Current Trainees

Trainees appointed in 2020

  • Lannah Abasi

    Lannah Abasi

    Undergraduate Institution: CSU Northridge
    Program: Chemistry & Biochemistry
    Advisor: Galia Debelouchina

    Phase transitions of tau

    Tau is a microtubule-associated protein found in neurofibrillary tangles (NFTs) in the brains of Alzheimer's patients. While tau forms amyloids inside the NFTs, it is unclear how fibrillization initiates in this strikingly soluble protein. Intriguingly, tau and several other amyloidogenic proteins can undergo liquid-liquid phase separation (LLPS), and it was also found that phosphorylation of tau enhances LLPS and that these droplets progress into viscous gel-like states and β-sheet rich aggregates, connecting this process with fibrilization. This has led to speculation that droplets and gels could represent an aggregation prone intermediate in this process, highlighting a need for structural and biophysical studies. My project is focused on understanding the phase transitions of tau.

  • Adarsh Balaji

    Adarsh Balaji

    Undergraduate Institution: UCLA
    Program: Chemistry & Biochemistry
    Advisor: Colleen McHugh

    Biophysical characterization of long non-coding RNA and protein complexes

    Long non-coding RNA (lncRNA) refer to a class of RNA molecules that contain more than 200 nucleotides and do not code for protein sequences. LncRNAs are currently extensively studied due to their observed dysregulation in human diseases and oncogenic or tumor-suppression function in cancers. LncRNA function is often dependent on the formation of a stable long non-coding ribonucleoprotein complex (lncRNP) for downstream regulation of target genes or proteins. Unfortunately, current computational modelling approaches for lncRNP identification are unable to provide residue-specific information regarding binding dynamics. In addition, there are very few biophysical studies for lncRNP identification in the current literature.3 Thus, a biophysics-based approach to study the folding, structure and binding of these novel lncRNPs will help address this gap in literature and shed light on the role of lncRNAs in human disease. The MALAT1/TDP43 lncRNP in human K562 cancer cells will be used as the experimental model. A tandem of biophysical techniques will be explored, including density ultracentrifugation, a combination of northern and western blotting, and microscopy for structural studies.

  • Andrea Dickey

    Andrea Dickey

    Undergraduate Institution: UC Berkeley
    Program: Biological Sciences
    Advisor: Samara Reck-Peterson

    Investigating the Role of LRRK2 in Regulating the Microtubule Cytoskeleton and Intracellular Transport

    The microtubule cytoskeleton and its motors are responsible for the spatial and temporal distribution of intracellular components, which are vital for cellular development and survival. Non-motor microtubule-associated proteins help to maintain this delicate distribution of intracellular components by stabilizing or destabilizing the cytoskeleton and disrupting or enhancing the motility of motor proteins. Leucine Rich Repeat Kinase 2 (LRRK2) is a large, multi-domain protein that has roles in intracellular trafficking and colocalizes with microtubules. Mutations in LRRK2 are the most common cause of familial Parkinson’s Disease. The Reck-Peterson and Leschziner labs recently showed that microtubule-associated LRRK2 can act as a roadblock for the motor proteins dynein and kinesin in vitro. However, many questions remain as to what impact this microtubule-associated LRRK2 has on intracellular transport and microtubule dynamics. The goal of my work is to understand the mechanisms by which LRRK2 filaments form on microtubules, their impact on microtubule dynamics, and their role in modulating intracellular transport. I am using biochemical and in vitro reconstitution methods combined with single-molecule imaging to dissect the mechanisms of LRRK2’s interaction with the cytoskeleton. I aim to provide molecular-level insights into LRRK2’s functions and how they are perturbed in disease.

  • Nesreen Elathram

    Nesreen Elathram

    Undergraduate Institution: UNC Charlotte
    Program: Chemistry & Biochemistry
    Advisor: Galia Debelouchina

    Elucidating the role of histone H4 K20 methylation in chromatin compaction

    Chromatin is a complex of protein and DNA that works together to guard and regulate genetic information in eukaryotic cells. As the fundamental building block of chromatin, the nucleosome comprises 147 bp of DNA wrapped around an octamer of the histone proteins H2A, H2B, H3 and H4. The nucleosome is the active site of many different post translational modifications (PTMs) such as acetylation, methylation and ubiquitylation. These PTMs function in the recruitment of chromatin effector proteins and by directly impacting chromatin’s biophysical properties. I am investigating two post-translational modifications involved in gene silencing. This includes histone H3 lysine 9 methylation which is recognized by a key gene silencing protein called heterochromatin protein 1α (HP1α), and histone H4 lysine 20 trimethylation which is known to compact chromatin.  To understand how these modifications, regulate chromatin structure and dynamics, I am developing advanced solution and solid-state NMR tools that allow us to probe chromatin environments at atomic resolution. My project aims to uncover how small chemical modifications such as methylation can influence gene regulation and nuclear organization on a much larger scale.

  • Ximena Garcia Arceo

    Ximena Garcia Arceo

    Undergraduate Institution: UC Santa Barbara
    Program: Chemistry & Biochemistry
    Advisor: Brian Zid

    Stochastic Modeling of mRNA Localization to Mitochondria

    Over 99% percent of yeast mitochondria proteins are transcribed in the nucleus and translated in the cytoplasm, yet some key proteins are encoded by the mitochondrial genome. The simultaneous translation and localization of nuclear-encoded mitochondrial mRNAs attunes the cytoplasmic to the mitochondrial translation program but the gene-specific and condition-specific localization behaviors have not been modeled quantitatively. We have created a stochastic model that incorporates molecular binding, translation and morphology to predict the localization patterns of mRNAs, which are known to be sensitive to mitochondrial size. Given the inherent competition between diffusion and biochemical affinity of some mRNA-ribosome complexes for the mitochondrial surface, the model also illuminates the limits as well as the opportunities for differential localization of various classes of mRNAs. Since mitochondria increase in volume as respiratory flux and cellular doubling time increase, our model informs our experimental endeavor to elucidate post-transcriptional regulation of metabolic flux and cellular growth rate.

  • Israel Juarez Contreras

    Israel Juarez Contreras

    Undergraduate Institution: UC Merced
    Program: Chemistry & Biochemistry
    Advisor: Itay Budin

    Uncovering the biophysical basis for sterol evolution and diversification in cell membrane 

    The pathways for sterol synthesis have been studied extensively in vertebrates, fungi and land plants. However intriguing questions remain:  

    1)    Spectroscopic assays show that the main sterol in fungi, ergosterol, has a larger ordering effect on lipid bilayers compared to the main sterol in vertebrates, cholesterol. Yet, yeast can utilize either for growth. What specific advantage do fungi acquire from ergosterol?  

    2)    The sterol intermediates: lanosterol and cycloartenol are likely to have been the initial sterols utilized by the last eukaryotic common ancestor (LECA). What sort of tradeoffs resulted in the LECA to switch between them?  

    3)    It is hypothesized that sterol biosynthesis appeared and evolved because of the oxygenation of the atmosphere. However, there are known sterol-like molecules in bacteria and some eukaryotes that do not require oxygen for their synthesis. Information regarding the biophysical properties and cellular effects of these molecules are limited.  

    In order to answer these questions, I will be characterizing how different sterol chemistries affect membrane structure and organization through the use of synthetic lipid vesicles and engineered yeast strains. This will lead to fundamental insights on the function of these lipids in membranes and the evolution of eukaryotic cells. 

  • Emily Pool

    Emily Pool

    Undergraduate Institution: Butler University
    Program: Chemistry & Biochemistry
    Advisor: Susan Taylor and Jin Zhang

     Elucidating the regulation and functional role of RIβ liquid-liquid phase separation

    Protein kinase A (PKA), the prototypic kinase model for signal transduction, is a tetrameric holoenzyme of two regulatory (R) subunits and two catalytic (C) subunits. PKA is activated when cyclic AMP (cAMP), a second messenger, binds to the holoenzyme and releases active C subunits. Both R and C subunits have functionally nonredundant isoforms with variable tissue expression. The RIβ isoform’s specific function and localization is not well-characterized. Interestingly, RIβ knockout and mutant mice have shown learning defects, nociceptive pain, and decreased inflammatory responses, which are phenotypes associated with aberrant cAMP signaling. The RIβ isoform, expressed mainly in hippocampal neurons and retinal tissues, has been shown to engage in liquid-liquid phase separation, which challenges our understanding of how cAMP is spatiotemporally regulated. I am using FRET-based biosensors to elucidate how cAMP is buffered and regulated by these RIβ phase-separated compartments. These insights will contribute to the current model of signaling specificity within the cAMP/PKA pathway and provide an improved understanding of cAMP-regulated functions and associated diseases.  

  • Brandon Rawson

    Brandon Rawson

    Undergraduate Institution: UC Irvine
    Program: Physics
    Advisor: Doug Smith

    DNA packaging dynamics in bacteriophage T4 and Lambda

    Many eukaryotic viruses and bacteriophage are reliant on an ATP-powered molecular motor during assembly to package their genomes into preformed procapsids. These motors bind and hydrolyze ATP to package double stranded DNA to near-crystalline densities against high resisting forces due to confinement. Previous work on bacteriophage T4 has produced a model of the motor protein with two distinct domains, one of which grips the DNA via positively charged residues. Electrostatic interactions drive a conformational change of the two domains which moves the DNA ~2 bp into the viral prohead. I will probe the role of residues in the supposed flexible hinge region between domains, predicted to grip the DNA and that of the residues involved in coupling between the ATP hydrolysis cycle events and translocation. Effects of residue changes in various T4 mutants will be observed using optical tweezers to monitor the packaging kinetics for individual complexes. I am also currently investigating the dependence of lambda’s motor velocities, pauses, and slipping on ATP and slowly hydrolyzed residue concentrations and will look to extend similar measurements to T4 mutants of interest. 

Trainees appointed in 2019 and reappointed in 2020

  • 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.