Hi.
My name is Taekjip Ha.
I'm a professor of biophysics and biomedical engineering
at Johns Hopkins University.
I am also an investigator with the Howard Hughes Medical Institute.
Today, I'm going to tell you
how single molecule measurement technologies
are changing the way we study
and understand nature's nano-machines.
You may have heard people say that
proteins can be viewed as nano-machines.
Why?
First, because they are unimaginably small.
They are only a few nanometers across.
How small is a nanometer?
Well, the smallest object that human eyes can see
is about 80 [micrometers] across,
which is a human hair.
That means that you can put 20,000 proteins
across a single human hair.
So, it's really, really small and you need special technologies to be able to
view and measure them.
Proteins are also called nano-machines
because they can perform jobs
that human-made machines can do.
For example, they can convert chemical energy
into mechanical energy
to power proteins along DNA or other molecules,
or you can store energy into a protein
just like you can wind up a wind-up toy
to store mechanical energy.
Here is an example:
a virus can package its genomic material,
the DNA,
into a very small volume using
a protein nano-machine
called DNA packaging motor,
and this packaging motor can use the energy coming from ATP consumption
to move the DNA into the capsid
one base pair at a time,
and it can do this until it can build
a pressure that matches the pressure
inside a champagne bottle.
So it's an amazing machine that can,
you know,
cram a large piece of DNA into a very, very small volume.
Here's another example of a nano-machine.
This is a protein called kinesin
carrying a cargo inside a living cell,
and kinesin is one of the molecular motors
that can move on the cellular highways directionally,
using the energy coming from ATP.
Just like cars are moving on the highway
using gasoline as a fuel,
these molecules can move on microtubules
using ATP as the fuel.
So, we wanted to study the mechanism
of the movement of these motor proteins.
One way of achieving this is to
label the motor
using a single fluorescent molecule
and measure the movement of a motor protein
at the single molecule level
using a microscope in a dark room,
as shown here.
The labeling can be done by
attaching a small fluorescent molecule
to the site of your interest.
In this case, let's say a molecular motor
called myosin V
is moving on the actin filament,
and it's known that the center of mass of the motor
moves on the track
with a step size of 37 nanometers.
Here, you can put a fluorescent molecule
on the foot of the motor
and you can image that single molecule
as a function of time in a dark room
using a microscope,
a very sensitive microscope
using CCD-based detection.
Now, if you image a single molecule
using your camera,
you get a pixelated image shown on the left,
and here the pixel size is about 80 nanometers,
but the motor protein itself is
much, much smaller than even a single pixel size,
about 10 times smaller,
but because of a process called light diffraction
-- actually... this is actually
based on Heisenberg's Uncertainty Principle --
when you take an image of a single molecule,
you know, using even the best optical microscope,
this molecule appears to be much bigger,
about 250 nanometers across,
which is about the half of the wavelengths of the light
used to image the molecule.
However, if you have a good enough
signal-to-noise ratio in your imaging,
then you can fit this pixelated image
using a 2-dimensional Gaussian function
and then find the center
with a much better precision
than the width of the image itself.
In fact, mathematics tell you that
you can do it down to about 1 nanometer precision,
and in practice it has been shown that,
yes, you can even approach that level
by, you know, going down to
about 1.5 nanometer precision.
So, it's amazing that you
can actually follow the movements of these motor proteins
in the lab,
down to single nanometer precision.
So, here's an example
where we imaged a single myosin V motor protein
moving on the track
and, as you can see,
as time goes on,
this spot, coming from a single molecule,
moves diagonally across to the top left,
and you can visualize individual stepping motions
as discrete movements,
you can find the center,
and then plot that position as a function of time,
and you can see beautiful steps,
and the step size turns out to be 74 nanometers,
which is twice the step size of the center of mass.
And this actually showed, conclusively,
for the first time,
that the step size of...
actually, that the molecule,
instead of crawling on the track,
it walks like a human walker, a bipedal walker,
and it proved a model called the hand-over-hand model
in the most definitive way.
So, to recap what I just told you,
what we did with the myosin V molecule
was just this:
you know, if you want to follow the movement
of your favorite soccer player on the field,
you can put a fluorescent molecule
on a body part of the player
and then follow its movements as a function of time.
But this measurement alone does not tell you why
-- you know, my favorite soccer player, here, Messi --
is such a great player, not a merely average player.
Soccer players, as you can imagine,
have to undergo multiple conformational changes...
actually, conformation is a jargon that
biophysicists use to denote the shape change of a molecule,
so a soccer player has to change his body shape
and posture,
you know, repeatedly
to perform the amazing action
of kicking and moving the ball.
And you are blind to the conformational changes
if you just follow one spot
in a body part.
In addition...
so, that's basically what you will be seeing,
you know, just one spot,
you know, nothing else.
So, we have, actually, another technology
that can be used to address this issue.
It's called FRET for
fluorescence resonance energy transfer.
Here, instead of just using one dye,
we use two dye molecules,
and one is called "donor", in green,
and the other is called the "acceptor", in red.
Now, when the two dyes are very far away from each other,
you use a laser light to excite the green molecule
and then you get emission from the green molecule,
so you get green photons from the molecule.
But if the acceptor moves closer to the donor,
then there is more and more
energy being transferred from the green molecule
to the red molecule.
As a result,
now you get photons coming from the red molecule,
of a red color.
So, by measuring the relative intensities
of the green and red molecules,
you can deduce how far they are away from each other
and if you know where the dyes
are attached to the molecule,
you can, for example,
distinguish between different conformations of a molecule.
Now, this technique
has a very strong distance dependence
and it can go from 100% energy transfer to 0
over a very few nanometer length scale change,
between 3-8 nanometers,
which is about the size of many proteins
and many other important biological molecules.
So it's the perfect technique
to measure the shape changes of
all kinds of important nano-machines inside our body.
Here's an example.
Imagine there's a protein
-- it's a butterfly that I'm using as an example --
you can put two dye molecules, green and red,
on two different locations,
so that in the closed conformation
energy transfer is efficient,
you get many red emissions,
whereas in the open conformation over there,
you get mainly green emission,
because the energy transfer efficiency is not high.
Here's another illustration
using the world famous using the world famous rap singer
Psy's Gangnam Style as an example.
So, he's going between different conformations,
The Sunglasses,
The Cowboy,
The Horseback Riding,
and The Finish,
and if you have dyes on the two hands,
then you can go in between intermediate distance,
a large distance,
and small distance,
and different, you know,
intensities of two different dyes
can be measured as a function of time.
Now, unlike what the cartoon suggests,
in a microscope you cannot see these two dye molecules
as two distinct spots because they are so close to each other,
much closer than the diffraction limit,
you know, 250 nanometer resolution of the microscopy
that we are using,
so they appear as a mixture of green and red,
in yellow, as shown here.
And when you move to the next conformation,
then it appears as green,
and then as red and so on,
so you can go back and forth between two conformations,
and you can read out the movement,
how quickly this movement occurs,
and how big the amplitude of the movement is
by just looking at the color change
as a function of time.
So, this is the idea,
and here's the final reaction.
So, if you measure intensities of green and red fluorescence
as a function of time, as you can see,
you can see beautifully anti-correlated changes
that report on the conformational changes
during the molecular dance.
Here are the results...
in addition to intramolecular conformational changes
that you can measure, as shown here,
you can use single molecule FRET
also to measure intermolecular changes,
interactions between two different molecules.
You can put a green dye on a protein
moving on the DNA lattice,
let's say, powered by ATP,
and then put the red dye at the destination end of the DNA,
then as time goes on,
as the protein approaches the red fluorescent molecule,
FRET increases and you'll see green signal going down
and red signal going up.
So, this is the idea behind using FRET to measure
movements between two molecules.
This animation is not the actual data,
it's what I prepared using a PowerPoint,
but our data is not that bad, it's almost as pretty.
So, here's an example where single molecule data
show that green and red intensities go up and down,
you know, green goes down initially, red goes up initially,
as shown in the cartoon.
The surprising observation here was that,
instead of falling off at the end of the DNA track,
when the molecule reaches the end of the DNA track,
it somehow reappears at the beginning of the track
and goes back to a low FRET value very quickly,
and then it then shows this gradual increase in FRET
and then repeats this process many, many times.
So, we have done a lot of different measurements
to understand the physical basis for this phenomenon
and how the molecule is able to perform
such acrobatic activities,
and also to deduce how this type of interesting,
you know, repetitive movements
can be useful for biological functions of the molecule.
So, after many months of research of this kind,
we eventually asked a magazine
to publish our research,
and after a usual back and forth
they eventually agreed to publish the study.
So... and then we asked an artist to,
you know, make a cover illustration
to suggest how, you know,
this enzyme may be performing the function.
So here's an example:
we proposed that this protein can sit on the DNA
and then reel in the single-stranded DNA
using the ATP-powered movement,
and then remove proteins bound to the DNA
to clear the DNA of unwanted proteins.
Well, we submitted this illustration to the magazine,
hoping that they would show it on the cover,
but unfortunately they just said, "No,"
and instead they showed a picture of a diabetic mouse,
and, well, that's fine...
you win some, but, you know, lose some,
so we just move on.
Here's another illustration that we paid an artist to create...
same idea, now we have a superhero,
my student Jeehae Park, who did the work,
sitting on the DNA and reeling in the DNA,
and removing asteroids bound to the DNA
using a powerful motor function.
Again, the magazine did not pick this cover either,
so I have to, you know,
use these images whenever I give public lectures.
Alright, so let me show you some real data
using DNA repair as an example.
As you know, DNA is an important genetic material
required to make proteins,
and proteins are the actual players
of the living cells in our body,
but DNA is under constant threat of damaging reagents.
If you smoke or if you go out to the sun in California,
then your DNA gets damaged
because of the smoke and also sunlight,
and DNA damage can cause
aging or cancer or other genetic disorders.
I see that I'm getting older every time I look at myself in the mirror,
so this is an important topic for me as well.
Do you know how much DNA you make in your lifetime?
It turns out that,
in your own body at this moment,
you have about 100 trillion cells,
and each cell contains about 2 meters of DNA,
and during your lifetime you,
you know, regenerate the cells
about 100 times on average.
So, if you connect all of the DNA molecules
that you produce back-to-back,
it can actually span a distance of one light year.
One light year is not a unit of time,
it's a unit of distance,
it's how far the light travels in one year,
so it's an enormous, actually, distance
and you have to make a lot of DNA.
And each cell in the body
can produce more than 1000 damages
even in a single day,
so unless you are able to repair the damages efficiently
you can inevitably accumulate
enough DNA damage to cause cancer.
You know, without proper DNA damage repair,
you'll actually get cancer at a much younger age.
So, there are several ways
to repair DNA damage,
and one is called mismatch repair,
another is called nucleotide excision repair.
Today, I'm going to tell you briefly about
a repair process called
homologous recombination.
Homologous recombination is
about as close as you can get to having sex,
if DNA can have sex.
So, there's a DNA and you have,
you know, DNA damage
causing a break in the middle of the DNA,
producing what's called a double-strand break.
Then, the cell has machineries to
digest one of the two strands of DNA
to produce single-stranded DNA,
and that single strand then finds a matching sequence,
you know, another copy of the same DNA inside the cell,
called homologous DNA.
And then once you find the matching copy
then you can use the information stored
in the other copy
to reproduce the original DNA without any mistakes.
This process requires a protein called RecA
to form a filament around single-stranded DNA,
and this is for bacterial cells,
and for humans we have its own counterpart
called RAD51.
Now, this RecA filament formed on a DNA sequence
has to find the matching sequence
in the sea of millions of different base pairs,
even in a small E. coli bacterium,
so, you know, this is equivalent to finding a soulmate,
your future husband,
you know, when you have millions of people
living in New York City, for example.
How do you actually achieve that?
More like finding a needle in a haystack.
One mechanism that has been proposed is called the 3D search.
Here, the filament is diffusing in solution
and then it randomly collides with the DNA
at random locations.
Unless it has a perfect match of sequence,
it dissociates quickly
and then goes to look for another sequence,
and this is more like dating a random person on the street
to find your soulmate,
and if you have a lot of actually
competing potential partners,
this can take a long time.
In this animation, I got tired,
so I just stopped it in the middle, so...
the filament found the correct target,
but you can imagine that if you millions of possibilities
it's going to take a long time.
Another possibility is called 1D sliding,
and this is equivalent to joining a book club.
So, instead of dating a random person on the street,
you join a club to meet people
who share a common interest,
and then you can date maybe 20 people within the club
to see whether you have a good fit,
and if you don't find the perfect match
then you can join a different club,
like a knitting club,
and then do the sampling again.
So, here's an illustration:
the single-stranded filament can bind
and then slide on the DNA, 1D sliding,
to sample,  you know, 200 or 300 base pairs
before falling off,
so you can actually sample multiple potential partners
in one binding event,
and this can make the process much, much faster.
So, we perform single-molecule FRET measurements
of the process
by putting a green dye on the filament itself,
and the red dye on the DNA.
In this experiment,
DNA, target DNA,
actually does not have the matching sequence,
so the filament will still bind to the DNA
and then show sliding motion,
and that sliding motion,
if it indeed occurs,
then it will be visualized as fluctuations in,
you know, single-molecule FRET,
where green and red intensities go up and down
in an anti-correlated manner, very rapidly.
And this is the actual signal that we see
from single molecules,
indeed showing that RecA filament
can slide on the DNA,
possibly in search of a target sequence.
So that was really, really exciting,
but does it really
find the matching sequence while sliding?
So, we performed another experiment, shown here.
So, we designed a DNA sequence
that contains a matching sequence
for the filament DNA,
but the sequence is not very long,
it's only seven base pairs long,
so it's not a perfect match,
so it's as if, you know, you...
you find a near match,
but here in this case we put
two copies of the same near match.
So, you know, again,
to use that example of dating,
you may be dating twin brothers, okay?
Well, both are near matches,
but you spend some time with one of the two twin brothers,
but the match is not perfect,
so you slide off to the other twin brother,
and that's why you see
a change in FRET efficiency,
shown on the top,
and then you then leave the other brother
and you go back to the original brother
and so on.
So, this data showed
very clearly
that the sliding on the same DNA
can be used to sample
different sequences in search of
a matching sequence.
So, overall, data of this type
showed us that 1D sliding
can make the target search process,
you know, 250 times faster than 3D search,
and in principle this sliding activity
that we discovered in this study,
you know, can make DNA repair
that much faster.
Okay, so I have told you about how
single molecule fluorescence imaging,
tracking of single fluorescent molecules
and measuring the interaction between
two different molecules
to, you know, to measure
the activities of single protein molecules
in real time.
There is another technology
that is extremely popular in the single molecule biophysics community.
It is called the optical trap
or optical tweezers technology.
If you want, this can be viewed as
chopsticks made of light.
So, you can use a tightly focused laser light
to grab a single particle in solution
and then if you move the laser beam around back and forth
you can move the particle,
and if the other end of the DNA
linking the particle to the surface
is an enzyme that is transcribing the DNA
-- RNA polymerase --
then the distance between the particle and the enzyme
will change, it will become shorter and shorter,
and you can measure this with Angstrom-level precision,
and you can apply forces that are relevant
to physiological conditions
and measure the mechanical response very precisely.
So, my laboratory and many others
have decided to combine these two different technologies,
single-molecule FRET and the optical trap,
so that you can use fluorescence
to measure conformational changes of a single molecule,
but use the optical trap to apply
a defined level of force
and understand how the tension, or force applied,
can change the activities of a single molecule.
And this is equivalent to
combining two different features of these measurements:
in the optical trap measurements,
you are using your hands to
manipulate a single molecule
and measure its response mechanically,
but with your eyes closed;
but in the FRET measurement,
you are using your eyes to make observations,
passive observations,
with your hands tied in the back.
By combining the two,
we can actually hope to sample
the best of both worlds
and this is going to be the topic of my second talk.
Alright, so I'd like to acknowledge
my former and present colleagues
and collaborators.
We collaborated with Yale Goldman's lab
and Paul Selvin's lab
on the myosin V studies.
And Kaushik Ragunathan, Chen Liu,
and Chirlmin Joo, and Jeehae Park,
and Sua Myong
worked on many of the
single molecule FRET studies I told you about.
And Ahmet Yildiz and Sean McKinney
worked on the
tracking of a myosin V molecule.
Thank you very much.
