Current Trainees

Trainees appointed in 2019

  • Aileen Button

    Aileen Button

    Undergraduate Institution: University of Vermont
    Program: Chemistry & Biochemistry
    Advisor: Colleen McHugh

    Characterization of interactions between Xist long non-coding RNA and transcriptional regulators of gene expression

    Long non-coding RNA (lncRNA) and protein interactions play important roles in cellular function. Increasing numbers of these interactions are being identified by high throughput methods. However, more detailed structural characterization of these complexes is needed to determine the molecular basis of RNA-protein interactions, and to understand how these molecules achieve specificity in the complex cellular environment. Prediction of RNA-protein interactions is complicated by a relative lack of sequence conservation in RNA molecules, and secondary and tertiary RNA structures seem to be conserved and important for function.

    A major challenge in the field is that we still do not understand the rules governing lncRNA-protein interactions. To start addressing this question, we are studying the interaction between Xist (a lncRNA) and SHARP (a transcriptional repressor). This interaction was recently identified by RAP-MS (RNA affinity purification with mass spectrometry) and has been shown to be required for X chromosome inactivation in female mammals, including humans. Interestingly, SHARP also binds another lncRNA, the steroid receptor RNA activator (SRA)5. Furthermore, SHARP contains four RNA recognition motifs (RRMS), which are well-established domains for RNA binding. For these reasons, the Xist-SHARP and SRA-SHARP interactions are an excellent gateway into understanding what makes a lncRNA-protein interaction specific.

  • Quinn Cowan

    Quinn Cowan

    Undergraduate Institution: University of Southern California
    Program: Chemistry & Biochemistry
    Advisor: Alexis Komor

    Multiplexing base editing for the functional investigation of linked-SNVs

    Although sequencing has identified 4.6 million missense variants, only 2% have a defined clinical interpretation in ClinVar and over half remain variants of uncertain significance. These variants often occur in tandem, requiring multiple editing events for functional analysis. Traditional CRISPR/Cas9 genome editing methods rely on DNA double-strand break (DSB) induction and the disfavored homology-directed repair pathway. The simultaneous introduction of multiple DSBs is cytotoxic, yields low efficiencies, and forms unwanted genetic alterations. However, a new class of genome editing tools, base editors, were recently developed that circumvent DSB induction. The two existing base editors employ an inactivated Cas9 and specific deamination chemistries to catalyze the conversion of C•G to T•A base pairs or A•T to G•C base pairs. Engineering a multiplexed base editing system to precisely install multiple point mutations throughout the genome will allow researchers to endogenously characterize of the effects of linked-single nucleotide variants (SNVs). This novel system would be transformative for the study and potential treatment of human genetic diseases.

  • Alexander Hoffnagle

    Alexander Hoffnagle

    Undergraduate Institution: University of Pennsylvania
    Program: Chemistry & Biochemistry
    Advisor: Akif Tezcan

    De novo design of dimeric metalloenzymes

    Metalloenzymes fulfill diverse cellular roles and are capable of catalyzing a wide range of chemical reactions. As such, there is considerable interest in engineering metalloenzymes to carry out functions for applications in synthetic chemistry, biotechnology, and more. Recently our lab has developed a new approach to designing a protein metal binding site, dubbed Metal Active Sites by Covalent Tethering, or MASCoT. Unlike other engineering approaches, which largely rely on a preexisting site in the interior of a tertiary or quaternary structure, MASCoT allows for a metal binding site to be created along a protein-protein interface. Crucially, these metal binding sites tend to be coordinatively unsaturated, raising the possibility of using them as active sites in catalysis. My goal is to use rational design and directed evolution to develop these MASCoT-designed metal binding proteins into novel, functional metalloenzymes.

  • An Hsieh

    An Hsieh

    Undergraduate Institution: Case Western Reserve University
    Program: Chemistry & Biochemistry
    Advisor: Tatiana Mishanina

    Human mitochondrial transcription and DNA replication

    Transcription of DNA to RNA by RNA polymerase (RNAP) is an underlying foundation of biological processes. However, an important place where transcription occurs that is still not well understood is in human mitochondria. The mitochondrial genome (mtDNA) encodes essential proteins required for oxidative phosphorylation (OXPHOS), the cell’s major source of energy. To keep up with the needed energy production, mitochondria must replicate their genome and divide constantly – a process closely coupled to transcription.  Mutations that compromise OXPHOS and replication can lead to devastating disease. Therefore, my project is to gain a thorough understanding of mitochondrial transcription and its coupling with mtDNA replication and other gene expression processes, particularly when the mitochondrial RNA polymerase (mtRNAP) pauses transcription. I will test a pause site of mtRNAP and determine its potential role in switching from transcription and mtDNA replication.

  • Calvin Lin

    Calvin Lin

    Undergraduate Institution: UC Santa Barbara
    Program: Chemistry & Biochemistry
    Advisor: Elizabeth Komives

    Structural characterization of ubiquitination in Cullin-RING E3 Ligases

    Our cells have evolved to recycle proteins and use the ubiquitin (Ub) proteasome system as the main mechanism to target specific proteins for degradation. Protein complexes known as E3 ligases initiate this process by tagging proteins with Ubiquitin chains, which are recognized and degraded by proteasomes. Cullin-RING complexes are the largest group of E3 ligases and are involved in neurodegenerative diseases and various cancers. However, a lack of structures of multi-protein Cullin-RING complexes limits comprehensive understanding of the E3-mediated ubiquitination mechanism and rational drug design. My goal is to obtain structures that capture the full complex before and during Ub transfer using cryo-electron microscopy (cryoEM). I will complement my structural studies of Cullin5-RING complex with information about the dynamics between substrate and E3 using crosslinking and hydrogen-deuterium exchange mass spectrometry (HDX-MS).

  • Hoang Nguyen

    Hoang Nguyen

    Undergraduate Institution: Arizona State University
    Program: Chemistry & Biochemistry
    Advisor: Mark Herzik

    Structural and biophysical insights into mitochondrial preprotein receptor plasticity

    Mitochondria are the central hub of eukaryotic cells and are critically involved in many cellular processes including metabolism, energy production, heme and lipid biosynthesis, and apoptosis. Of the ~1500 proteins that necessary for normal mitochondria function in humans, 99% of those proteins result from nuclear-encoded genes that must be translated in the cytosol as precursor proteins (preproteins) and subsequently imported into mitochondria. Importantly, mitochondrial dysfunction is a hallmark of a wide variety of diseases, including cancer, cardiovascular disease, diabetes, aging, and neurodegeneration, with most of these diseases exhibiting some form of impaired mitochondrial protein import. The translocase of the outer membrane (TOM) complex is the primary preprotein gateway locating to the outer membrane of mitochondria and imports nearly all mitochondrial preproteins. The TOM complex comprises 7 subunits, including two primary preprotein receptors, Tom20 and Tom70, that recognize mitochondrial preproteins possessing either a N-terminal signaling sequence or an internal targeting signal, respectively, for import into mitochondria. Amazingly, these receptors recognize, bind, and deliver ~1300 different preproteins to the TOM complex despite the considerable sequence variation that exists within these targeting signals. Furthermore, Tom70 possesses a single preprotein binding site that must adapt to the vast sequence space necessary for preprotein engagement and import. Although these complexes have been studied biochemically for decades, a myriad of open questions remain due to a lack of structural information. Using a combination of high-resolution single-particle electron cryomicroscopy (cryo-EM), hydrogen-deuterium exchange by mass spectrometry (HDX-MS), molecular dynamics simulations, and biochemical assays, I aim to provide molecular-level insights into Tom70 preprotein engagement and import.

  • Angel Payan

    Angel Payan

    Undergraduate Institution: Rochester Institute of Tech.
    Program: Chemistry & Biochemistry
    Advisor: Mark Herzik

    Utilizing Molecular Dynamics and in silico Electron Microscopy Data to Better Understand Conformational Heterogeneity in cryo-EM

    A molecular-level description of the complex conformational landscape a biological macromolecule must traverse to perform its function is critical to understanding its role in the cellular context. Electron cryomicroscopy (cryo-EM) is an increasingly powerful tool for visualizing these conformationally complex macromolecules in near native environments. Indeed, a single cryo-EM dataset often enables us to view multiple conformations of macromolecules that can then be “in silico” purified using current data processing methodologies to yield multiple conformationally distinct major classes. Unfortunately, however, these processing strategies often yield the most populated, low-energy states of a given protein with local dynamics being lost. The major challenge is distinguishing between these subtle minor conformations in order to gain a better insight into both the local and global dynamic behavior of proteins. Toward this end, I will generate in silico simulated EM data derived from long-scale molecular dynamics simulations of several proteins whose structures have been determined to high-resolution using cryo-EM. Using these data, I will develop methodologies to better quantify local and global conformational heterogeneity that can then be applied to all cryo-EM data. Achieving this will allow for the thorough investigation of these protein ensembles and their roles in protein function, further pushing the boundaries of cryo-EM.

  • Christine Stephen

    Christine Stephen

    Undergraduate Institution: UC Santa Barbara
    Program: Chemistry & Biochemistry
    Advisor: Tatiana Mishanina

    Structural and Mechanistic Study of a pH-Responsive Riboswitch

    Riboswitches are 5’-untranslated regions (UTRs) of mRNA that control the fate of their respective transcript in response to a ligand-binding event.  However, this ligand-based model of riboswitch function has been challenged with the discovery of a “pH-responsive element” (PRE) riboswitch at the alx gene locus in E. coli.  At neutral pH, the PRE folds into a translationally inactive structure with an occluded ribosome binding sequence; however, at alkaline pH, the PRE adopts a translationally active structure. PRE folding is kinetically controlled: RNA polymerase (RNAP) pausing – a temporary inhibition of nucleotide addition during transcript elongation – is enhanced by alkaline pH at two specific sites on the alx gene, promoting folding of the PRE into the active structure.  Although it is known that transcriptional pausing mediates these distinct structural outcomes, the mechanistic basis for this pH-responsive pause behavior remains unknown. With the support from the training grant, I will investigate the molecular mechanisms underlying this novel, pH-responsive riboswitch, by utilizing a combination of structural approaches and biochemical assays. Ultimately, elucidating the mechanism of PRE folding and its control by RNAP pausing will drive design of pH-responsive sensors for microbial engineering, which has diagnostic and therapeutic applications.

  • Ryan Weeks

    Ryan Weeks

    Undergraduate Institution: North Carolina State University
    Program: Chemistry & Biochemistry
    Advisor: Jin Zhang

    Direct detection of intracellular Ras protein

    Ras is a key signaling protein that controls numerous cellular outcomes such as growth and differentiation.  Activated downstream of epidermal growth factors, and upstream of the Raf-MEK-ERK axis, this protein plays a key role in controlling growth in cells.  As such, activating mutations of Ras are frequent in many cancers.  Currently I am developing and characterizing a genetically encoded biosensor that interacts with activated Ras and reports on its activation directly.  Biosensors to date, such as the Ras-Raichu sensor, contain full-length Ras protein within the design, and technically report on the activity of upstream regulators.  With direct detection, endogenous levels of Ras may be sensed, and direct detection of Ras inhibition by small molecule drugs can theoretically be tracked live within the cell.

Trainees appointed in 2018 and reappointed in 2019

  • Bryce Ackermann

    Bryce Ackermann

    Undergraduate Institution: University of California: Davis
    Program: Chemistry & Biochemistry
    Advisor: Galia Debelouchina

    Interrogating genome packaging

    The human genome is compacted into cell nuclei in the form of chromatin, a giant polymer of protein and DNA that spatially organizes to allow for timely execution of nuclear processes. On a crude level, chromatin separates into transcriptionally hindered and transcriptionally active regions dependent on local chemical modifications and chromatin interacting proteins that lead to differential compaction densities. The effects can be visualized at the nuclear level but difficult to study in molecular detail. Therefore, our lab will focus on advancing methodology for structural biology in cells. We will develop chemical biology and nuclear magnetic resonance spectroscopy tools to investigate the structure and dynamics of differentially packaged chromatin in human cells.

  • Joshua Corpuz

    Joshua Corpuz

    Undergraduate Institution: UC San Diego
    Program: Chemistry & Biochemistry
    Advisor: Michael Burkart

    Understanding PCP-mediated protein-protein interactions in NRPS systems.

    49% of FDA approved anticancer drugs are natural products or derivatives of natural products. Natural products also have a variety of bioactivities in humans and other organisms, including antibiotic, antiviral, and antifungal activities. A major family of enzymes that synthesize these natural products are nonribosomal peptide synthetases (NRPS). Since they are prevalent in natural product synthesis, NRPSs have become targets for protein engineering to create novel pharmaceutical drugs and novel biosynthetic pathways of the drugs.

    NRPS are modular enzymes that act like an assembly line to create peptide products.One of the core domains in the NRPS is the peptidyl carrier protein (PCP); its role is to transport the growing peptide chain between multiple domains in a specific order. The molecular mechanism by which the PCP recognizes the different domains is not well understood. My project helps advance our understanding of PCP-mediated protein-protein interactions through biochemical and structural analysis of the PCP and partner proteins. With increased understanding of the interactions, we can better engineer NRPS systems to create novel pathways and pharmaceutical drugs.

  • Mounir Fizari

    Mounir Fizari

    Undergraduate Institution: Univ. of Illinois Urbana-Champaign
    Program: Physics
    Advisor: Doug Smith

    Single molecule studies of bacteriophage phi29 DNA packaging

    A critical step in the lifecycle of many bacteriophages and some eukaryotic viruses is the packaging of the viral genome into a prohead by an ATP-powered molecular motor. The bending rigidity and electrostatic self-repulsion of the DNA and the entropic penalty of confinement make this process energetically unfavorable. In bacteriophage phi29, previous studies have found evidence that the nonequilibrium dynamics of the confined DNA affect packaging by providing a resistive load on the motor and triggering an allosteric feedback mechanism. Using optical tweezers to monitor the force exerted and rate of DNA packaged by individual phi29 complexes, I will vary the prevalence of nonequilibrium conformations by reducing the rate of packaging, nicking the genome to reduce the DNA’s bending rigidity, and using a larger mutant phi29 prohead to probe the relationship between nonequilibrium conformations and packaging kinetics.

  • Sonjiala Hotchkiss

    Sonjiala Hotchkiss

    Undergraduate Institution: Univ. of New Orleans
    Program: Chemistry & Biochemistry
    Advisor: Gourisankar Ghosh

    My proposed research will focus on characterizing and elucidating how the binding of small molecule inhibitors of human IKK2/b affect its protein dynamics and therefore its function. IKK2 is a catalytic subunit of the heterotrimer IkB Kinase (IKK). The two additional subunits are IKK1/a, also a catalytic subunit, and NF-kB essential modulator (NEMO/IKKg), a regulatory subunit. IKK is an essential kinase in the activation pathway of the nuclear factor kB (NF-kB) family of transcription factors. This family consists of five proteins that can combine to form homo- or hetero-dimers: p65/RelA, p50, p52, c-Rel, and RelB. The NF-kB dimers remain inactive in most cells which can be activated by a large number of stimuli including cytokines, pathogens and radiation though a cascade of reactions that mostly starts at the cell surface. NF-kB activation occurs along two pathways, the canonical pathway and the noncanonical pathway. As an enzyme in the canonical pathway, IKK2 is activated by phosphorylation of its activation loop serines, S177 and S181.

  • Dominic McGrosso

    Dominic McGrosso

    Undergraduate Institution: CSU San Marcos
    Program: Biomedical Sciences
    Advisor: Geoffrey Chang

    My current research in the lab of Dr. Geoffrey Chang utilizes directed evolution techniques to generate and characterize small molecule binders to investigate the physical properties and biomolecular structure of small molecule pollutants and their binders. Small molecules, especially pollutants such as polycyclic aromatic hydrocarbons, polybrominated diethyl ethers, and triclosan, have been found in many environments including those essential for humans, like water used for drinking, ocean fisheries, and farmland. Many of these molecules have been demonstrated to have negative effects on human health and environmental robustness. There are multiple methods used to detect small molecule pollutants in use today, including mass spectrometry and other various chromatographic and spectrophotometric methods, however each of these methods have inherent weaknesses that preclude them from direct field applications.

  • Elizabeth Porto

    Elizabeth Porto

    Undergraduate Institution: Univ. of Missouri - Kansas City
    Program: Chemistry & Biochemistry
    Advisor: Alexis Komor

    Expanding the Scope of DNA Base Editing

    The use of the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system has become the standard method for genome editing. The system relies on the ability of Cas9 to introduce a double strand break (DSB) at a desired DNA sequence, followed by precise repair from a donor template through homology-directed repair (HDR). However, DSB-reliant genome editing results in stochastic mixtures of unwanted genome modifications, making it less reliable for commercial use. Base editing is an alternative technique that enables the direct, irreversible conversion of a single target DNA base in a precise, programmable manner without introducing a DSB or requiring a donor template. This methodology is currently limited in its scope by only facilitating C•G to T•A or A•T to G•C base pair conversions. My project is focused on engineering new DSB-free genome editing tools that expand the types of DNA base pair transformations researchers can cleanly and efficiently introduce into the genome of living cells. This project will advance basic scientific understanding of genome editing enzymes as well as accelerate novel organism engineering efforts, with the potential to be used commercially in improving therapeutic efforts.

  • Douglas Zhang

    Douglas Zhang

    Undergraduate Institution: UCLA
    Program: Chemistry and Biochemistry
    Advisor: Thomas Hermann

    Functionalization of Nucleic Acid Nano-Structures

    Nucleic acids have traditionally been recognized for their role in carrying genetic information, however, their ability to self-assemble into a variety of shapes based on simple Watson-Crick base-pairing rules makes nucleic acids an ideal material for nanotechnology. Combining the structures of RNA as architectural joints with DNA as functional modules, the Hermann lab has created a variety of nucleic acid nano-architectures. We are now focused on expanding the types of nano-structures that can be formed as well as the functionalization of these nano-structures for the purposes of protein-protein interaction studies, biosensors, and catalysis.