
imidazole functionality,
tryptophan has this
indole functionality,
so start memorizing
this right away, OK?
That would really help
your understanding
of what's going on.
In addition, there are
other functionalities
that are fully hydrophobic.
These are the aliphatic
function--
aliphatic amino acids
shown here.
OK. And then, the last
of our 20, there are some
that contains sulfur,
this includes methionine
and cysteine, and
then some other ones
that are hydrogen
bonding polar amino acids.
Some of these, two of these
in fact have a second
stereocenter at the beta carbon.
Notice that we're going to be
calling the carbon that's alpha
to the carbonyl, we're going
to call that the alpha carbon,
and then if you go alpha to beta
the second carbon away is called
the beta carbon, and tredine
[phonetic] has a second
stereocenter at this
beta carbon.
I didn't point it out
but the other amino acid
that has a second stereocenter
at its beta carbon
is isoleucine.
OK. So, isoleucine
has an stereocenter
in the Cahn-Prelog-Ingold
Rules for assigning S
and R stereochemistry,
[inaudible] has an R
stereocenter in that position,
those two you just
have to memorize,
there's no way around it, OK?
But good news, for the other
19 of the 20 amino acids,
the stereocenter at the
alpha carbon is S, OK.
So, every single one of
these has an S stereocenter
at this alpha carbon.
The one exception is glycine,
which just not have
a stereocenter
at its alpha carbon, OK?
Makes sense?
OK. I expect that you might have
seen this in Bio 99 or something
like that, if not don't panic
just go ahead and memorize these
as quickly as possible, OK?
Any questions?
All right, let's move on.
OK. So, when we refer to the
amino acids our convention
in this class and the convention
in biochemistry and chembio,
is that we're going to refer
to them using the 1-letter
codes followed by a number,
that number designates the
position of this residue,
residue is another way
of saying amino acid
within the larger protein.
OK. So, if you had a protein
that has 180 amino acids in it
and the position 171 the amino
acid was a histidine you would
designate this as
H-171, OK, and so on.
And obviously this is manganese
ion and so it's just going
to get the regular
elemental designation.
OK. So anyway, that's the
convention we're going
to follow, pretty
straightforward.
OK. So here's what I want to do
over the next couple of days.
I want to understand the forces
that take a set of amino acids
like what I showed on the
previous two slides or,
you know, two slides back,
and then understand how these
amino acids force a protein
to adapt a particular shape,
OK, because the sequence
of amino acids that
encodes a protein
in turn specifies one
predominant shape, OK,
and this is actually kind
of a pretty wild concept
if you stop and think about it.
But again, if you have
a particular sequence
of amino acid, that in turn will
dictate one particular shape,
OK?
There are some proteins
that refuse
to have any particular shape,
they're largely disordered,
those are kind of the
special exceptions.
So for this class where it's
kind of foundational level,
we're only going to
talking about proteins
that adapt one predominant
shape.
And if you start to look
at things more closely
there are lots of exceptions
but we're not going to
worry about that, OK?
So again, our goal is to
understand how you go from this
to something like this, OK,
understand the forces
behind protein structure.
OK. So, the first thing we have
to talk about is what happens
when you string together
the amino acids.
So, first, the backbone
of the peptide
or protein has some
directionality associated
with it.
And by the way, I'm going
to use the words peptide
and protein interchangeably
but they're not.
OK, so peptides are short
sequences of amino acids
that are connected
together by amide bonds
and in turn these are so short
that they don't adapt
any one-fold
or one particular structure.
Proteins are long enough so
that the amino acids force the
peptide biopolymer into
a single confirmation.
OK. So, it takes to a
certain minimum length.
Short peptides that
has say 20 amino acids
or less are typically
largely disordered.
When we put them in an NMR tube
we see all kinds of things.
OK, there's all kinds of
different confirmations
and these things are just kind
of flying all over the place.
If we make the protein
longer, if we add amino acids
on often times we can get
to something that's more
structured, OK, at least--
especially for barring
[phonetic] from sequences
that are known to be structured.
OK. So now we have to talk
about an important convention
in designating these sequences
of peptides and proteins.
We're always going to list the--
we're always going to list
the sequences in order
from N-terminus on the left
to C-terminus on the right.
Notice that the C-terminus
is the carboxyl end
of the amino acid.
So, in each case of these amino
acids over here we're going
to have the carbonyl or the
carboxylate on the right
and the amine on the left.
And, something that's really
important is the sequence.
These sequences that go from
N to C cannot be reversed
without resulting in a
different peptide sequence.
The two sequences, the
one on the top and the one
on the bottom are constitutional
isomers of each other.
They're not diastereomers,
they're nothing more
than just isomers.
And, the way you can tell
is notice that the positions
of the carbonyls and the NHs
are all wrong between these two.
OK, in fact it's very likely
of these two will have
different binding profiles.
It's very likely that
these two are going
to have say different flavors
if we were looking at a peptide
that had a sweet taste for
example, because the arrangement
of the carbonyls,
the arrangement
of the NHs will really
determine hydrogen bonding
to the backbone of the peptide.
And typically, say a taste
receptor if we're talking
about sweetness, a taste
receptor hydrogen bond donors
and acceptors that will
interrogate that backbone
and want a certain
arrangement of that backbone,
and the wrong isomer is likely
not to bind to the target.
I'm using the word likely
because molecular recognition
is very hard to predict,
but it's very likely that
it's not going to bind.
OK. So, these two
molecules are different,
and that's why we have
to have a convention
that we're always going to
write these with the N-terminus
on the left and the
C-terminus on the right.
Notice here that I'm not
listing the N terminus
and the C-terminus.
It's kind of like when we
talked about DNA sequences,
we have a 5 prime
end and a 3 prime end
but often times we didn't list
the 5 prime or the 3 prime,
right, we just assumed
that they were there.
So similarly with amino acids,
sometimes people write H2N
on this side and
CO2H on this side,
but most of the time actually
people just leave it out.
OK. It's kind of understood,
N-terminus is always
on the left, C-terminus is
always on the right, OK?
OK. So, that's the convention
that we're going to follow.
Here's something else that's
extremely useful for you to know
to the point where I'm
kind of tempted to ask you
to memorize it, which
are these pKas.
I haven't asked you to memorize
a lot of pKas if you took a 51C
with me or if you took, you
know, earlier courses with me
but these actually
are very useful.
And, the pKas that you need to
memorize are all shown here,
and you don't have
to memorize them
to the nearest decimal
place, just round them up.
OK. So, just come up
with something that's
roughly equivalent.
So, what I mean by
that is just know
that these carboxylate
side chains will have pKas
on the order of four to five
or so, somewhere in that range.
And the reason why the exact
numbers are not so important is
that the environment that the
functionality finds itself
in can dramatically
alter the pKa.
And this is a property
that proteins are notorious
for taking advantage of.
You can have, for example,
a carboxylic acid side chain
that's positioned in a way
that makes it much more acidic
than the pKa that's written
behind me might suggest, OK,
so proteins will
tailor the environment
that these functional groups are
going to found in, and in doing
so drastically alter at times
the pKas functionalities.
So, I want you to have just
the range of the pKas in mind.
You should know for
example that when a peptide
like this one is dunked in pH
of water which is obviously,
you know, pH7 being neutral,
here's what we would expect, OK.
We'd expect to see these
carboxylates deproteinated,
we'd expect this tyrosine to
be proteinated, it's only 0.3--
1.3 [phonetic] percent
deproteinated
at this pKa-- at
this pH et cetera.
OK. So, the C-terminus is
going to be deproteinated,
the N-terminus will be
proteinated at this pH, OK?
Gee, I realized I
wrote this last year,
but it's kind of true.
OK, when I think about
proteins first thing that comes
to mind are charge
functionalities.
Charge functionalities
control a lot of the ability
of the protein to
catalyze things,
to bind to different
things, so the first thing
that you absolutely need to
know is what is a charge?
OK. And you kind
of need to be able
to predict that quickly, OK?
So, I'd like you again
to memorize these pKas,
just the range, OK, not exact
numbers but just the range.
OK. So, these side chains
are going to dictate--
as the side chains
interact with each other,
that in turn is going to dictate
the folding of the protein.
OK, so this goes back
to the nature challenge
that I set up earlier.
How do we go from amino acids
to this complicated
three-dimensional structure?
What we find is that the side
chains are going to be packing
against other and
that in turn is going
to largely dictate
particular folds of a protein.
OK, and added into this
could be found in the fact
that the trends in side
chain-side chain interactions
mirror the strengths
of these interactions.
OK, so the side chain-side
chain interactions
that are found most
frequently are also the ones
that are the strongest.
OK, so for example, at
the very top over here,
we have phenylalanine
binding D-phenylalanine.
These are two amino acids that
have benzyl functionalities.
These benzyl functionalities,
large aromatic phenyl
groups can pack
against each other
quite readily.
And so for this reason, this
side chain is highly enriched
when we look at sort
of statistical analysis
of side chains found
in structures, OK.
So this is very strong
interaction
and it's therefore used
quite a bit by proteins
to set their structures.
On the other hand,
the weak interactions,
this is one that involves
the amino acid glycine,
these are very rare.
OK, so glycine doesn't
have a side chain.
Glycine is the one amino acid
that had no stereocenter,
it has two hydrogens
at its alpha carbon
and this one does not
form good interactions
with the other side chains.
And so for reason,
its interactions
with other amino
acid is very rare
and its interaction
is very weak, OK?
Makes sense?
OK. So, let's talk a little
bit about the specifics
of those interactions from
one side chain to another.
What we find when
we looked at this is
that the strongest ones are
most common, that's the point
of the previous slide, and the
strongest ones are typically
things like aromatic
functionalities binding
to aromatic functionalities.
And note that I'm showing this
as an edge to phase interaction.
That's a very common
mode for side chains
to interact with each other.
Another way is through
a dispersive force,
this was at Van der Waals
interaction that I introduced
to you back in Chapter 2.
Again, these are common
because they provide a lot
of binding energy and this sort
of binding energy can be used
to drive protein folding.
Less common and perhaps more--
perhaps surprisingly so are the
ones that are more predictable.
OK, so you can readily predict
a charge-charge interaction
on say the surface
of the protein.
But it turns out these
are relatively weak,
and do you know why?
So why are these
weak interactions,
charge-charge interactions?
[ Inaudible Remark ]
Depends on pH?
Yes.
[ Inaudible Remark ]
Yes. I heard a couple of
people say aqueous or in water.
In these aqueous or in water
environments often times there's
counter ions which can
partially shield these charge
functionalities or the
water itself is competing
for hydrogen bonding to
these charge functionalities,
and that has the effect
of weakening the charge,
lessening the charge and making
these charge-charge interactions
much weaker than they might
otherwise be expected to be.
And so, let me show
one example of this.
This primary amine
functionality would be found
on a lycine side chain
and nearby chloride ions
from sodium chloride
could readily interact
with this primary amine.
If the chloride ion is
interacting very closely,
that's going to partially
neutralize this positive charge,
right, chloride is negative,
the positive charge is positive,
the two of these are going to be
neutral and so the net effect is
that this interaction
is much weaker
than it would otherwise be.
Over here, the hydrogen bonding
ones are also unexpectedly weak.
And again, that's
because they're going
to be competing with water.
Water is the ultimate hydrogen
bond donor and acceptor,
and it's capable of readily
forming hydrogen bonds,
and this has the effect
of weakening a designed
in hydrogen bonds, in ways that
are very bonding [phonetic].
OK, if you're a medicinal
chemist somewhere
and you design the
perfect hydrogen bond,
you might be shocked to find
that you're only gaining say,
you know, half a kcal per
mole of binding energy,
which is very aggravating
because you might have
done a lot of work
to install that hydrogen bond.
OK. Let's take a close look
at two proteins binding
to each other.
And what I'm showing
you is one half
of a protein-protein
interaction.
At the top, this is protein 1,
and in blue down here
this is protein 2.
I've coloring in the atoms
according to their identity.
So for example, oxygen
is colored here in red,
nitrogen in blue and
sulfur in yellow, OK?
Notice that the interface of
the second protein, protein 2,
is dominated by hydrophobic
functionalities, OK?
There's a lot of carbons
and hydrogens down here.
There's no hydrophilic
functionalities, carboxylates,
hydrogen bond donating
carboxamides, et cetera.
It's almost entirely
hydrophobic stuff.
So at the interfaces
between a receptor
and its ligand the big inter--
the big money in terms of
interaction energy is dominated
by these aliphatic or
aromatic functionalities, OK?
They're the ones that
are dominating down here.
Up here at the interface
with water on the outside
of a protein or outside of a
protein-protein interaction,
there's lots of charge in that--
lots of charge functionalities
such as this carboxylate side
chain from a glutamic acid,
this amine functionality,
there's lots
of hydrophilic functionalities
that can interact
with the water.
This has the effect of orienting
the protein in this interface
between one protein
and water, OK?
Makes sense?
It should make sense, right?
We also alluded to this earlier
of the quarter when we talked
about the hotspot of binding
energy, when we talked
about the non-covalents
interactions.
So this is kind of a concept
that we've seen a
couple of times now.
OK. I want to talk to you
next about peptide structures
and why peptides are useful and
then we'll get on to proteins.
OK. So, here's a antibiotic
called cyclosporin A A,
this is actually given
to transplant patients,
patients who have had
liver transplants,
other types of transplants
as a way
of suppressing their
immune system.
It's an immunosuppressant.
Notably, this is kind of a
big exception amongst peptides
because it could be
given orally, OK,
meaning that you give it
to someone in pill form
and they can take it that way.
I should note that
in the United States,
culturally we prefer pills
that are given or we prefer
to give pharmaceuticals
in pill form
but that's not true
in other countries.
Other countries prefer
things like sublingual,
suppositories, et cetera.
OK, there's lots of ways
at giving pharmaceuticals.
In this country though,
we prefer to develop drugs
that can be given
in pill form orally.
The problem though is that when
you give someone a pill orally
it drops into their stomach,
where there're all kinds
of proteases to hydrolyze
amide bonds.
OK. And so if you give them
a peptide that's unstructured
those proteases will
catalyze the hydrolysis
of the amide bonds, OK?
And that in turn will
result in the destruction
of the pharmaceutical making
it ineffective, right?
If the pharmaceutical
gets digested
up readily it's not going
to be useful as a drug,
maybe it's a good amino acid
supplement but it's not going
to be useful as a therapeutic.
And so, the kinds
of big exception
to these are peptides
that are cyclized.
OK, notice that this does not
have any free or N or C termini.
The N and C termini of this
peptide has been hidden away
as the peptide has been
made into a ring, OK?
It's entirely ring shapes
that's been cyclized.
It turns out this
is fairly general
that you can take peptides and
connect up their N and C termini
if they're close to
each other in space.
And in doing so, make the
peptide highly resistant
to being chewed apart
by the peptidases
by the proteases
found in the stomach.
OK, this actually
works pretty well
and it's used very commonly
in drug development.
Some other little things
about this that happen
to make it even more
resistant are--
notice that each one of
these nitrogens are largely
with this exception
over here and this one,
these are largely mentholated,
the little line here,
that indicates a
methyl functionality.
That in turn also makes it
very hard for the proteases
to digest the amide
bonds of cyclosporin A.
So this is really
a major exception.
The other thing is that these--
the laws of these
NHs turns this amide
into being something
that's largely hydrophilic
into something that's a
little bit more hydrophobic.
And this helps the peptide
slip through the membranes
that are found on the
surface of the cell, OK,
those plasma membranes.
OK. So, let's talk a little bit
about what you can
do with peptides.
Peptide binders to
particular target,
is relatively easy to discover.
There's many source of this.
Some sources we have
talked about,
you can use phage display,
you can use a variant
of aptamer display, a messenger
RNA display, you can also search
through organisms
found in the planets
like this pit viper shown here
and identify venom
peptides in the pit viper.
So this peptide works by binding
to a particular active site.
OK. So, you now-- if you
can solve the structure
of this bound to
active site or do a lot
of structure activity
relationship studies
where you mutate these side
chains that identify one half
of the peptide that's working,
you can make an analogue
to the original starting
peptide.
OK, let me explain
that a little bit.
So, you have your pit viper
somehow and I don't know how
but you do this part
very carefully,
you collect the venom, OK?
And then, you start looking at
various fractions of the venom
to identify the active
ingredient that's making it
effective as a-- I think in
this case is antiinflammatory
or pain suppressing peptide.
OK. So you look through that,
you identify its active peptide,
which happens to be this
long peptide over here,
you then make a bunch
of versions
of this long peptide
that are shorter, OK?
So you make one that
has the five amino acids
at the C-terminus, one that
has the five amino acids
at the N-terminus and you
test each one of those.
OK, this is some work,
OK, but it's not so hard.
It's pretty easy to
synthesize peptides.
The book provides
more details on that.
OK. So, if you do that
you will eventually find
that you don't need this big
long peptide instead you could
get away with just the
shorter analogue peptide.
And then, if you-- and if you
spend a little but more time
in this you might find some
amino acid substitutions
that make it more effective.
So replacing of example
this ring
like amino acid side
chain proline,
within this case alanine.
OK, so you're making
a little bit simpler,
you're lowering the molecular
weight, next step here is
to do make analogues of this
lead peptide over here and try
to find a simpler
variant of it, OK?
So, you do down here
to the simpler variant
and then this thing that turns
out is not so orally available.
But if you make a--
this version over here,
this actually is orally
available as a drug.
OK, so this is actually
prescribed pharmaceutical,
and this actually
survives in the stomach.
OK, so even though
it has an amide bond,
it's not getting chewed apart
by peptides by proteases,
it's surviving in the high
protease rich environment
of the stomach.
OK. So all of this stuff over
here, this is what I was talking
about earlier today when I was
talking about all those stuff
that medicinal chemist are doing
and then this chemical biologist
and biologist, that's
the stuff that we do.
OK, we're going to start
with some large peptide--
peptide lead, try to cut it
down and then try to take it
and turn it to something
that's orally available.
And it's typically this is
done in teams of like 20
to 100 people working in
a project like this, OK?
This is not done by one
soul person who, you know,
kind of makes that their mission
in life to workout this details.
Typically that person
would be part of a team
with the computational
chemist, structural biologist,
chemical biologist to do the
assays, medicinal chemist
to synthesize these
things, it's a big team.
OK, so it's a big team
effort to get to here.
And then this compound over
here might sell, you know,
hundreds of millions of
dollars if not billions
of dollars a year in sales, OK?
OK. So, let's talk very briefly
about the synthesis
aspect of this.
This is all I'm going
to be showing you
and after this I would like you
to review this very
briefly this topic
and then scheme through
the book, OK?
So here's what I
would like you to know
and this is essential knowledge.
What I would like you to know is
that if you mix together
two amino acids
in a high concentration,
what will happen is
you'll get an exchange
where the amine functionality
of the one amino acid
and the carboxylate--
carboxylic acid functionality
of the other amino acid
will exchange protons.
OK. So, the proton n the
carboxylic acid will protonate
this amine giving you ammonium
ion and then also a carboxylate.
OK, so that exchange happens
very, very rapidly, OK,
but no amide bond results.
You can mix these two together
for a million years,
nothing will happen.
Furthermore, you could heat this
up for very long time and, yeah,
you'll get a little bit
of amide bond coming out,
but you largely get a lot
of other junk as well.
OK, you get all kinds
of side effect--
side products when you
start heating things
up to high temperatures.
OK, so instead what we do in
the laboratory is use some sort
of carboxylate activation
reagent.
This is analogous to the tRNA
that form that activated ester.
The aminoacyl-tRNA
that I showed last week
that activates the amino acid,
this is analogous to that.
And the way we do this is we
use an activation reagent that's
typically a carbodiimide.
OK, this is the-- this is an
isopropyl [phonetic] variant
of the dicyclohexylcarbodiimide
that you learned
about back in Chem 51C.
OK, so this is equivalent
of DCC.
Remember DCC?
It's like DCC, OK?
So this thing is a
fantastic electrophile.
It's an even better electrophile
if it's proteinated.
OK, this then access an
electrophile to be attacked
by the nucleophilic carboxylate
to give you this
key intermediate
which is acylisourea.
OK, this acylisourea is
now an activated carbonyl.
OK. So, this is all nicely
teed of to form an amide bond
because you've turned this
oxygen adjacent to the carbonyl
into the world's
greatest living group.
OK, key step.
This means then when
you add the amine,
you can then readily form the--
you can have the amine
and attack this carbonyl,
electrons get kicked up
here, electrons come back
down kicking off a
good living group.
And that good living group that
kicks-- kicked off is a urea.
Notice that this gives you
a carbon oxygen double bond
which as we've talked
about is very strong.
OK, so this goes back to when
we talked about tautomerization
of the DNA basis, and I
pointed out the strength
of a carbon oxygen double bond.
Here we see it strike
again, that strength is key
to understanding how
this reaction works.
OK. It also helps if in the case
of DCC, the urea precipitates
out of solution driving
the reaction toward
by Le Chatelier's is principle.
OK. In this case though,
this diisopropyl urea
not so insoluble, OK?
Oh, and by the way, for the
aficionados in the audience,
if you're wondering why we're
no longer using diisopropyl
carbodiimide often
times we switch to the--
from the dicyclohexyl to the
diisopropyl because chemists
that spend a lot of time
doing this reactions--
and believe me that you
end up spending a lot
of time doing these stuff.
When you spend a lot of
time doing this stuff,
small quantity is a
DCC that get on your--
in your skin turn into
a potent or allergen.
OK, so this will also react
with carboxylic acids found
on the surfaces of the proteins
in your cells and on your skin.
And in turn, that will lead
to a massive immune reaction.
So what we do is we switch
around between the
dicyclohexyl variant
to the diisopropyl variant.
And we also try to be
extremely, extremely meticulous
about not inhaling this
stuff, about not allowing it
to contact our skin et cetera,
that is really, really
essential.
This stuff is really nasty.
OK. And, you know, I have
friends who can't even go
into sort of laboratories
because that sensitize
to this type of chemical.
OK, so this is the level of
detail that I would like you
to know to understand
peptide synthesis.
The text provides a whole
lot more level of detail,
and I'd like you to just to
kind of scheme through it.
OK. Don't stress about it,
just scheme through it.
But I'd like you to know
this particular reaction,
this one is useful.
This is the reaction
that I've done a lot
that I think almost everyone
at Chembio has done--
has used many, many times.
OK. So, after the peptide is
synthesized, how are you going
to get to a larger protein?
OK, two ways, way number one
is to coax bacteria cells
to synthesize the large
protein directly for you.
OK, we talked about
that in the context.
I believe this was week
one where we talked
about E. coli [phonetic]
acting as little factories
to synthesize proteins
for you, OK?
So that's one way to do it.
A second way is to
stitch together a bunch
of short peptides.
And in doing this, then you
get to a much longer peptide
that eventually becomes
its own protein.
OK, it turns out that's
actually kind of nontrivial.
Each one of the peptides has
functionalities dangling off the
side chains, et cetera, so it's
not as easy to do that kind
of thing as one would like.
So instead, we've invented
a series of reactions
that allow us to do this
kind of convergent synthesis
to make a larger protein.
And the way this works,
here's one example.
This is one called native
chemical ligation discovered
by Steve Kent.
Here's the way this works,
you have a thioester
at the C-terminus.
OK, so this would
be the carboxylate,
instead of a hydroxide
over here there's a sulfur
and then an R. R
could be something
like a phenol functionality.
OK, so that's called
a thioester.
This happens to be
even more reactive
than the regular esters.
OK, and we know esters
a reactive
because we saw an example of
that with the aminoacyl-tRNAs
which were esters, right?
OK, so we start with this
very reactive functionality.
And this can do something
called transesterification
or thioester exchange where this
SR functionality is kicked off,
this thiolate functionalities
kicked off
to give is a free thiol and in
turn, a cysteine side chain,
the sulfur of that cysteine side
chain nucleophilically attacks
this carbonyl.
Why would it bother doing this?
Well, part of it is driven
by the tremendous
nucleophilicity of sulfur.
Sulfur is the king
of nucleophilicity.
It loves being a nucleophile.
Part of that is if we look over
here at the periodic table, OK,
so sulfur is one
row below oxygen,
these electrons are a little bit
further away from the nucleus.
And being a little
bit further away,
they can be more
readily given up.
They can be-- They're a little
bit further out of the orbit
so they're more effective
as a nucleophile.
So again, sulfur is the
champion of nucleophilicity.
Going down the periodic table,
selenium is even better,
tellurium oxidizes so
quickly, we don't even see it.
Selenium though is even
better than sulfur,
but sulfurs are grand
champion at least in Chem 128.
OK, so sulfur attacks
this activated thioester
that in turn gives
us this intermediate
and this intermediate can then
do a attack where this amine,
the lone pair in the amine
attacks the carbonyl giving you
a-- giving us-- kicking
electrons up to the oxygen
which in turn collapse down and
in turn give us a new amide bond
and booting off the sulfur
of the cysteine side chain.
OK, now why is this useful?
This is useful because
it gives you a way
of stitching together
two big peptides.
OK, peptide one over
here is indicated
by this little squiggle that can
have 20 amino acids going off
to the left.
So imagine 20 amino
acids going this way,
peptide 2 again indicated
by a little squiggle,
why do the amino
acids going this way?
And here's the thing, if
you mix these two together,
you will get an amide
bond out of it uncontrol--
you know, without even
thinking about it, all you have
to do is mix the two
together, maybe control the pH
with little pH buffer and
boom, you get amide bonds
out of it, it works that well.
OK, so that gives us a way
then of taking 21 over here,
21 over here, put them
together and you get 42.
If you do that a couple
more times you're talking
about really big
assemblies of proteins.
This is how you could synthesize
really big structures,
structures that start to
have some real consequence
for your experiments.
OK, so this works really well.
It's used very commonly.
It's often used to
incorporate natural amino acids.
A little bit different than
the suppression technology
that we talked about
in the context of RNA.
But in this case, you could just
chemically synthesize whatever
you want including natural amino
acids, including weird backbones
that I'm not talking
about today, OK?
Chemistry, this elegant must
have a natural analogue.
And it turns out that when we
look closely at cells we find
that cells can also do a
cell splicing reaction,
where proteins newly synthesized
by the ribosomes will actually
cut and paste themselves apart
without any intervention
from other proteins.
OK, so there's no other
catalysis that's available.
Here's the way this works.
So, some proteins been
synthesized by the ribosome,
here's the N-terminus,
here's the C terminus.
And by a mechanism
that I'll show
on the next slide called protein
splicing and insert intermediate
between the two exteins
is cut out is actually,
you know, physically removed.
This is analogous to the introns
of the messenger RNA, right?
We talked about splicing
messenger RNA.
Here we're seeing
something analogous to this,
but instead of having
the RNA backbone,
this has the protein backbone.
And again, this also
is something
that happens spontaneously
on its own.
You synthesize this
thing, you put it in water,
and before you do it the
intein has dropped out.
Why don't we take a look?
The mechanism recalls what I
showed on the previous slide
that we call native chemical
ligation it involves a similar
rearrangement of the
backbone of the protein.
OK. So, let's see, protein
splicing to remove intein.
Here's the way this works.
Flanking on the N-terminus
of the intein,
there's always a
cysteine functionality
that gives us the
wonderful nucleophile
of the thiolate functionality
of cysteine.
OK. So this thiolate can
now attack the backbone
of the extein.
OK. When that happens, you get
this intermediate structure
and then by an N to S acyl
transfer this other flanking
cysteine can attack
this thioester.
OK. So this is kind
of like, I don't know,
it kind of reminds me of
like a trapeze artist.
OK, we're going to basically
be passing off an activated
carboxyl carbonyl.
We're passing it off from
one person to another
to another, et cetera, OK?
So high wire act.
OK. So, that gives-- this
is the thioester exchange,
this is analogous
to the first step
of the native chemical
ligation that I showed you,
very analogous thioester
exchange reaction, OK?
So again, thioester exchange
over here that has the effect
of bringing these two
exteins next to each other,
and then the final step,
asparagine functionality,
the side chain of asparagine
functionality attacks the
carbonyl backbone giving--
freeing up the intein down here
and giving you this
imide functionality.
OK. And notice now that the
backbone has been restored,
the two exteins are bound
to each other covalently
through amide bonds.
This works really well, OK,
and it's actually found
naturally occurring in something
like 300 different proteins
in various organisms.
In fact New England Biolabs has
a whole website that's dedicated
to tracking this.
So if you're interested you
can look those up on Google
and you can find a big listing
and all kinds of examples.
In order for this
to work however,
there is conserved sequence.
This isn't going to work
with just any protein, OK,
this really requires for example
the cysteine at the N terminus
of the intein and another
cysteine at the C terminus
of the intein, and then a key
is the asparagine right here.
Furthermore, the intein has
to bring the two ends kind
of close to each other, right?
If the intein is set up so the
two ends can't find each other
then structurally this
cannot happen, right?
If the two ends are not, you
know, wandering around nearby,
no reaction is possible.
So, for these reasons we find
lots of conserve sequences
within the inteins and
certainly all of the amino acids
that I've depicted on this slide
because that's its mechanism.
OK, makes sense?
Questions about anything
we've seen so far?
OK. In that case,
let's talk next
about conformational analysis.
When we look at proteins,
we find that they're not just
flopping all over the place.
Amide bonds for example
which hold together proteins
occupy only a small amount
of three-dimensional space
that there's really limits
on rotational angles
within the protein.
And this topic we're going to
call conformational analysis,
and we're going to look
at it in some detail
over the next 15 minutes
and then the next
lecture we'll cover it
in much greater detail, OK?
So that's our plan.
I want to talk to you about
how to look at structures
and be able to predict their
predominant confirmation.
I'm not going to call it the
one and only one confirmation
because in fact what
we find is kind
of a bell-curve of
confirmations.
This is shown by this
upper graph over here.
So, this is a histogram, meaning
that higher numbers over here,
higher on this Y axis
over here or Z axis
over here means more
representation.
And notice that there
is a predominant peek
but there is some
population that exist
in these other otherwise
flawed rotation angles, OK,
where I to find some
rotation angles down here.
So, yeah, there's going
to be some dominance
of one particular confirmation,
but for the most part there's
also some other rotations
that are possible.
The goal again though is for
you to be able to predict what
that dominant confirmation
is going to be.
And it turns out that actually
just using exactly what you
learned back in sophomore
again in chemistry,
you can make predictions
about what the amino acid
side chains are going to look
like ad then in turn
we can start
to understand how
proteins can fold
into particular confirmations.
OK, that's our goal.
Let's talk first
about hydrogen bond.
OK, let's start basic.
It turns out that hydrogen
bonds are not flying all
over the place.
And this is set by the fact
that the hydrogen bond acceptor
are the lone pairs on oxygen
with [inaudible] look like
this Mickey Mouse ears.
OK, so here's the oxygen,
here're the orbitals
that encompass these
two lone pairs.
That is that they're not
going all over the place,
they're like Mickey Mouse ears,
they have only one
particular orbitals that--
or two particular orbitals
that are involved up here.
The electrons are highly
confined to those orbitals.
They're not going
all over the place.
And so for this reason, when
we look at hydrogen bonds
to this oxygen we find that
the hydrogen often times can be
found at an angle that sets it
up to be occupying these
orbitals or to overlap
with these orbitals, OK?
So, yeah, we've talked
about this before
but the restrictions adherent
to hydrogen bonding
limit the flexibility
of the protein backbone.
It turns out that
the flexibility
of the protein backbone is
largely dictated by this--
by hydrogen bonding by the amide
functionalities of the backbone.
Let me show you an
example of that.
OK, so this is just a backbone.
I've removed all of the
amino acid side chains,
and in dash lines these
are the hydrogen bonds.
Notice that many of these
hydrogen bonds are coming off
to oxygen at an angle.
That's to take advantage of
the Mickey Mouse ear shape
to the lone pairs on the oxygen.
That's not totally
perfect, right,
this hardly looks
Mickey Mouse shape.
But, you know, they kind
of look Mickey Mouse shape,
this one looks little
straighter,
some of these look a little more
Mickey Mouse shape than others.
OK. So, again, you know,
this is more of a guideline
and there is considerable
other angles
that are available as well, OK?
These are not definite,
you know, set in stone kind
of things, these are
more like preferences.
OK. So, this is a
structure of a protein,
a beta sheet protein called
concavalin A. I'll talk more
about beta sheets in a moment.
But it demonstrates the
variation of the geometry
of hydrogen bonds that's
possible all the way
from straight to angled, but in
general angled predominates, OK?
And notice that the hydrogen
bonds we're talking about are
between the NHs of an amide from
the backbone to the carbonyl
of a backbone, where again,
the carbonyl is the
hydrogen bond acceptor,
the NH is the hydrogen
bond donor,
exactly what we've seen before.
This kind of to be looks
like a zipper, right?
So this is kind of like
the zippers enclosed.
And so, these hydrogen
bonds are relatively weak
but when you get enough of
them together you get a very,
very strong interaction,
like a zipper, right?
You know, each one of those
little hooks in the zipper,
not so strong, but
you zip the thing up
and you get enough
strength out of it.
OK. So that's a very
similar concept
to what we're seeing here.
OK, each one of this maybe
half to 1 kcal per mole,
nothing to write home about.
You put a bunch together though,
now you're talking
about big money.
OK. Now, another canonical mode
for protein backbones
is the form helices.
So we saw on the previous slide
that the backbones
can form sheets,
these sheets are
typically curvy.
We'll talk more about
that in a moment.
In addition, the hydrogen
bonding preferences can force
the backbone of the protein
into a curvy helix
that looks like this.
If the helix has a pitch
such that the I residue
is interacting
with the fourth residue down
away from it [phonetic], OK,
so if we count-- actually
let's start at the top.
OK. So over here if the NH
of an amino acid is
forming a hydrogen bond
with its own carbonyl that would
give us this weird structure
over here.
OK, so in pink, these dash lines
indicate the hydrogen bonding.
We don't see this.
OK. So this would be
I to I's interaction.
We don't see this at all
in protein structures, OK?
Why? Well, this kind of kegs
[phonetic] the whole thing
into a confirmation
that's not so comfortable
and hydrogen bonds down here can
better satisfy the requirement
for overlapping with those
lone pairs on oxygen.
OK, notice over here the
lone pairs on the oxygen not
so Mickey Mouse eared, more
like coming off on the sides.
OK. So, if we interact with the
next amino acid the carbonyl
interacting with the
I plus one amino acid,
that gets us again a kind
of twisty looking structure,
not so strong, notice now
that the hydrogen bonds
are at terrible angles.
OK. Oh, something else I
should have pointed out.
I didn't notice that the
NHs are pointing directly
at the carbonyls, again,
within those angles.
That angle really
matters a great deal.
When the angle is broken,
that sets up a much
weaker hydrogen bond.
And I know that's something
we've talked about but I want
to remind you, over here
weak hydrogen bonds, right?
The angle is here set up
a very weak hydrogen bond.
That H is going off in space
one way, the oxygen carbonyl is
over here, does not set
up a good hydrogen bond,
this is two angled.
Similarly over here, two
angled where things start
to get interesting is when
we get to interactions
between a carbonyl on I
residue and an NH on I plus 3,
so that's 3 residues away.
That sets up a hydrogen bond
that's nearly the right angle.
And this kind of very twisty
helix is called the 310 helix.
It's observed.
We could find 3 10
helix-- helices.
They are important in cell
signaling for binding two
for example SH3 domains.
OK, so we can find those
and we'll talk more
about those later.
And the reason we can find
this is that the hydrogen bonds
for the 310 helix, not
so bad actually, right?
They're not so bad because
actually the NH is kind
of pointing the right direction.
There's a kind of a
nice-- the oxygen--
the lone pair on the
oxygen gets to keep--
stay in its Mickey
Mouse orbital.
This is not so bad.
OK, so those actually
do happening-- happen.
So, these 310 helices
are a very twisty helix.
This is a really tightly wound--
a very tightly wound helix.
More common helix is called
an alpha helix and it consists
of an interaction between
a carbonyl on the I residue
and the NH of an I plus 4 and
this gives you hydrogen bonds
that have nearly
perfect geometry,
nice and straight
with each other.
And so on these alpha helices
are very calmly found that's an
important motif of protein
secondary structure,
very, very common, OK?
Yeah, question over here.
>> The [inaudible]
of the interaction
or it's always like [inaudible]?
>> Yeah, OK that's
a great question.
What is your name again?
>> Oh, Ashley [assumed
spelling].
>> Ashley.
OK. So Ashley's question is,
is it always one
thing or the other?
Do you sometimes see the
two things interconverting?
And the answer is, we
largely see dominance
of these alpha helices.
And when we look at 310 helices
often times we'll see some
flickers of going to I plus 4
and then going back to I plus 3
and I plus 4 and I plus 3.
And so, yeah, there can
be a mixture of the two.
And there's techniques that you
can use like circular dichroism
to follow those two
and separate out.
What percent is 310?
What percent is alpha helical?
So thanks for asking.
OK, our goal here is to maximize
the number and the quality
of hydrogen bonds
between backbone atoms.
If we do that we're going
to have a much more stable
secondary structure, OK,
so that's kind of the
ultimate goal for everything
that I'm talking
to you about today.
Let's talk next about of the
alpha-helices in general.
Alpha helices often
clamp together
with other alpha helices
into large assemblies.
And it turns out that the
alpha helix itself has a dipole
associated with that.
OK. And, you can see this,
up here notice that all
of the carbonyls are pointing
in the same direction,
each one of these carbonyls has
a tiny little dipole associated
with it.
We talked about dipoles
when we talked
about dipole-dipole
interactions,
much earlier in the quarter.
At the time I told you that
dipoles were separations
of charge, where in this case
the oxygen being a little more
electro negative has a little
bit more negative up here
and a little bit more positive
at the carbon of the carbonyl.
And so this little dipole
over here, this little dipole,
this little dipole, all these
little dipoles are all pointing
in the same direction.
So, if you sum up all those
little tiny dipoles you end
up with a much larger
net dipole.
So the helix itself has a fairly
large net dipole, and that means
that one end has
a negative charge
and the other end has
a positive charge.
And, I would tell you that when
I first learned about this,
it was kind of-- I thought
of it as kind of myth,
I've come around entirely.
And I can show you
the best example
of a helix dipole in action.
This is the outer surface
of the filamentous bacteriophage
that's used for phage display.
It consists of a
series of alpha helices.
OK, so it's like these 50
amino acid long alpha helices
that are coiled around
each other.
They're actually coiled
in an unusual arrangement.
And this is a cross
section through the virus
and you can see all the alpha
helices stack in each other.
In the very center down here
is the DNA of the virus,
OK and it's not shown.
I've left it out to
make this simpler.
OK, but notice that these
actually form a right-handed
coiled coil, OK?
Unfortunately I hate to
do this, but I'm starting
with the major exception
to all coiled coils.
Every other coiled coil that
we're going to see this quarter
and in fact 99.999 percent
of all coiled coils bound
in nature are left-handed
coiled coils.
This one just happens
to be right-handed.
But here's the thing,
because each one
of these alpha helices
has a dipole
and all these coils
are pointing in this--
all these alpha helices are all
pointing in the same direction,
the virus ends up with
a massive negative--
sorry, net positive
charge up here,
net negative charge down here.
And the fact is that the
virus itself can be oriented
in a magnetic field.
OK, it has enough of a
dipole that if you put it
in a NMR tube, it goes
boing [phonetic] an orient
in the magnetic field
of the NMR tube.
And to me that's pretty
persuasive evidence
that actually this alpha helix
stuff might have a dipole
associated with it.
Furthermore, when we looked
at lots of protein structures,
we find that the positive end
of this alpha helix dipole
is oftentimes pointed
at the negative charge of
functionality in the protein.
And I'll show you
an example of this.
OK, so here is a sulfate atom
and a sulfate binding protein,
and look at this alpha helices
that are pointing
directly at the sulfate.
That extra little bit of
positive charge could come in
and stabilize that
negative charge.
We also find this when we
look very closely at enzymes
that have to do things
like transfer phosphate
functionalities,
which also have lots
of negative charge.
OK, let's stop here.
When we come back next
time, we'll be talking
about protein structure
and my favorite topics.
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