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Colin Heyes Arriving fall 2008 |
Degrees:
- B.S. Chemistry - Loughborough University, United Kingdom 1997
- Ph.D. (Bio) Physical Chemistry - Georgia Institute of Technology, 2002
- Postdoctoral: Alexander von Humboldt Fellowship, University of Ulm, Germany 2002-2003
- Human Frontiers Science Program (HFSP) Fellowship, University of Ulm, Germany 2003-2006
- McGill University, Montreal, Canada 2007-2008
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
Research:
Single Molecule Biophysical Chemistry
Most biochemical processes are complex and intrinsically heterogeneous. Biomolecules, such as nucleic acids and proteins (e.g. enzymes, signaling proteins such as neural receptors and G protein coupled receptors, transport proteins and structural scaffolding proteins) all adopt a wide range of conformations to perform their specific functions. These conformations are constantly changing with time and with environmental conditions. Two examples are given below.
(A) One example of such a process is the folding of a protein molecule once it has been synthesized in the ribosome of a cell. Each protein molecule may take different routes to find its specific structure but they all generally find their way there. Furthermore, considering the huge number of conformations that a protein could possibly adopt, they find this structure rather quickly. Nature apparently provides a mechanism so that a protein may start out from a huge number of initial unfolded conformations but find its way to a specific structure quickly. However the details underlying this mechanism are still not very well understood.
(B) Another example is the transport of proteins in a cell membrane. Often, signaling processes are initiated by the interaction of 2 or more protein molecules upon a certain stimulus. This raises a few questions. How do these protein molecules find each other in a reasonable timescale? Once they find each other, how do they interact? Once they interact, how exactly do they signal the cell? Does each molecule signal the cell in the same way or are there a number of mechanisms?
In each of these 2 examples, the molecules may start from a range of possibilities, and may progress through many different pathways, but end up in a specific state (or subset of states) which results in a specific function. However, if each molecule is doing something different, measuring the averaged signal from many molecules will hide the details of what each molecule is doing. By looking at each molecule one at a time, and building a statistical picture of each event, we can begin to understand how nature biases the system so that the specific structure and function can be achieved in a reasonable time.
An additional impact of this type of research is that nature is not always perfect. Sometimes a protein will not fold to the correct structure and, in some cases, may “misfold” into another structure which could be dangerous. Certain diseases such as BSE, CJD, Alzheimer’s and Parkinson’s have been connected with the occurrence of “misfolded” proteins. Also, certain neurological disorders have been connected with the inability of proteins to find their way to specific locations in a synapse (hence hindering signaling function). If we can understand how nature makes these mistakes, we can attempt to counter them.
To tackle these questions, we use single molecule fluorescence microscopy. The biomolecule is labeled with a bright fluorophore. This allows us to track the location of the molecule as a function of time, and to determine how and when it interacts with other molecules. It is also possible to label the protein with two fluorophores of different colors (e.g. green and red). When the fluorophores are in close proximity, excitation of the higher-energy fluorophore (green) will result in the transfer of energy to the lower-energy fluorophore (red). This process is called Förster Resonance Energy Transfer (FRET), and the efficiency of the energy transfer is extremely sensitive to the distance between the fluorophores. Therefore if a protein molecule changes conformation and the distance between the two fluorophores changes, the FRET efficiency will change, and we can use this change to determine which conformational changes have occurred. Here is an example of FRET applied to protein folding.
By measuring the changes in FRET efficiency with time, we can determine on which timescale individual proteins fold, and whether they form “intermediate” structures on their way there. The same technique can be used to infer structural changes in a protein upon the addition of a substrate or signaling molecule.
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, which limits the maximum time that a single molecule can be observed. One possible solution is to use fluorescent inorganic nanocrystals, such as direct-bandgap semiconductor nanoparticles, which are also called quantum dots (QDs). Since they are crystals, they are more stable than organic bonds, thus less prone to photobeaching. Also, 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 several 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:
Scanning Confocal Fluorescence Microscopy (SCFM)
Total Internal Reflection Fluorescence Microscopy (TIRFM)
Simultaneous measurement of Atomic Force Microscopy (AFM) images and single molecule fluorescence images
Fluorescence Lifetime Imaging (FLIM
Transmission Electron Microscopy (TEM)
Single molecule Surface Enhanced Raman Scattering (SM SERS)
Data are analyzed using the following techniques:
Förster Resonance Energy Transfer (FRET) Efficiency
Fluorescence Correlation Spectroscopy (FCS)
Image Correlation Spectroscopy (ICS)
Statistical analysis of time durations (Histogram binning)
Colocalization and Cross-Correlation analysis
Single Particle Tracking (SPT)