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Research Interests:
Single Molecule Biophysical Chemistry Development and Applications of Nanocrystals
for Biophysical Applications Spectroscopic Properties of Single Nanoparticles Novel Techniques Applied to Single Molecule Research Single Molecule Biophysical
Chemistry Most
biochemical processes are complex and intrinsically heterogeneous.
Biomolecules adopt a wide range of conformations to perform their specific
functions. These conformations are constantly changing with time and
environment. We are studying these changing conformations to more thoroughly
understanding biological mechanisms. Single
molecule fluorescence allows us to study the heterogeneity of the various
conformations, as well as quantifying the timescales of the conformational
transitions by using Fluorescence Resonance Energy Transfer (FRET). FRET uses
the fact that fluorophores can transfer energy when they are in close
proximity, and the efficiency of this transfer is extremely sensitive to
distance – therefore it can be used as a ruler to measure very small
distances (1 – 10 nm) between fluorophores that are placed at specific sites.
With our microscope, these distances can be measured on a single molecule as
a function of time, so, as a biomolecule changes conformation, the distance
between the fluorophores change, and we can determine how long these
transitions take, and how many different conformations are present. The great
thing about doing this at the single molecule is that we see the transitions
directly – we don’t need a mathematical model to extract the data. Also, we
can do the experiments under equilibrium since, at the single molecule level, molecules are constantly fluctuating between
conformations. We don’t need a “trigger” to start the process. This
technique is highlighted in the figure below, which allowed us to follow the
folding-unfolding pathway of a protein (For more details on the protein
folding work, see our publications).
We are now using such
techniques to address important Health and Energy issues, specifically cancer,
neurological disorders and plant photosynthesis. (A)
protein interactions related to the onset of cancer
(proteins known as growth factors) and how anti-cancer drugs interact with
their targets. Small
(often benign) tumors need nutrients in order to grow into larger, more
dangerous tumors. They get these nutrients by forming blood vessels which
then connect to the blood supply. This process is called Angiogenesis and, when regulated, is critical to allow new healthy
cells to grow. Cancer cells can break down this regulation, allowing them to
grow faster than normal cells, which can then lead to tumor growth and
eventually metastasis. Angiogenesis
is initiated by a growth factor interacting with a growth factor receptor,
which starts a complicated signaling process in the cell. By regulating this
interaction, either naturally (autoregulation) or
with drugs, angiogenesis may be controlled. This may be an effective way of
treating cancers at very early stages. If we are to do this, we need to
understand the process of angiogenesis and how to improve drug interaction
with these potential targets. The particular proteins that we are studying are
fibroblast growth factor (FGF) interacting
with its receptor (FGFR) at the single molecule level. What factors regulate
the strength of this interaction and what are the
underlying protein dynamics involved. (B)
Structure of neurological receptors (glutamate and glycine receptors) in live
cells and organisms. Many
neurological disorders can be related to the improper recognition of
neurotransmitters such as glycine
and glutamate by their receptors. This recognition is highly
dependent of the structure and arrangement of the various subunits that make
up the receptor, and is highly variable depending on the function, organism
and environment. We are elucidating the structure-arrangement function
relationships between these various glycine and glutamate receptors in living
cells and even living organisms using our single molecule fluorescence techniques.
We
recently published a paper on using specific subunit
labeling by genetically engineering them with green fluorescent protein (GFP)
and used single molecule fluorescence together with stepwise photobleaching of the GFP to literally count the number of different types of
subunits present in the human glycine receptor expressed in Xenopus Oocytes. (C)
Mechanisms of how plants transport and organize the proteins involved in
photosynthesis. Photosynthesis
in plants requires the specific organization of proteins that bind
chlorophyll in the thylakoid membrane – imaginatively called light-harvesting chlorophyll-binding
proteins (LHCP). This specific organization of proteins is called the light harvesting complex, and allows
photosynthesis to be very efficient at focusing the light energy to a
reaction center so that photosynthesis can occur – by which the plant
produces chemical energy from solar energy. If we can learn how plants
organize these proteins in such a way, we can perhaps learn how to mimic the
process for our own needs in renewable solar energy. We may even be able to
use the plant targeting proteins themselves to arrange light harvesting
complexes for us. We use our single molecule fluorescence techniques to study
how plants use multiple proteins (at least 5 of them) to target the LHCP into
the thylakoid membrane and then insert it at the correct time and place. Development and Applications of Nanocrystals for Biophysical Applications One
of the limitations of single molecule fluorescence experiments is that high
excitation powers are necessary to detect the fluorescence from a single
molecule. This means that the fluorophore will spend a lot of time in an
excited state. When in this excited state, oxygen or other chemicals may
react with it and cause it to become non-fluorescent. This process is called photobleaching, and organic fluorescent dyes suffer from
it significantly, and it limits the maximum time that a single molecule can
be observed. We
are overcoming this limitation by using fluorescent inorganic nanocrystals, which are also called quantum dots (QDs).
These nanoparticles (QDs) have other advantageous optical properties: They
have a narrower fluorescence spectrum compared to organic molecules, can be
excited at any energy above their bandgap (a result of the band structure of
the energy levels) and, due to the effects of quantum confinement, the
emission spectrum (color) can be tuned simply by changing their size. These
properties are highlighted below.
However,
the quantum dots are synthesized in organic solvents, and if we plan to use
them for labeling biomolecules, they must be made water-soluble. We are
developing techniques to enable this, which include the design of
water-soluble ligands which can bind to the surface of the quantum dot. After
binding, the quantum dots take on the chemical properties of the ligand that
we have attached. However, adding these ligands increases the overall size of
the nanoparticle, and we must be careful not to make them too large for the
biomolecule that we want to attach to it. The general schematic of this
quantum dot-bioconjugate is shown below.
In
order to make the conjugation reaction specific to a particular biomolecule, we
vary the connection between the quantum dot and the biomolecule depending on
the bioconjugation reaction which we want to perform. We bind the quantum dot
to proteins, DNA or lipid molecules depending on the system that we are
studying. Once
the nanoparticles are conjugated to a biomolecule, we can use the optical
properties of the nanoparticle to study the biological questions of the
biomolecule. Alternatively, we can use the biomolecule to assemble
nanoparticles into specific higher order structures. Biomolecules are able to
bind specifically to other biomolecules in well-defined geometries and stoichiometries. One example of such a system is the
strong, specific binding of a single-stranded DNA molecule to its
complementary strand to form a helical double-stranded DNA by the use of
Watson-Crick base pairing. Other examples include protein-ligand interactions
such as the strong binding of 4 biotin molecules (also known as Vitamin H or
B7) to a streptavidin protein molecule in a tetravalent, tetrahedral geometry.
We are using these biological interactions to assemble nanoparticles into
pre-defined geometries. By assembling them in such a way, the nanomaterials
couple to each other, and their optical properties are affected. We are
taking advantage of this for the next generation of optoelectronic and
biosensor applications.
Spectroscopic Properties
of Single Nanoparticles Semiconductor
nanoparticles, or quantum dots, are very interesting but complicated systems.
If we want to use a single quantum dot as an optical probe for a single
biomolecule, we must also understand the spectroscopic properties of a single
quantum dot. One particular property that single quantum dots possess is a process
known as “blinking”. Upon constant illumination, the quantum dots switch “on”
and “off” – i.e. they go from “fluorescent” to “dark” – for which the
mechanism is not yet fully understood. We are investigating the underlying
mechanism of this blinking so that we can either eliminate it, or at least
take it into account, when we are interpreting our single molecule
fluorescence data. An example movie of single blinking quantum dots
immobilized onto a glass slide is shown below.
By
analyzing the timescales of this blinking process under different conditions,
we can determine which parameters affect it, and thus attempt to eliminate
it. Also, by knowing how various parameters affect blinking, we can take this
into account when we need to analyze the quantum dot-biomolecule conjugate
under certain conditions (such as inside a cell). By
simultaneously analyzing the fluorescence image of single quantum dots and
the topography of the quantum dots using atomic force microscopy (AFM), we
have found that there are a fraction of quantum dots that are permanently
“dark” – they never fluoresce. An example of a fluorescence image and a
simultaneously measured AFM image is shown below. The
fluorescent quantum dots are circled on the AFM image, but it is clear that
there are more quantum dots physically present than the fluorescence image
shows. We are investigating the causes underlying this phenomenon using
combined AFM and fluorescence microscopy (see below).
Novel
Techniques
Applied to Single Molecule Research The
following techniques are used for our single molecule experiments:
Data
are analyzed using the following techniques:
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Last Update: Sept 22nd, 2012 |
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