University of Chicago Scholars

2016-17

Ellen Iverson
CRISPR as an Innovative Tool for Studying Senescence-Associated Aging in the Lung
Stephen Kron Group

In recent years, studies have linked cellular senescence to pathologies associated with aging tissue, including serious lung conditions like chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Improving understanding of the factors that accelerate the development of senescence may help suggest new treatment options for such diseases. In particular, compounds that may be able to delay or prevent the onset of senescence have exciting potential. To this end, I purpose to develop a CRISPR-Cas9 construct capable of inducing a dose-dependent amount of pure DNA damage without any of the secondary effects associated with drugs or radiation. This tool can then be tested in murine lung fibroblasts for its ability to induce senescence alone and in combination with reactive oxygen species (ROS), allowing new and important insight into the relationship between DNA damage, ROS, and senescence. I will then use these data to design and demonstrate the efficacy of a screen for possible modulators of the onset of accelerated senescence in the lung.


Andrew Molina
Molecular Nechanism of CNS Myelinogeneis: Self-Assemblyof Lipid Nanotubesand Nanotube Bilayer Dynamics
Ka Yee Lee Group

My research interests center around the biophysical process of self-assembled structures formed by lipid and lipid/protein mixtures associated with Central Nervous System (CNS) myelinogenesis. The myelin sheath is a layer of insulation consisting of a mixture of proteins and lipids that surrounds the axons of neurons, and allows for the rapid transmission of electrical impulses throughout the body. This functionality is disrupted in patients suffering from demyelinating diseases such as multiple sclerosis, in which the myelin sheath breaks down and the propagation of the electrical impulse is significantly compromised. Specifically, my research objective is to re-examine the mechanism of myelin sheath formation and elucidate the role of plasmalogen, phospholipids, glycolipids, cholesterol, galactocerebrosides, sulfatides, and myelin-specific proteins in the formation of tubular structures recently observed around developing CNS myelin sheaths. Using transmission electron microscopy, I aim to find the optimal conditions and ratios of myelin constituents for spontaneous tubule self-assembly in an attempt to recreate in vitro tubule structures observed in vivo. I further plan to use atomic force microscopy to examine the driving forces behind the transition of these self-assembled structures from tubules into multilamellar structures characteristic of a mature myelin sheath.


2014-15

Theresa Hwang
Photoswitchable affinity clamps: Modulating protein activity with light
Shohei Koide Group


Currently at MIT's Biology PhD Program, Keating lab

Optogenetics, the study of using genetically-encoded, light-controllable molecules to manipulate living systems, provides biologists with an extremely powerful way to control and observe cellular behavior. Hence, the challenge to broaden the range of cellular processes optogenetic tools can address is an interesting and pressing one. I propose the creation of a light-controllable affinity clamp whose binding affinity towards a target, can be modulated by light. Affinity clamps are novel binding proteins developed in the Koide lab that feature a unique clamshell like architecture. These clamps are a transformative solution in the quest to generate high affinity reagents that can bind to short, unstructured peptide segments present in biologically significant protein molecules, such as post-translation modification sites (PTMs). Hence, the coupling of light to allosterically control affinity clamps presents the possibility of a very potent tool that can link cellular processes with cellular behavior. The immediate goal of this project will be proof-of-concept, as light-controllable affinity clamps have never been produced. The creation of such a protein, and ultimately being able to control its behavior in cells through light dependent association and dissociation of the clamp to its targets, will allow us to determine a reliable method to rationally and systematically convert clamps into powerful cellular perturbation tools. This project will be accomplished through three aims: (1) the cloning and expression of a light-controllable affinity clamp, (2) modulating its binding affinity in vitro, and (3) testing its light-controllable activity in vivo. We can then extend the conclusions gained from this project to create a wide variety of clamps that can modulate PTM-dependent signaling.


Trevor Roberts
Product Branching of CH2CH2ONO Radical at 193 nm Using BrCH2CH2ONO
Laurie Butler Group

The experiments I proposed are designed to determine whether BrCH2CH2ONO can be utilized as a photolytic precursor to generate the CH2CH2ONO radical, a radical of potential importance in atmospheric chemistry, and to probe the unimolecular dissociation channels of that radical. Unlike at 351 nm excitation, which only cleaves the O-NO bond in BrCH2CH2ONO, we expect that excitation at 193 nm to raise BrCH2CH2ONO to an excited state repulsive in the C-Br bond that should allow us to photolytically cleave the C-Br bond. Radicals are difficult to study under bulk conditions due to their high reactivity, so employing collision free conditions allows for the study of particular radicals like CH2CH2ONO. If our initial experiments show that C-Br photofission occurs in some of the photoexcited molecules, we can then study the subsequent unimolecular decomposition of the CH2CH2ONO radical. In particular, two different product channels might occur, one producing NO and the other producing NO2. NO and NO2 are atmospherically abundant and responsible for facilitating numerous reactions; they play an important role in the ozone cycle. My proposed experiments use a crossed laser-molecular beam scattering apparatus with electron bombardment detection of the photofragments, resolving their velocity distribution to allow us to separate the NO formed in primary O-NO photofission from the NO produced from the unimolecular dissociation of BrCH2CH2ONO radicals. The measured recoil kinetic energies allow us to first determine the internal energy distribution of the nascent radicals, using conservation of energy and momentum. Then we can probe the energetics of the CH2CH2ONO radical decomposition product channels. The experiments, thus, give a deeper scientific understanding of potentially significant atmospheric mechanisms.


2013

Mara Farcasanu
Exploring the Structure and Biochemical Activity of the Free-State Anthrax Edema Factor Protein
Wei-Jen Tang Group

Edema Factor (EF) is an exotoxin secreted by the anthrax-causing bacteria Bacillus anthracis and plays an essential role in the progress of the disease within a cell. Upon entering the cell, EF is activated as an adenylyl cyclase by binding to calmodulin (CaM). Although the structure of active EF has been determined, the structure of inactive EF is not definitively known. To study both the structure and biochemical function of EF, it is important to begin with an understanding of the inactive EF's structure. Because the protein must unfold in order to enter the host cell, EF is recalcitrant to crystallization without the stability provided by CaM. Thus, it will first be important to find specific chaperones that will stabilize the protein. I propose to perform in vivo biotinylation and purification of EF, followed by a search for an antibody that would bind to EF with high affinity, made possible by collaboration with the Shohei Koide group. The stability provided by such high-affinity binders should aid in the crystallization of EF. Once appropriate antibodies and buffers are identified and crystallization has been successful, diffraction data for the crystals will be obtained at Advanced Photon Source at Argonne National Laboratory. Furthermore, the antibodies' effect on EF's function and activity can be analyzed by measuring in vivo cAMP concentrations of affected cells and through an enzymatic assay. Success in these aims will thus yield not only a structure of the CaM-free EF, but also provide insights towards a possible EF-based therapeutic against anthrax infection.


Isaac Larkin
Respiration and a Putative Carbon Monoxide Dehydrogenase in Caulobacter crescentus
Sean Crosson Group

Aerobic carbon monoxide dehydrogenase is an oxygen-stable enzyme that enables some bacteria to use carbon monoxide as an energy and a carbon source, and has potential applications to the sensing and degradation of CO in homes and industrial pollution. We have found genes that code for the L and S subunits of a putative carbon monoxide dehydrogenase in Caulobacter crescentus. We propose a project that uses protein engineering tools to purify the L and S subunits and pull down and characterize the missing M subunit. We will then use an anaerobic hemoglobin-based assay to determine the activity of the putative carbon monoxide dehydrogenase. In parallel, we will grow knockout and overexpression strains of the putative carbon monoxide dehydrogenase in C. crescentus, and determine via outgrowths in gas-tight ampoules containing different mixtures of air, argon, and CO whether C. crescentus can consume CO as a carbon or an energy source, and whether it requires the putative carbon monoxide dehydrogenase to do so.


2012

Kathleen Bohanon
Identification of 5-Hydroxymethylcytosine Binding Proteins in Mammalian Brain Tissue
Chuan He Group

Epigenetic DNA modifications play an essential role in brain function and are altered in various neurological disorders. The exact function of 5-hydroxymethylcytosine (5hmC), a recently discovered DNA modification that comprises 40% of modified cytosine in the brain, remains obscure due to inadequate techniques for precisely mapping it in genomic DNA. Utilizing the recently developed Tet-assisted Bisulfite Sequencing (TAB-Seq) method, it is possible to examine previously detected 5hmC-enriched loci with single-base resolution. The sequences found to be enriched with 5hmC will be used to design nucleic acid probes in order to pull down proteins that selectively bind to 5hmC. Since the presence (or absence) of this modification may affect the recruitment of various proteins, these results may illuminate the role that 5hmC plays in transcriptional regulation.


Christopher Delaney
Engineering Phosphotyrosine-Specific Affinity Clamps and using them to Dissect Cancer Signaling Pathways
Shohei Koide Group

The goal of this project is to enhance our understanding of phosphotyrosine (pY) signaling by developing engineered proteins and using them to dissect individual pY signaling pathways. Modern protein engineering focuses on creating novel biomolecular tools with scientific and medical applications. However, generating useful binding proteins with high affinity and specificity is challenging when targeting short epitopes or distinguishing between similar peptide motifs. Engineered proteins (termed affinity clamps) being developed in the Koide group, provide an innovative solution to these problems in that they demonstrate both exquisite binding affinity and specificity to typically elusive targets. For instance, affinity clamp technology could more easily produce binding proteins that recognize specific phosphorylated tyrosine residues, or pY sites, than could general protein engineering. Such pY-target affinity clamps would greatly benefit medical and biological research given the implications of rogue pY signaling in many cancers. Specifically, pY-targeted affinity clamps will help piece together the intricacies of pY signaling by binding to and blocking specific pY residues.


2009

Jason Hao
The Synthesis of Scaffold-Unbiased Small Molecule Libraries
Kozmin Group

This project proposes to use the methods of diversity oriented synthesis (DOS) to produce a library of compounds aimed at the discovery of new bioactive chemotypes. The chemical scheme begins with a 1,6-enyne, which through catalyst enabled diversification and selective [4+2] Diels Alder reactions may yield 25 cycloadducts, all of which have an unsaturated double-bond suitable for epoxidation. This project intends to further diversify these adducts via two possible pathways. In the first scheme, read more...


Vasilios Kalas
Exploring the Molecular Bases of Substrate Recognition and Degradation by Human Insulin-Degrading Enzyme
Wei-Jen Tang Group

Insulin-degrading enzyme (IDE) is a metalloprotease that demonstrates a remarkable ability to recognize and degrade functionally and structurally diverse bioactive peptides such as insulin and amyloid-beta (Aβ) yet exhibits high selectivity and affinity to the targeted substrates. While many of this protease's unique structural properties have been studied extensively in understanding its catalytic versatility, the mechanism by which IDE binds and degrades structurally diverse peptides remains elusive. To gain a better understanding of this intriguing mechanism, this project will investigate two read more...


2008

Carl Brozek
The Search for a Terminal Ni Oxo Complex
Gregory Hillhouse Group
Current Institution: MIT
Program: Ph.D. in Chemistry
Honors: National Science Foundation Graduate Fellowship

While early-metal oxo complexes (M=O) are common and demonstrate interesting reactivity, late metal complexes are unobserved. Recently, the Hillhouse group synthesized the d8 imido complex 1,2-bis(di-tert-butylphosphino) ethane)Ni=NAr. Attempts to synthesize the analogous d8 oxo led to rapid decomposition to an oxidized phosphine. Though unsuccessful, these results strongly suggested a Ni(II) oxo intermediate. This project will focus on the isolation of a Ni=O complex by introducing a more rigid backbone in the chelated phosphino ligand to prevent intramolecular migration of phosphorus to oxygen. read more...


Dan Houle
Chemical Cross-Linking to Study the Electrostatic Switching Mechanism of E. coli N-Ada
Chuan He Group

In order to prevent mutagenic damage to the genome by cellular or environmental alkylating agents, many organisms have evolved a set of DNA repair proteins that search for and repair these covalent modifications. Escherichia Coli N-Ada is an exemplary DNA repair protein, serving a dual role in methylation resistance: it first acts to repair methyl phosphotriester damage by directly transferring a methyl group from the DNA backbone to its tetrathiolate zinc(II) active site. Upon this methylation, N-Adas affinity to DNA increases 100-1000 fold, allowing it to bind sequence specifically to the ada promoter, initiating transcription of a collection of methylation resistance proteins. read more...


2007

Donnie Bungum
Synthesis of  61Ni compounds for Mossbauer spectroscopy
Gregory Hillhouse Group
Honors: Goldwater Scholar, Marshall Scholar

Mossbauer Spectroscopy is one of the most powerful tools available for characterization of metal-containing compounds. Based on a Nobel Prize winning principle of high-energy physics discovered by Dr. Rudolf Mossbauer, this technique allows researchers to determine the oxidation state, electronic environment, spin state, symmetry, and magnetic properties of compounds containing one of the 100 or so Mossbauer-active metals. Nearly 90% of the literature on Mossbauer spectroscopy published to date concerns Fe species, as this isotope of iron can be studied in a wide range of chemical environments under varied experimental conditions.
read more...


Emily Jane Glassman
Investigation of the Unimolecular Dissociations of CH3OSO and CH3SO2 Radicals

Laurie Butler Group
Current Institution: University of California, Berkeley
Program: Ph.D. in Chemistry
Honors: National Science Foundation Graduate Fellowship

The reactions of the CH3SO2 and CH3OSO radicals are particularly important processes to understand in the fields of combustion and atmospheric chemistry. As coal-burning technologies expand, it becomes imperative to understand the dynamics of reactions involving sulfur, one of the major components of coal. The radicals and their products, CH3, CH3O, SO, and SO2 play a significant role in the atmospheric oxidation cascade of dimethyl sulfide, a major natural source of sulfur. However, the energy barriers for the unimolecular dissociations of CH3OSO and CH3SO2 have never been positively determined. read more...


2006

John Anderson
Exploring the Biologically Inspired Chemistry of Nickel(I)

Gregory Hillhouse Group
Current Institution: MIT
Program: Ph.D. in Chemistry
Honors: National Science Foundation Graduate Fellowship

My research is focused on using nickel systems involving the chelating ditertbutylphosphinoethane (dtbpe) ligand system to reproduce some proposed biological intermediates in the laboratory. This system has already been used extensively to explore a number of interesting nickel complexes ranging in oxidation state from zero to three. I will be studying nickel one in particular due to the biological significance of this oxidation state.

Nickel is believed to be an important oxidation state in both the acetyl coenzyme a synthase/carbon monoxide dehydrogenase (ACS/CODH) and the nickel superoxide dismutase (NiSOD) enzymes. read more...


James Fitzgerald
Optimal Coordinate Systems and the Backbone Dependence of Energy Functions used for Protein Folding Simulations and Structure Prediction
Tobin Sosnick Group
Graduation Date: 2007
Current Institution: Stanford University
Program: Ph.D. in Physics
Honors: National Science Foundation Graduate Fellowship

Our overall goal is to create an energy function to address currently inaccessible problems in protein folding and binding. Statistical potentials specify the "energy" of a protein structure based upon statistics obtained from a library of protein structures. The energy is assigned according to the Boltzmann distribution. The benefits of this approach are immediately clear. Rather than explicitly including physical interactions between atoms or groups of atoms, statistical mechanical concepts are used to relate the probability of occurrence to the energy, so the precise forms of the interaction potentials are irrelevant. Moreover, by using a statistical potential, realistic modeling of the solvent becomes unnecessary, thereby providing another enormous computational simplification.

However, in order for statistical potentials to be computationally useful, the read more...


Amy Winans
The Effects of GM1 Ganglioside- and Cholesterol- Containing Membranes on Amyloid-β Binding and Aggregation
Ka Yee Lee Group
Current Institution: Stanford University
Program: Ph.D. in Biophysics
Honors: National Science Foundation Graduate Fellowship

My works focuses on how the addition of gangliosides (specifically monosialic ganglioside: GM1) and cholesterol to a model cell membrane mediates the interaction between the membrane and the amyloid-β protein. (A-β). A-β has been identified as the protein composing the amyloid plaques found deposited in the brains of Alzheimer's patients. Under physiological conditions, the A-β protein folds into a random coil. In amyloid plaques however, A-β is found deposited in cross-linked β-sheet conformation. A growing body of research has provided evidence for a link between cell membranes and A-β aggregation into β-sheet fibrils. Interaction with the cell membrane may also be the way in which A-β exerts its neurotoxic effects. read more...


2005

Doran Bennett
Ground State Nonadiabatic Dynamics in Combustion Intermediates: A Study of the Vinoxy →H + Ketene Product Channel
Laurie Butler Group
Graduation Date: 2007
Current Institution: University of California, Berkeley
Program: Ph.D. in Chemistry
Honors: Goldwater Scholar , National Science Foundation Graduate Fellowship

The vinoxy radical (CH2CHO) has shown itself to be of both practical and theoretical interest. Its practical interest arises from combustion chemistry where vinoxy is an intermediate for a number of important reactions:

  • (1) O(3P) + C2H4i
  • (2) O(3P) + C2H3ii
  • (3) OH + C2H2iii
  • (4) OH + C2H4Oiv

These reactions are important because they are commonly found in the mechanisms for the combustion of larger aliphatic and aromatic compounds. The research I propose will probe the dissociation dynamics of the vinoxy radical by way of the potential energy surface (PES), illuminating the role vinoxy pays in the overall combustion process.

Vinoxy is of theoretical interest because it exhibits nonadiabatic effects in its ground state. Often in textbooks the read more...


Michelle Rengarajan
Using molecular modeling and structural screens to identify potentially novel inhibitors of Saccharomyces cerevisiae cyclin-dependent kinase Cdc28
Steve Kron Group
Current Institution: Stanford University
Program: M.D. / Ph.D.
Honors: Medical Science Training Program Fellowship

A vast body of protein structural data and sequence information has in recent years been made available for a wide variety of organisms. Before attempts to process this information experimentally, molecular modeling can be a powerful tool to mine and utilize this otherwise overwhelming body of data. In particular, molecular modeling of proteins can be used to search for structural components conserved across species or components which, if mutated, result in a compensatory mutation in an interacting partner. I propose to use ab initio modeling, homology modeling and structural screens to search for inhibitors of Cdc28 Saccharomyces cerevisiae cyclin-dependent kinase (CDK), which is involved in the transition between the G1 and S phases of the cell cycle. read more...


2004

Joshua D. Tice
Nanoliter droplet-based microfluidic system for evaluating protein crystallization conditions with on-chip diffraction

Rustem Ismagilov Group
Graduation Date: 2005
Current Institution: University of Illinois, Urbana, Champaign
Program: Ph.D. in Chemistry

Growing high-quality crystals of proteins and other biological macromolecules plays an important role in the determination of tertiary structure. Crystallization conditions are usually identified by screening a large number of assays with variable ratios of solutions of the protein, precipitants, and other additives. Microfluidic technology presents an opportunity to improve this process by reducing the amount of protein needed, reducing labor, and making it economically feasible for use in individual laboratories. A microfluidic system, comprising a network of channels fabricated in poly(dimethylsiloxane) (PDMS) and a glass x-ray capillary, was used to perform protein crystallization trials in nanoliter-sized droplets.
read more...