Current and Past Molecular Biophysics Trainees
Molecular Biophysics Trainees Receive a Salary Supplement from the College of Sciences at Georgia Tech. Five students are selected each year by the Molecular Biophysics Steering Committee. The research statements and publications of current and previous trainees are described below.
Christina Hampton (advisor, Fernandez). The most fundamental problem facing mass spectrometrists today is how to convert a dissolved neutral molecule into a gas-phase ion. This is of particular concern with the analysis of biological molecules such as peptides and proteins that commonly undergo conformational changes during the ionization process and are generally expensive or difficult to isolate. To that end, the development of novel ion generation devices has experienced considerable growth in the past few years and the aim of my project is to further the development of three ionization sources that have been recently introduced to the scientific community. The Array of Micromachined Ultra-Sonic Electrosprays (AMUSE) is a device that is capable of producing micrometer-sized droplets by mechanically pushing liquid in a sealed analyte reservoir through an array of nozzles. The pressure used to drive this process is a direct result of the deformation of a piezoelectric transducer due to the application of an RF potential to the surface. Desorption Electrospray Ionization (DESI) is an ambient method in which a charged stream of very-fine droplets is directed at a surface causing desorption of charged species from the surface. We are using DESI to directly analyze tissue segments to determine the blood-brain barrier permeability of a drug that has shown to reduce neuronal degeneration. This method requires minimal sample preparation and provides high throughput. Direct-Analysis-in-Real-Time (DART) is also an ambient ionization method but uses a charged stream of helium gas molecules to ionize volatile molecules on a surface. The screening of pharmaceutical drugs is an ideal application of DART, and we have used it to analyze counterfeit antimalarial drugs and to determine the active ingredients in drug cocktails, which are sold in Asia and Africa with the claim that they can reduce the symptoms of malaria. Each of these ionization methods can be used with any mass analyzer and provides information that complements the results obtained using conventional ionization sources such as electrospray ionization or matrix-assisted laser desorption ionization. The use of the AMUSE in the field of proteomics enables the analysis of native protein structures in a wide variety of solvents. DESI and DART can be used to analyze almost any surface with high throughput and minimal sample preparation. The goal of my thesis work is to test the limits of these novel ionization methods in the hopes of improving the application of these methods to very specific real world problems.
Adam Offenbacher (advisor, Barry). My current research
focuses on the investigation of the biological significance and role of
a post-translational covalent linked tyrosine-histidine moiety in the
binuclear active site of cytochrome c oxidase (CCO). There is much controversy
over the significance and/or role of this modification in the CCO reduction
of molecular oxygen to water. Some speculate that this tyrosine with the
aid of the covalently linked histidine plays a key role as an electron
and a proton donor. A few studies utilizing small organic-derived models
have tested this hypothesis. However, most of these studies have not implemented
copper into their "active site" model compounds. Using three
models (a copper complex, zinc complex and a metal-lacking complex), all
synthesized by our collaborator, Prof. Olof Einarsdottor from U.C. Santa
Cruz, I will attempt to shed light on the functional significance of the
cross-linked amino acid. In my studies, I am using electron paramagnetic
resonance (EPR), which identifies unpaired electron spins (i.e. radical
formation), and difference Fourier transform infrared (FT-IR) spectroscopy,
which provides information about structural differences (i.e. changes
in force constants) throughout the molecule upon laser-induced photo-oxidation.
My future research project concentrates on the ribonucleotide reductase
enzyme and the elucidation of the details of the catalytic mechanism.
In collaboration with Prof. Joanne Stubbe at MIT, I am developing rapid
stopped-flow FT-IR spectroscopy, which will be used to monitor and identify
the rate-determining step. This rate-determining step has been hypothesized
to be a conformational change within the protein.
Chris Richards (advisor, Dickson). As scientists seek to
better understand biological processes, the study of intracellular dynamics
such as protein-protein interactions has become an increasingly important
area of research. Single molecule fluorescence is a powerful technique
that enables the monitoring of these cellular events; however, these types
of experiments require extremely bright fluorophores that are also photostable.
Many biological processes of interest happen on time scales that are much
longer than the time scales at which organic dyes bleach, thus limiting
the current application of single molecule fluorescence. To address this
problem we are developing new single molecule probes. Fluorescent silver
nanoclusters have shown unique photophysical properties that would make
them ideal candidates as single molecule labels. These clusters, on the
scale of just a few atoms, have size tunable emission that is extremely
stable over long periods of time. Oligonucleotides, such as RNA and DNA,
are being used as a scaffold to encapsulate silver clusters. Additionally,
the use of small oligonucleotides allows for the fluorescent probe to
be water-soluble and for easier modification for cell permeability. In
order to make them viable in biological applications, optimization of
the cluster characteristics is being pursued by modifying oligonucleotide
sequences and solvent conditions. Analyses of the effects these modifications
have on the single molecule photophysical characteristics of the silver
nanoclusters is being done on immobilized molecules and in aqueous solutions.
Typical single molecule techniques such as correlation spectroscopy and
photon counting statistical analysis are utilized to determine the optimum
design of the oligonucleotide scaffold. The goal is to create a series
of water-soluble single molecule labels that are both bright enough and
robust enough to be utilized in biological applications.
Ashley Ringer (advisor, Sherrill). The nature and strength
of p-p interactions, and the forces that stabilize these interactions,
have been the focus of much theoretical and experimental investigation.
These noncovalent interactions are involved in a host of chemical and
biophysical processes, ranging from self-assembly of synthetic molecules
to drug intercalation in DNA. In 2004, our group described the first ever
high-level quantum mechanical determination of pi-pi interaction energies
in substituted-benzene dimers. Notably, our results showed that all substituted
dimers had a greater interaction energy than the benzene-benzene dimer,
a result the Hunter-Sanders rules, which rely on electrostatic considerations,
would not predict. My research seeks to further characterize the effect
of substituents on pi-pi interactions and develop a new model describing
how substituents effect pi-pi interactions generally. If a substituent
is substituted in multiple locations on an aromatic ring, it has a greater
affect than if only one substituent were added. For face-to-face configurations
of substituted-benzene dimers, this additivity is linear. However, for
T-shape interactions, the additivity is only linear through two substitutions,
and then interactions between the substituents on one ring and the hydrogens
on the other begin to make significant contributions. Because information
about multiply-substituted T-shaped dimers can not be simply extrapolated
from information about mono-substituted dimers (as is possible for sandwich
configurations), we have developed a multi-linear model to predict the
interaction energy for T-shaped dimers. This model includes parameters
corresponding to electrostatic and dispersion interactions as well as
direct interactions between the aromatic rings.
Ashley Tucker (advisor, Hernandez). Apoferritin, a 24 mer
protein shell, possesses an outer diameter of 12-13nm and inside diameter
of 7-8nm. The twenty-four subunits are all equivalent except for the presence
of a ferroxidase center on the heavy or major subunit. The twenty four
subunits will spontaneously assemble and dissociate in solution depending
on the pH conditions. Utilizing this characteristic of apoferritin, it
is possible to trap molecules from the solution in the protein interior.
This property of apoferritin makes it extremely attractive for drug delivery
systems. In order to further understand this system, molecular dynamics
and Brownian dynamics simulations of the multi-time step process of protein
dissociation/reassembly and subsequent dynamics through a viral suspension
have been carried out. This work is being compared to other computational
work done on the dynamics of apoferritin.
Heather Bean (advisor, Hud). In order to form duplexes and
higher order structures, nucleic acids require the associated cations
to screen electrostatic repulsions between their negatively charged phosphate
groups. Close analyses of DNA and RNA crystallographic structures have
revealed that several helical parameters vary with the properties of the
coordinated cations. Similarly, molecular dynamics simulations predict
that the coordination of cations in the grooves of nucleic acid helices
actively alters groove width. Other researchers have shown that nucleic
acid structure is principally determined by base sequence or backbone
stereochemistry, suggesting a rather minor role of cations in determining
nucleic acid structure. The impossibility of creating nucleic acid structures
devoid of cations means that the potential structural role of cations
might only be probed by altering the way in which ions interact with nucleic
acid structures.
The structural properties of numerous modified oligonucleotides have been
studied, including those containing alternate sugars, modified bases,
and non-ionic or cationic backbone analogues. However, few of these studies
have addressed how changes in counterion association alter nucleic acid
structure. In natural nucleic acids hydrated divalent cations have been
shown to bridge the phosphates across the minor groove of B-form helices
as well as across the major groove of A-form helices. It has been proposed
that these cations play an active role in determining the groove widths
of the helices. In the laboratory of Dr. Hud, I have designed and successfully
synthesized an alternative nucleic acid backbone where the phosphate groups
in a nucleic acid duplex are replaced by glyoxylate, which we term gNA.
Molecular modeling of a gRNA linkage shows that the electrostatic properties
of the carboxylate group are very similar to that of phosphate, yet the
presence of stereocenters in the glyoxylate linkers allows the distances
between the carboxylate groups across the major groove to be varied by
as much as 3Å. We hypothesize that the narrowing or widening of
the distance between the ion-coordinating carboxylate groups in the backbone
will affect the number, type, and location of ions associated with the
helix.
William Blackburn (advisor, Lyon). My main research interests
center on controlling interactions between micron- and nanosized hydrogel
particles and biological systems. The specific hydrogels used in this
research are environmentally responsive, such that they undergo swelling
or conformational changes in response to environmental stimuli. We synthesize
these hydrogels into unique constructs called core/shell microgels, where
the core and one or more shells may have different responsivities and/or
chemical functionalities, thereby allowing for the synthesis of "programmed"
multiresponsive nanomaterials. As described above, these projects provide
links between polymer chemistry and biological systems. In one project,
core/shell microgels designed in the Lyon group will be used as a targeted
and triggered chemotherapy system in collaboration with the Chmielewski
group at Purdue University. The specific system under investigation here
is the folic acid-folate receptor system that has been shown to be effective
in cancer targeting, as tumor cells often overexpress folate receptors
on the cell membrane surface. The second project involves the study of
cellular adhesion to thin films composed of core/shell microgels. We have
demonstrated, in collaboration with Andres Garcia (GT-ME) that thermoresponsive
poly(N-isopropylacrylamide) (pNIPAm) microgel particles cross-linked with
PEG display reduced protein adsorption and therefore reduced cellular
adhesion. Continuing this work, we will optimize the non-fouling properties
of these films and then, again using the core/shell construct, the particles
will be designed to display specific cellular adhesion moieties and/or
contain proteolytically degradable linkages that will allow for enzymatically-triggered
release of cytokines or other signaling molecules. The fundamental questions
here are how do ligand density, spacing, and clustering influence cellular
adhesion, and can cellular adhesion and recruitment be modulated by specific
macromolecule release events. If successful, these efforts will dramatically
impact the surface design of implanted materials such as monitoring electrodes
or drug delivery devices.
Amanda McCook (advisor, Harvey). I am interested in developing
computational biophysics methods and software. The use of predictive computational
methods is certainly useful for reducing cost and time associated with
experimental work, and I am interested in such applications. For example,
software is commonly used to predict energetics of a polymer to determine
whether synthesis is worthwhile. Another example is the use of computational
docking and interpretation thereof to focus synthesis efforts on the most
likely drug candidates. However, improvement of theory is a major driving
force behind experimental scientific progress, and the use of computer
to formulate and test new theories is, when performed responsibly, and
excellent tool for such. In particular, I am quite interested in writing
programs to extract information from databases such as the PDB for generating
hypotheses about sequence-structure relationships. I am also interested
in writing a program to mine RNA sequences and secondary structures to
predict potential signals of genomic viral RNA relative to other types
of RNA, since viruses are known to preferentially package their own genomic
RNA when placed in a solution of mixed RNAs. Clearly if rules for such
signaling can be determined, there is potential for creating a predictive
software to parallel mfold, with parameters modified to predict secondary
structure specific to viral packaging. Currently, I am writing a program
to determine helical properties of a polymer by searching potential conformations
as defined by free torsion values. The application is intended for both
amino acid and non-amino acid polymers. The program creates, by way of
grid search, a torsional profile for helicoidal properties such as twist
and rise. For any given conformation of the polymer, as defined by its
free torsion values, the polymer will form a helix of a given twist and
rise, and the profile for all possible conformations is plotted in a manner
similar to a Ramachandran plot. Underlying this method is the physical
requirement that, in order for the polymer to viably bind to DNA, the
polymer twist and rise must match the DNA twist and rise. This project
has potential applications for creating nanowires on a DNA template as
well as for generating potential conformations of basic DNA-condensing
proteins such as spermidine. Previously, I have generated a method, combining
the use of several existing programs, to predict RNA secondary structure
in regions of the RNA where neither thermodynamic nor comparative analysis
methods were previously able to reliably predict structure. It primarily
exploits the comparative analysis portion of the method used by Alifold.
Prediction methods relying on the sequence of interest (non-diverse) or
the broadest available multiple alignment of related sequences (maximally
diverse) are both incapable of predicting structure in certain regions
of mitochondrial rRNA. However, we have seen that consensus structure
at intermediate levels of diversity is able to predict additional structure
that can be validated by experimental methods such as cryo-electron microscopy
density. I have also previously developed web-interactive software for
pharmaceutical library design. The software automated the creation and
analysis of a chemical library, using an existing library and a user-defined
template.
Reagan McRae (advisor, Farhni). Copper serves as a cofactor
for many enzymes involved in biological processes. However, excess copper
is deleterious to cells because cooper (I) ions may catalyze the production
of reactive hydroxyl radicals, which can damage lipids, DNA, proteins,
and other biomolecules. The cell is therefore required to maintain buffer
sites not only as a defense against deficiency, but also as protection
from abnormal copper concentrations. Several copper chaperones, which
function to transport copper to its destination and maintain cooper homeostasis,
have been recently identified. A number of disease including Menkes' syndrome
and Wilson's disease are caused by impaired copper transport and regulation
by these copper transporters. Although many of these copper chaperones
have been identified and characterized, the nature of the cellular structures
and organelles that may act as transient storage places and thus intimately
participate in cellular copper homeostasis remains elusive. To investigate
how the intracellular copper distribution is altered in Menkes' disease,
we are utilizing both a mottled embryonic mouse fibroblast cell line (802-1)
that is defective for the Menkes gene as well as a cell line deficient
for the copper transporter, Atox1 (Atoxl-1), which directly interacts
with the Menkes' ATPase. We are employing microprobe synchrotron x-ray
fluorescence (micro-XRF) to visualize the intracellular copper topography
with submicron resolution, and thus to identify possible locations for
copper storage.
Robin Sibert (advisor, Barry). Photosystem II harvests energy
from sunlight and uses it to oxidize water and reduce plastoquinone. A
key component of water oxidation is a redox active tyrosine residue. Upon
absorption of light, tyrosine is oxidized by the primary donor, P680+,
and reduced by the manganese cluster for the catalytic site for water
oxidation. In the oxidized form, tyrosine exists as a neutral radical.
In our lab, Fourier transform infrared (FT-IR) spectroscopy and electron
paramagnetic resonance (EPR) spectroscopy experiments have been performed
which support the hypothesis that the tyrosyl radical is stabilized via
interaction with neighboring amino acids. This interaction is conformationally
dependent and may be altered by varying the peptide sequence. Similar
studies have also been used to study pentapetides that serve as models
for the peptide sequence of photosystem II near the redox-active tyrosine
residue. The results of those studies indicate that the tyrosyl radical
is stabilized through interactions with nearby functional groups. Although
the relationship between tyrosyl radical formation and peptide sequence
has been investigated for dipeptides and pentapeptides, no studies have
been performed on polypeptides that have secondary structure. Such studies
are relevant because the introduction of secondary structure means that
non-covalent interactions may occur between the side chains of the component
amino acids. Therefore, the ability to oxidize the tyrosine residue may
increase or decrease, depending on the stability or lack of stability
imparted on the resulting neutral radical by the non-covalent interaction.
I will test this hypothesis by performing reaction-induced FT-IR spectroscopy
and EPR spectroscopy on designed beta hairpin polypeptides, among which
the cross-strand interaction partner of the tyrosine residue will be varied.
The secondary structure of the designed polypeptides will be verified
using one-dimensional proton NMR and two-dimensional NMR techniques such
as TOCSY and NOESY.
Publications
"Conformational free energies of 1,2-dichloroethane in nanoconfined methanol," J. A. Gomez, A. K. Tucker, T. D. Shepherd and W H. Thompson; J. Phys. Chem. B 109, 17479-17487 (2005).
"Redox-active tyrosine residues in pentapeptides," I. Vassiliev, A. Offenbacher, and B. A. Barry. J. Phys. Chem. B 109, 23077-23085 (2005).
"Imaging of the Intracellular Topography of Cooper with a Fluorescent Sensor And by Synchrontron X-ray Fluorescence Microscopy," L. Yang, R. McRae, M.M. Henary, R. Patel, B. Lai, S. Vogt, C.J. Fahrni. Proc. Nat. Acad. Sci. USA 102, 11179-11184 (2005).
"Glyoxylate as a Backbone Linkage for a Prebiotic Ancestor of RNA," H.D.Bean, F.A.L.Anet, I.R.Gould, N.V. Hud. Origins of Life and Evolution of Biospheres 36, 39-63 (2006).
"A Structural Model for the Large Subunit of the Mammalian Mitochondrial Ribosome" J A. Mears, M R. Sharma, R.R. Gutell, A. S. McCook, P.E. Richardson, T.R.Caulfield, R.K. Agrawal, S.C. Harvey. J. Mol. Biol. 358, 193-212 (2006).
"DNA-directed assembly of polyanilines: Modified cytosine nucleotides transfer sequence programmability to a conjoined polymer," B. Datta, G.B. Schuster, A. McCook, S.C. Harvey and K. Zakrzewska. J. Am. Chem. Soc. 128, 14428-14429 (2006).
"Characterization of solid counterfeit drug samples by desorption electrospray ionization and direct-analysis-in-real-time coupled to time-of-flight mass spectrometry," Facundo M. Fernandez, Robert B. Cody, Michael D. Green, Christina Y. Hampton, Rose McGready, Sivong Sengaloundeth, Nicholas J. White, and Paul N. Newton. Chem. Med. Chem. 1, 702-705 (2006).
"Correlative Micro-XRF and Optical Immunofluorescence Microscopy of Adherent Cells Labeled with Ultrasmall Gold Particles," R. McRae, B. Lai, S. Vogt, C.J. Fahrni. J. Struct. Biol. 155, 22-29 (2006).
"Aliphatic C-H/pi Interactions: Methane-Benzene, Methane-Phenol, and Methane-Indole Complexes," A. L. Ringer, M. S. Figgs, M. O. Sinnokrot, and C. D. Sherrill, J. Phys. Chem. A 110 10822-10828 (2006).
"The Effect of Multiple Substituents on Sandwich and T-shaped pi-pi Interactions," A. L. Ringer, M. O. Sinnokrot, R. P. Lively, and C. D. Sherrill, Chem Eur. J. 12 3821-3828 (2006).
"Proton-coupled electron transfer in a biomimetic peptide as a model of enzyme regulatory mechanisms," R. Sibert, M. Josowicz, F. Porcelli, G. Veglia, K. Range, and B. A. Barry. J. Am. Chem. Soc. 129, 4393-4400 (2007).