At heart I am a biochemist who loves the knowledge and detail gained by the understanding of systems at the molecular / atomic detail. As a graduate student in the Biophysics program I am able to take advantage of the interdisciplinary mindset of the faculty to explore the many facets of how proteins behave. In the lab of Dr. Kevin Kubarych I have worked on developing and programming new instrumentation for acquiring 2D-IR spectra, the development of new multidimensional spectroscopies, as well as the synthesis of new molecular probes for 2D-IR spectroscopy of biological samples. All of these projects promise new exciting ways for us to gain a molecular understanding of what makes biology tick.
I am conducting research into the mechanism of methylation by S-adenosyl methionine methyltransferases. S-adenosyl methionine is the most common source of methyl groups for the modification of proteins, carbohydrates, and nucleic acids, with enzymes being able to catalyze transfer to carbon, oxygen, nitrogen, and sulfur atoms. The Trievel laboratory has previously shown that there are CH…O hydrogen bonds between the AdoMet methyl group and both a backbone carbonyl and tyrosine hydroxyl in the active site of SET7/9 that align and facilitate methyl transfer. My research is focused on other classes of AdoMet methyltransferases to determine if the hydrogen bonding represents a convergent form for methyl transfer, possibly by providing stability to the carbocation-like transition state. Characterization of the CH…O hydrogen bonding will be conducted by an assortment of biophysical techniques. Specifically, computational modeling for NMR prediction will be paired with NMR spectroscopy to observe the shift of methyl hydrogens, providing an indication of the electronic environment of the ligand when in complex with the enzyme. Infrared spectroscopy will allow direct observation of the bonds utilizing perdeutero-methyl AdoMet as the C-D stretch will be unique to the ligand. Point mutations of tyrosines in the enzyme’s active site, by incorporation of para-aminophenylalanine or phenylalanine, will allow the probing of the bonding interaction. ITC will provide binding data that will be useful in comparison of mutants with their native forms, which will also be paired with kinetic characterization of the enzymes to determine the role of specific bonds. Both neutron and X-ray crystallography will be used to interrogate active site structure, as well as to determine methyl hydrogen positions. This research will reveal the possible broader mechanism of AdoMet-dependent enzymes, and will also provide useful contacts for inhibitor design.
I am using crystallography, NMR, and kinetics to elucidate the enzymatic mechanism and molecular recognition determinants of both the plant and human mitochondrial RNase P enzymes. Transfer RNAs (tRNAs) are transcribed with extraneous nucleotides on their 5’ and 3’ ends which must be removed by tRNA processing enzymes. RNase P is the enzyme responsible for 5’ end processing of precursor tRNAs and is conserved in all domains of life. Traditionally, RNase P enzymes are ribozymes, utilizing catalytic RNA and magnesium for phosphodiester bond hydrolysis. However, recently it has been shown that a protein form of RNase P exists in plant and human mitochondria.
I have obtained the first high resolution crystal structure of mitochondrial RNase P and have begun structure-function studies to understand the enzymatic mechanism. I have also performed metal-soak experiments with various divalent metals on the crystal to determine metal binding sites. The metal-soak experiments in combination with metal reconstitution assays have revealed that Mg and Mn activate catalysis where as Ca and Zn do not. Binding and cleavage assays with various precursor-tRNA substrates are also being performed to reveal regions of substrate specificity. Preliminary binding assay data has revealed a tight binding tRNA substrate which subsequently has been used to set up cocrystal screens in hopes of determining the structure of a protein-tRNA complex. Preliminary crystals have grown and will soon be analyzed at the Advanced Photon Source at Argonne National Laboratory. I have been trained in data collection and analysis of protein crystals, fluorescently labeling techniques of tRNAs, and transient and steady state kinetics. These techniques, their theoretical bases, and recent advances in molecular biophysics have been introduced to me through my course work and have significantly complimented my thesis work.
During my time on the Molecular Biophyiscs Training Grant, I have been actively involved in understanding the interaction taking place between β-amyloid (Aβ) and lipid bilayers. Aβ is the amyloidogenic peptide implicated in the pathology of Alzheimer’s disease (AD) and is believed to play a principal role in neuronal toxicity; however, the exact mechanism by which it does so is still uncertain. It has been suggested that Aβ can bind to and disrupt membranes, leading to cellular Ca2+ uptake that triggers an apoptotic cascade. I performed a series of experiments using fluorescence spectroscopy demonstrating that membrane disruption by Aβ involves a two-step mechanism: (i) Aβ oligomers bind to the membrane to form ion permeable pores and (ii) the process of Aβ fibrillization causes membrane fragmentation via a detergent-like mechanism. In demonstrating this two-step process, we found that gangliosides are the key component that mediates the second step of the proposed mechanism. This work was published in Biophysical Journal in June of 2012 and is the basis for my current research.
While this work suggested a mechanism for Aβ toxicity, there is little known about specific Aβ-lipid interactions. I plan to continue using fluorescence spectroscopy as well as the combinational technique of solution- and solid-state nuclear magnetic resonance spectroscopy to further characterize the structure and dynamics of the Aβ-ganglioside interaction by specifically elucidating how gangliosides alter the structural morphology of Ab at early and late stages of amyloid aggregation. In doing so, I hope to shed light on the definitive role membrane composition plays in toxic amyloid formation, with a specific focus on the Aβ-ganglioside interaction.
While on the training grant, I have had the opportunity to try two distinct fields of research. My first project was to computationally model networks of neurons while rotating in Dr. Zochowski's lab. The purpose of this study was to understand how neurons form connections during brain development. To make the problem more tractable, we used a Kuromoto harmonic oscillator as the model neuron and observed the behavior of the network when each neuron had a distance-dependent positive and negative coupling function. Using this model, we explored what combination of parameters would cause oscillating modes, traveling waves, or random oscillation. I studied the theory of dynamical systems, how to model them in the C++ programming language, and how to perform large-scale computations on a computing cluster.
After that rotation, I realized that I prefer experimental work and I joined Dr. Ogilvie's ultrafast 2D spectroscopy group. My current project is to develop a setup to study the Stark shift in two-photon absorption (2PA) spectra of GFP derivatives. It was recently reported that the 2PA spectra are strongly dependent on local electric field and should exhibit large Stark shifts. I want to exploit this dependence to make optical probes of the electric field in biological samples. With a probe like this, one could observe the field in a firing neuron at biologically relevant time-scales, measure pH with an membrane-embedded fluorophore, and even perform high-resolution imaging using the field-enhancement of an atomic field microscope in combination with two-photon fluorescence.
To accomplish these goals, I have built electronics to control the high voltage power supply, designed and built sample holders, and performed some preliminary measurements on samples. I have been trained to work with ultrafast optical setups, the machine shop, cryogenics, and basic wet-lab work. I am also taking course work in nonlinear optics, quantum mechanics, statistical mechanics, and biophysics to aid me in the analysis of my data.
My research goal is to characterize the dynamic behavior of microtubules, intracellular polymers that play crucial roles in cell division, motility, and transport. Microtubules are assembled from heterodimeric αβ-tubulin subunits in a process known as dynamic instability whereby microtubules switch abruptly between phases of slow growth and relatively rapid shortening. Proper microtubule functions depend on these unique dynamics, which can be modulated by cellular proteins (e.g. tau) or drugs (e.g. taxol). Tau is a protein implicated in Alzheimers Disease that is thought to stablize microtubules. Taxol is a chemotherapy drug that disrupts the ability of microtubules to properly orchestrate chromosome segregation. However, the effect of these drugs at therapeutic concentrations is not well understood. My aim is to study how substoichiometric binding of taxol and tau affect microtubule kinetics.
My early taxol experiments were performed using differential interference contrast (DIC) microscopy to obtain polymerization data at therapeutic taxol concentrations (0 – 100 nanomolar). However, more recent data has been collected using total internal reflection fluorescence microscopy (TIRFM), which yields improvements in spatial and temporal resolution. Using TIRFM, microtubule length changes are measured to within 25 nm at 8 Hz, and changes in the microtubule tip structure are also detected. Results show that taxol potently affects microtubule growth at concentrations as low as 10 nM, with higher variability in growth rate than control conditions, especially on longer timescales (on the order of 100 seconds). In some cases, microtubules switched between normal growth and periods of very slow growth. Further, we have found that 10 nM taxol almost completely suppresses rapid shortening, and beginning at 100 nM taxol, the tubulin on and off rates gradually decrease, though the net growth rate (excluding periods of very slow growth) remains constant.
My future work with microtubules will investigate the effects of tau, and, ultimately, results will be confirmed using optical tweezers to study microtubule polymerization at nanometer and millisecond resolution.
Natural products constitute a large portion of the medicinal compounds available today and understanding how these compounds are produced could enhance our ability to create new drugs or improve existing ones. These compounds are made by polyketide synthase (PKS) or mixed PKS-nonribosomal peptide synthethase (NRPS) biosynthetic pathways which are present in species ranging from archaebacteria to animals. The biosynthetic pathways are composed of modules, which consist of multiple domains, that each perform the addition and modification of a building block to the growing substrate in a process analogous to parts being added on an assembly line. Through the rearranging and/or modification of modules, pathways have evolved to produce a vast number of distinct compounds. Understanding how these modules function and how they interact with each other provides a stepping stone for engineering pathways to modify natural products or to create new compounds from scratch, the ultimate goal of the field.
During my time on the Molecular Biophysics Training Grant, my research interests have been focused on the apratoxin pathway which produces the macrolactone compound apratoxin. Found in a marine cyanobacterium, the apratoxin pathway is a great candidate for expanding our understanding of how synthesis is initiated. In addition, apratoxin has been shown to selectively target cancerous cells making its study of possible medical value. Synthesis is initiated by the first module of the pathway, called the loading module. In the apratoxin pathway, the loading module not only initiates synthesis, but produces a unique tertiary butyl group. To understand this process I am using structural biology, specifically X-ray crystallography and electron microscopy. The structural work is being supplemented by the use of biochemical assays to track the initiation of biosynthesis and the modification of the substrate. Complimentary to this work, I have been collaborating to develop a high-throughput screening technique utilizing biolayer interferometry to test multiple protein constructs under a range of lysis conditions to maximize yields.
The specific goal of my research is to use a form of Nuclear Magnetic Resonance (NMR) called relaxation dispersion to better investigate and understand RNA and DNA dynamics. Recently, there has been mounting evidence that in the cell it is common for these molecules to sample two or more states. Investigating and understanding these states could give a better understanding of how DNA is regulated, and how RNA is used to carry out certain cellular processes. In addition, while in the ground state (where the molecule spends most of its time in) biomolecules look similar to one another, in the excited state (which they are in for a minority of the time) they have a more unique shape, which could be used to specify which biomolecule a drug should bind to and affect. As many of the side effects of today’s drugs are due to them broadly affecting many different cellular processes, this opens up the possibility of developing safer, more effective drugs.
Currently, we have good methods for modeling and detecting two different physical states that an RNA or DNA molecule can obtain. However, three or more states are more difficult to detect, as they can easily be mistaken for a 2-state system, especially if the quality of the data is not sufficiently high. My research is focused on this area, working on the development of alternative data analysis methodologies, so that better discrimination of the various factors of the exchange system can be done, and the dynamics of the molecules we study can be better understood. As there is not a great body of literature on the theory of NMR for multi-site exchange for this reason, success will help not only our own investigations, but provide a new technique to other researchers doing research along similar lines.
RhoA, a Ras family GTPase and key actin cytoskeleton regulator, can be activated in response to G protein-coupled receptor signaling through Gq. By binding activated Gq, the effector p63RhoGEF assumes an active conformation and subsequently displays RhoA-specific GEF activity. The mechanism by which Gq activates p63RhoGEF is still unknown. The crystal structure of p63RhoGEF in its activated conformation has been determined, but attempts to crystallize the basal, inactive conformation have been unsuccessful. Additional biochemical data has also been inconclusive in determining how autoinhibition of p63RhoGEF is relieved through binding of Gq. We are using nuclear magnetic resonance spectroscopy to determine the solution structure of the p63RhoGEF catalytic core in its basal conformation and to identify residues within the protein that undergo large conformational changes upon Gq activation. The successful completion of this work would provide the molecular basis for regulation of an important link between heterotrimeric and small molecular weight G proteins.