During spring quarter, UCSD ACS-SA partners with the Department of Chemistry and Biochemistry to host an Undergraduate Research Symposium celebrating the research contribution of our student chemist community. The event provides our student researchers with a great opportunity to practice science communication using a poster presentation. Presenting a poster is required for department honors, and the best presenters within each division receive a symposium poster presentation award. More information on department honors and undergraduate student awards can be found below:
Due to the ongoing COVID-19 pandemic, ACS-SA’s Undergraduate Research Symposium will take place on Zoom. During the poster sessions, we will be utilizing breakout rooms to host poster presentations. It is important that you have the most recent version of Zoom installed, so that you can choose the poster presentations to see. More information on how to use breakout rooms and how to check your Zoom version can be found below:
Looking for an opportunity to enhance your undergraduate research experience by communicating your findings to a welcoming chemistry community? The ACS-SA Symposium this upcoming Spring 2021 is perfect for you! Present your findings via digital poster to fellow undergraduates, faculty judges, and the community at large.
-The winning poster from each division will receive a $200 scholarship.
-Fulfills the poster presentation criteria for departmental honors.
-Open to students of all majors conducting chemistry research.
Due to the nature of the Zoom format, attendees at the ACS-SA symposium may have the ability to record (i.e. screenshot) posters, and it is beyond our control. By submitting a poster, you are aware that sensitive data may be obtained without the permissions of the presenter and/or the organizers.
The deadline for abstract submission is May 7, 2021 at 11:59 pm.
Do you have a research project you’re really passionate about, or that you want to pursue in the future? Or do you have a chemistry topic you’d like to share with the world?
Our first annual Symposium Video Competition is designed to be more creative than competitive! We hope to encourage the development of science communication skills, even without a completed research project. The video should describe a research project, the motivations, experimental setup, predictions, and/or implications. Most of all, it should demonstrate your interest and passion in the subject. The video must be within 3-5 minutes of length.
-The winner of the video competition will receive $200 and the two runner ups will receive $75.
-You may submit both a video and a poster presentation.
-Video submission does not fulfill the poster presentation requirement for departmental honors.
-Open to students of all majors conducting chemistry research.
The deadline for video sign up is May 7, 2021 at 11:59 pm, and the deadline for video submission is May 21, 2021 at 11:59 pm.
Ubiquitin signaling regulates DNA organization in the developing brain
How DNA organization mediates gene expression and cell differentiation is a major outstanding question in biology. Furthermore, how protein turnover in the nucleus influences genome organization has been poorly defined. The anaphase promoting complex (APC) is a major E3 ubiquitin ligase whose degradative role in mediating mitotic progression is well-established. However, little is known about the role of the APC in human disease, its functions beyond mitosis and what specific substrates it targets. Recently, we identified a novel neurodevelopmental syndrome with impaired cognition that is linked to loss of the core APC component APC7. Proteomics evaluation in the brain of APC7 null mice identified the proliferation marker Ki-67 as a novel substrate of the APC. With immunohistochemistry, we discovered Ki-67 to be selectively elevated in the mutant postmitotic granule neurons of the cerebellum, suggesting Ki-67 may play an essential role in neurodevelopment. Additionally, Ki-67 was found to localize with constitutive heterochromatin, but not facultative heterochromatin. We plan to utilize the revolutionary method CUT&RUN (Cleavage Under Targets and Release Using Nuclease) to further examine distribution of Ki-67 chromatin-associated factors genome-wide. Through this approach, we will contribute to our understanding of how ubiquitin signaling regulates epigenetic state and chromatin organization during brain development.
Cotranslational mRNA localization has been identified as a mechanism for regulating protein synthesis because it facilitates the import of membrane proteins, including mitochondrial membrane proteins. Many nuclear-encoded mitochondrial mRNA are mitochondrial-localized but have no known binding partners and their methods of localization are not fully understood . Bioinformatics studies and experimental observations in yeast indicate that a mitochondria targeting sequence (“MTS”) is required but not sufficient for associating mRNA with mitochondria. Conditionally-localized mRNA contain an MTS but will only preferentially localize to mitochondria under certain conditions, e.g. complete inhibition of translation elongation. Preliminary analysis of HeLa and A549 mammalian cells has uncovered a pattern of genetic traits that distinguishes conditionally-localized mRNA from constitutively-localized mRNA. Cumulative distributions of gene length and translation elongation and initiation rates suggest conditionally-localized mRNA complete rounds of translation more quickly than constitutively-localized mRNA. Consequently, nascent peptides that contain an MTS signal remain exposed for longer timescales on constitutively-localized mRNA. A previous mathematical model indicates that long-lived nascent peptides favor mitochondrial association by increasing the fraction of binding-competent mRNA-ribosome complexes. We posit that these kinetic differences in translation give rise to gene-specific localization patterns. Using a Gillespie algorithm, we will stochastically simulate translation and mRNA diffusion to quantitatively predict mitochondrial localization in various mammalian cell types and in dynamic cell states, such as changing mitochondrial morphology. Elucidating the role of translation kinetics in MTS exposure and mRNA-mitochondria association would increase understanding of cotranslational import of mitochondrial membrane proteins in healthy and diseased cell states.
Quantitative determination of esterified eicosanoids and related oxygenated metabolites after Enzymatic Lipolysis
Eicosanoids and related metabolites (oxylipins) are products of enzymatic and nonenzymatic oxidation of arachidonic acid and related polyunsaturated fatty acids (PUFA’s). Phospholipids are enzymatically cleaved by c-phospholipase A 2 and these lipid tails are further to produce physiologically active lipid metabolites—e.g. prostaglandins, leukotrienes, Epoxyeicosatrienoic acids (EETs), and non-enzymatic oxidation products. Upon enzymatic activation, eicosanoids are secreted to induce critical physiological changes, including inflammation, fever, allergy, and pain. Interestingly, previous research has elucidated that most of eicosanoids are of the esterified form, and quantification of these lipid metabolites have served as biomarkers of NASH and NAFLD. The current method employs saponification to quantify total eicosanoids—however, this method has quantitative limitations due to the degradation of key metabolites, like prostaglandins. We have developed a sensitive method to quantify total eicosanoids by enzyme-induced lipolysis, followed by ultra-performance LC coupled with mass spectrometric analysis. Detailed evaluation revealed that there is little to no degradation of PGE2 and PGD2. Interestingly, we also discovered metabolites of the COX pathway present in the esterified form. While the alkaline method reports 38 analytes, we report the detection of 76 metabolites of the esterified form. We have determined that of the 38 analytes reported in the alkaline method, the percent-esterified eicosanoids remain consistent with the method of enzymatic lipolysis. Quantification of total eicosanoids by enzymatic lipolysis, may elucidate the role of total prostaglandins/metabolites of the COX pathway, in etiology and disease proliferation.
Pseudomonas aeruginosa infecting phage PhiKZ is a member of a special class of bacteriophages termed “jumbo phages.” These phages contribute towards a new group of DNA-dependent RNA polymerases (RNAP) with gene homologs close to the β and β’ subunits of active RNAP found in bacteria and eukaryotes. Infection of bacterial host by PhiKZ promotes propagation of phage genome through a two RNAP system: one with a virion RNAP (vRNAP) that is packaged as a protein with the phage’s DNA in a nucleus-like structure and injected into the host with the DNA, and a non-virion RNAP (nvRNAP) that is transcribed by vRNAP from the viral DNA. To understand the structure relationship of each RNAP to bacteriophage RNAP, plasmid for vRNAP and a dual-plasmid system containing only 4 subunits for the nvRNAP were transformed into BL21 (DE3) E. coli cells. Purification of vRNAP, a task not done before, was successful in low yields from size-exclusion chromatography and therefore could not be used for structural analysis through negative stain and cryo electron microscopy. Purification of nvRNAP, which has been successfully done, yielded a four-subunit (4s) inactive polymerase. The 4s complex cannot initiate RNA synthesis without the presence of the fifth subunit, which is responsible for recognition of the phage’s promoter sequences. Therefore, structural differences will be analyzed to determine the molecular basis of the fifth subunit’s function, the differences between the 4s and 5s nvRNAP, and between bacterial RNAP.
An in silico study of drug resistance of the SARS-COV-2 main protease through Viral Mutations
During the ongoing pandemic, new variants of COVID-19 with key mutations frequently emerge. These variants often vastly improve the efficiency of COVID-19. Scientists, of course, worry that some of these variants could potentially be immune to current vaccine treatments, or potentially develop drug resistance. In this study the drug resistance of the main protease of SARS-CoV-2 (3CLpro) was investigated in silico. 3CLpro is a frequently studied enzyme for drug development due to the absence of human homologs. First, an α-ketoamide inhibitor was bound to wild type 3CLpro using the Molecular Operating Environment (MOE) software package, and the α-ketoamide inhibitor binding affinity was computed with the GBVI/WSA dG forcefield-based scoring function as -9.4955 kcal/mol. The binding pocket was then mutated and the α-ketoamide inhibitor was bound to about 400 single mutants and about 400 double mutant forms of 3CLpro. Many of the mutant forms exhibited drastically reduced binding affinities indicating a systematic development of drug resistance of 3CLpro. The ChemBridge Coronavirus library was also scanned for potential ligands that bind the active site of wild type 3CLpro, and library compound S203-011 had a binding affinity of -8.7476 kcal/mol, similar to the α-ketoamide inhibitor. Compound S203-011 was also docked to all mutant forms, and overall, showed lower decreases in binding affinity suggestion that this drug is more robust against mutational induced drug resistance.
Heterologous Expression of the Columbamide Gene Cluster in Anabaena
Sebastian Rohrer, Arnaud Taton, Brienna Diaz, Raphael Reher, Lena Gerwick, William Gerwick, and James Golden
Cyanobacteria are prolific producers of diverse bioactive secondary metabolites including natural products which constitute a prominent source of novel drug leads. However, obtaining sufficient material for isolation, structure elucidation and biological assays from the native producers has presented a challenge. To address this issue for the columbamides, which are natively produced by the filamentous marine cyanobacterium Moorena bouillonii PNG, we expressed the columbamide biosynthetic gene cluster (BGC) in the model cyanobacterium: Anabaena PCC 7120.
Production of several columbamide analogs in Anabaena was indicated upon initial investigations by LCMS of the recombinant strains. In addition to previously characterized columbamide A, we saw production of putatively new analogs columbamides G, F, H, and I, for which preliminary structural characterization via LCMS/MS and NMR has been completed. Additionally, cultures of Anabaena carrying the columbamide BGC were subject to different growth conditions and the production of columbamide assayed. From these experiments we have found a strong positive correlation between the NaCl concentration in the medium and the columbamide produced, with consistently higher production seen in the 0.1 M NaCl cultures vs the 0.5 M NaCl and 0.0 M NaCl. In contrast, no difference was found in columbamide production between cultures grown with 0.1 M NaCl in constant light (24h) vs light/dark (12h/12h) conditions. Finally, cultures were grown in the presence of 0.1 M NaBr instead of 0.1 M NaCl. The LCMS/MS analysis indicates that Bromine can be incorporated into the columbamide structure in place of Chlorine, however only trace amounts of the brominated products were detected. Additional experiments are being done to further characterize the heterologous expression system and the putatively new analogs and their bioactivities.
Protein-Protein Interactions of the FAS II System in Mycobacterium Tuberculosis
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), employs the type II fatty acid synthase (FAS-II) metabolic pathway to produce long-chain fatty acids, precursors for mycolic acids and crucial structural components of the mycobacterial cell wall. Drug resistant TB infections continue driving the need for new antibiotics that target essential pathways. Our lab has used mechanistic crosslinking of the transient acyl carrier protein (ACP) with FAS-II enzymes in E. coli; here we aim to extend these studies to the Mtb FAS-II system. We investigate the protein-protein interactions between Mtb ACP (AcpM) and its dehydratase, HadBC. Results demonstrate that two fluorescently labeled probes previously used to crosslink E. coli FAS-II proteins do not react with HadBC, confirming the need to develop probes specific for Mtb FAS-II. Future work will investigate new probe development and crosslinking the Mtb HadAB protein with other probes evaluated in the E. coli system.
While cryogenic-electron microscopy (cryo-EM) has become one of the most powerful tools to determine the three-dimensional structures of macromolecular complexes, data processing in cryo-EM still remains largely subjective, with many critical decisions being made largely on a whim or based on anecdotal evidence. Furthermore, since most high-resolution cryo-EM structures require numerous (often 10+) distinct processing steps, a considerable amount of usable data often gets lost due to less-than-ideal parameter selection. Indeed, most high-resolution structures comprise <10% of the original particle images isolated from the raw data.
To circumvent this problem, and to provide standardized and objective parameterization of cryo-EM data processing, we used a ~1.7 Å cryo-EM structure of mouse apoferritin obtained by the Herzik lab, to systematically identify optimal parameters to isolate high-resolution apoferritin particles from contaminants. Specifically, we generated subgroups of data wherein each subgroup contains different amount of apoferritin particles that we then “poisoned” with “junk” images generated from known contaminants and/or pure noise. We then used the cryo-EM data processing package, RELION, to assess the ability of different parameter sets in both 2-dimentional (2D) and 3-dimentional (3D) classification steps to isolate intact apoferritin protein particles from the known junk images. From our efforts, we were able to identify conditions by which >90% of the apoferritin particles could be recovered for high-resolution structure refinement. These insights will immediately affect cryo-EM data processing within the Herzik lab and beyond to the entire EM community.
Structure-activity relationships of curcumin-like compounds as novel candidate treatments for Human African trypanosomiasis
Wenqian Yang, a, b Ludovica Monti, b Kaitlyn Watkins, b Rafay Syed, b Giuseppe La Regina,c Romano Silvestri c and Conor R. Caffrey b
aDepartment of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92092, USA; bCenter for Discovery and Innovation in Parasitic Diseases (CDIPD) and the Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA; cDepartment of Drug Chemistry and Technologies (CU019) Sapienza University P.le Aldo Moro 5, I-00185 Roma, Italy
Human African trypanosomiasis, also known as sleeping sickness, is a ‘neglected’ parasitic disease caused by the flagellate protozoan, Trypanosoma brucei. Millions of people in west and central Africa are at risk of this disease. As a disease of poverty, pharmaceutical company investment is low and the few current drugs available are toxic, require medical supervision for treatment and/or are less than effective due to drug resistance. New therapies are needed. Using in vitro whole-cell screens, we evaluated the antiparasitic activity of curcumin-like small molecules (Figure 1) against Trypanosoma brucei. Our screening effort identified a series of congeners with promising anti-trypanosomal activity. The synthesis and evaluation of a series of curcumin-like diarylpentanoid congeners leading to a characterization of the structure anti-trypanosomal activity relationship will be presented.
Biomolecules sample a range of conformational states to perform their cellular functions and a molecular-level description of these states is necessary to fully understand their biological roles. Recent advances in cryogenic-electron microscopy (cryo-EM) have led to significant improvements in image quality, now allowing for direct visualization of molecules in near-native states at high-resolution. Despite these innovations, there remains a critical need to develop methodologies that allow investigators to probe the dynamic, transient conformational states captured by cryo-EM so we can fully understand the dynamic conformational landscapes of biomolecules.
To bridge these gaps in our knowledge, we are developing modelling strategies utilizing high-resolution cryo-EM maps of dynamic molecules. Towards this end, we have coupled cryo-EM with molecular dynamics (MD) simulations to explore the conformational space sampled by biomolecules of interest and develop cryo-EM data processing strategies that extract the lowly-populated transient states. Together with a postdoc in the Herzik lab I used the MD software package, NAnoscale Molecular Dynamics (NAMD) to perform long-term simulations (2 µs) of rabbit muscle aldolase previously determined by Dr. Herzik, utilizing conditions that closely match the cryo-EM experiment. As a proof-of-principle we generated atomic snapshots to produce in silico simulated EM densities that we juxtaposed against the experimental EM data. Using Molecular Dynamics Flexible Fitting (MDFF) I worked to quantify the extent of dynamics within these data and identify distinct conformational states. From these experiments, we hope to demonstrate a unified cryo-EM and modeling pipeline, informing on protein dynamic and conformational heterogeneity in cryo-EM.
A key issue in ACL surgery is prolonged recovery time due to lack of vascularization to the newly grafted tendon; adequate blood flow typically is not established for nearly three months. Previous studies have shown that the VEGF promotes angiogenesis, however, it was also found that high concentrations of VEGF led to low density, highly vascularized tissues that resulted in decreased tendon integrity; likely due to procedures that have high initial concentrations of VEGF. To counteract this effect, I aim to deliver a substantially lower concentration of VEGF to the tissues over a substantially prolonged period of time,via a nanomaterial that can control release of the protein. The nanomaterial I prepared consists of porous silicon nanoparticles suspended within a cross-linked alginate/hyaluronic acid hydrogel. I prepared the 3% alginate solution by dissolving 0.300 grams of alginate powder into 10 mL of MES buffer at a pH of 5.5. Next, I prepared a 5% Hyaluronic Acid (HA) solution by adding 0.557g of HA powder to 10mL of KHP buffer at a pH of 4. After the HA was thoroughly dissolved, I added 0.216 mol/L ADH to the solution. The solutions were then mixed at a 1:1 ratio, yielding a gel with concentrations of 1.5% alginate and 2.5% HA by weight. Finally, all solutions were immersed in a solution of 1M CaCl2 and 100mmol L-1 EDC. After cross linkage, the chemical properties and performance of the hydrogels were analyzed.
The NONEXPRESSER OF PATHOGENESIS-RELATED GENES 1 (NPR1) protein is key to activating plant defenses. Upon infection, the transcription of NPR1 gene is induced leading to the activation of the expression of several defense-related genes. Despite the important role of NPR1 transcriptional regulation, the underlying mechanisms remain elusive. Using the plant model organism Arabidopsis thaliana, this project aims to identify those mechanisms. Recently, several transcription factors (TFs) that bind to the NPR1 promoter were identified and their role in inducing or repressing NPR1 promoter activity was determined. Interestingly, the only positive regulators of NPR1 promoter activity were transcription factors that belong to the WRKY family. Given that three WRKY TF binding sites (W-boxes) are encoded in the NPR1 promoter, I investigated the role of these W-boxes in recruiting WRKY TFs to the NPR1 promoter region. For that, I used available datasets to determine the binding preference of WRKY TFs and designed a combinatorial DNA-binding assay to determine the relative contribution of each W-box on WRKY TF DNA binding. Ultimately understanding how NPR1 expression is induced will inform the future design of NPR1 promoters that could produce higher NPR1 transcript levels upon infection, and thus increased defenses. Notably, the NPR1 gene is conserved across several plant species, thus my research could ultimately contribute to the rational design of pest-resistant crops in the future.
Targeted Delivery of Therapeutics, Cell-fate Mapping and Lineage Tracing in Directed in-vivo Neural Cells Transdifferentiation Using De Novo Molecular Design, and Single-Cell Omics Analysis
Neurodegenerative diseases are characterized by degeneration and death of specific classes of neurons. While therapies for some diseases have dampened symptoms, no therapy has been identified for any neurological disorder to replace lost neurons. Since the loss of neurons is an irreversible process, it has been a milestone to regenerate new neurons to replace lost ones. In this project, one-step direct conversion of one non-neuronal cell type (glia-like cells and other GFAP-expressing) into functional neurons has been reported by downregulation of the RNA binding protein Polypyramidine Tract Binding Protein 1 (PTB) by intrathecal injection of Antisense Oligonucleotide (ASO) into the cortex and dentate gyrus of mice model. Newly induced neurons mimicked mature neurons and successfully integrated them into endogenous circuits of the brain.
Moreover, molecular mechanisms and feasibility of direct in-vivo regeneration of functional neurons from astrocytes or other glial cells have been investigated. Further analysis is in progress by performing lineage tracing and cell-fate mapping using single-nucleus RNA sequencing (omics) data. Additionally, a uniquely expressed receptor in the blood-brain barrier (BBB) has been found and investigated thoroughly to find the potential characteristics of the ligand candidates that can interact with the receptor and cross the BBB. Lastly, by harnessing the power of de novo molecular design and identified molecular characteristics of the potential ligand, chimeric molecules will be developed for non-invasive, targeted delivery of ASOs to enable efficient, in-vivo neuronal transdifferentiation.
It has been proposed that codon repeats may have an effect on translation elongation within yeast cells. The Zid Lab has developed a quantitative way of measuring elongation rate in vivo. Plasmids were transformed with varying quantities of Lysine repeats. To measure both protein production and elongation rate, multiple elongation assays were performed upon different stall strains. Stall strains with codon repeats or synonymous codons have been shown to have an effect on translation, but the effects have not yet been qualified. Ribosome quality control has also shown to have an effect on translation elongation by initiating ribosome stalling at translation sites. Some points for discussion are the absolute effect of codons on elongation time, the elongation rate effect of codons, the threshold time to induce ribosome quality control, and if there is an effect from ribosome quality control machinery on elongation rate at stalls. Moving forward, the effect of codon repeats on mRNA levels and ribosome quality control during elongation will be further explored.
There are currently no approved inhibitors of SARS-CoV-2 viral proteases with specific treatment for post exposure
of SARS-CoV-2. Here, we have discovered inhibitors containing a key core group that maintains covalent cysteine
activity against the SARS CoV-2 Main Protease (Mpro) and SARS CoV-2 Papain Like Protease (PLpro). Our goal
was to find more potent inhibitors that target both viral proteases to reduce the necessary dosage and minimize the
adverse effects associated with these agents. We found that our derivatives RI172 and RI175 are the most potent
inhibitors from an enzymatic assay against SARS-CoV-2 Mpro and PLpro with IC50s of 330 and 250 nM
respectively. To further investigate these results, the identified protease inhibitors in this series containing the key
core group were also tested against SARS CoV-2 in a cell-based assay and toxicity assay. From these results, we
propose that the protease inhibitors containing this fundamental core group are key compounds that should be
further tested and implemented as dual inhibitors for treatment against SARS CoV-2.
My research focuses on inteins, which are a unique class of proteins that are capable of stitching other proteins together in a process known as protein splicing. Inteins are valuable tools for protein chemists as they allow for selective labelling and assembly of proteins. One caveat for using inteins is that they typically need the amino acid cysteine in order to stitch together the proteins. This cysteine is inserted into the newly assembled protein and results in an undesirable “cysteine” scar. This limits protein chemists, as not all assembled proteins function well with a new cysteine in their structure. In 2019, a cysteineless intein was discovered, which uses a serine instead of a cysteine to catalyze the protein splicing reaction. However, we do not know much about this intein, including its structure. My research goal is to determine the structure of this intein, and then improve upon its splicing efficiency. For the past year I have been computationally analyzing the intein from home, due to Covid. I have generated a homology model, or a hypothetical structure, of the intein by aligning it to an intein of similar sequence. I then ran molecular dynamics simulation on the homology model to gain an understanding of its behavior. I then took this a step further and mutated the intein’s active site to improve upon its promiscuity, followed by a simulation to verify its improvement. This research can be used in the future for improving the splicing efficiency of the real cysteine-less intein.
Investigating the Role of the Transactivation Domain of NF-KappaB and the coactivator CBP-KIX
NFkB is a family of transcription factors that are involved in the expression of more than 600 genes. Its canonical dimer p50-p65 (p50-RelA) contains its transactivation domain (TAD) in the p65 subunit. This TAD contains two regions called the TA1 and TA2 that are involved in the recruitment of coactivator proteins to initiate transcription. It is known that the p65TA1 (residues 521 to 549) subdomain interacts with the CBP-KIX domain of CREB-binding protein, but all the studies of this interaction have been done with a peptide rather than the full-length protein. To better understand this interaction, we have fluorescently labeled the truncated CBP-KIX protein (OG488-KIX) and used this to detect binding to p50-ReIA by performing fluorescence anisotropy. This results in a tighter binding of CBP-KIX to p50-ReIA relative to the reported results for the interaction with p65TA1 peptide only, suggesting that the presence of the RelA and the p50 rel-homology domains enhances the recruitment of coactivator proteins.In addition, different truncations of the p50-ReIA protein constructs are used to study if there are additional contacts between the KIX and NFkB that not involve the TA1, in order to understand the tighter binding that is observed.
cAMP-dependent protein kinase (PKA) is a Ser/Thr kinase that was discovered in 1968 and became the first kinase crystallized in 1991. In cells, PKA exists as an inactive tetrameric holoenzyme with a Regulatory (R) subunit dimer bound to a Catalytic (C) subunit dimer. When cAMP binds to the R-subunits, the holoenzyme undergoes conformational changes that enable the C-subunits to phosphorylate downstream substrates. There are four R subunit isoforms RIα/β, and RIIα/β and three C subunit isoforms Cα/β/γ. The different holoenzymes are functionally non-redundant and have different quaternary structures. Almost all of the C subunit and holoenzyme structural and biochemical studies have been done with Cα, relatively very little is known about Cβ and its holoenzymes. Cβ4, specifically, is strongly expressed in the brain where it accounts for much of PKA signaling and is expected to be potentially connected with Alzheimer’s Disease.
The goal of this project is to express and characterize Cβ and Cβ holoenzyme, which will be accomplished in two specific aims.
Aim 1: Elucidate the structure of Cβ4 holoenzyme complexes, both wildtype and mutant.
RIIβ:Cβ4 holoenzyme complexes are purified and formed into a crystal structure, through crystallography as well as small angle X-ray scattering analysis. With the compositional data, computer MD simulations are performed to understand the effects of the mutations.
Aim 2: Perform assays to understand biochemical properties of these complexes.
Fluorescence polarization competition assays are done to quantify how easily the Cβ holoenzyme is activated by cAMP relative to Ca holoenzymes.
The urokinase-type plasminogen activator (uPA) is a serine protease that activates the inactive plasminogen to plasmin. In turn, plasmin activates uPA and therefore results in a positive feedback loop and the subsequent cascade. The uPA-plasmin system is involved in cancer metastasis. The focus of this project will be on the uPA because the activity of the uPA is essentially the rate-limiting step in the system.
The uPA has essentially four “domains” or regions: The N-terminal EGF-like domain, a Kringle domain, a linker region that links the EGF-like domain and the Kringle domain to the C-terminal protease domain. Active plasmin activates inactive single-chain uPA by cleaving it at the linker-protease domain region and creating a two-chain uPA protein. The two chains are attached via a cysteine disulfide bond formed between the linker region and the protease domain. The main research questions of this project are what the effects of the linker region on uPA protease activity are and how the linker communicates with the protease domain of the uPA.
Fluorescence quenching of AzW48 apoprotein via copper(II) incorporation
With a particularly stable neutral radical, the protein azurin is the subject of spectroscopic studies regarding the role of amino acid radical intermediates in long-range electron transfer. Azurin is capable of single-electron redox reactions when excited, resulting in either electron transfer or vibrational relaxation leading to a distinct tryptophan fluorescence peak at 308nm when excited. While the fluorescence spectra of apo and zinc substituted azurin have the signature 308nm peak, this feature is hypothesized to be absent in the copper holoprotein. The purpose of this study was to verify the results by Argo, et al. showing that the 308nm peak is roughly 40% of the intensity of the apoprotein. A titration of apoazurin with stochiometric amounts of copper sulfate was conducted, showing that copper azurin has a fluorescence of effectively zero. With this observation we conclude that excited copper azurin molecules exclusively undergo an intramolecular electron transfer rather than photoemission. The results are explained by the fact that the copper(II) metal center is redox active, and can hence facilitate intra-molecular ET from Trp to Cu(II) in azurin while the zinc(II)-bound azurin and apoprotein cannot.
Examining the Salt Dependence of Amino Acid Buffering Capacity in Sea Spray Aerosols
Sea spray aerosols (SSA) are small particles ejected into the atmosphere through wave breaking and bubble bursting at the air-sea interface. These aerosols present a novel environment for chemistry as they can have salt concentrations reaching 5 M NaCl and they are rapidly acidified. Furthermore, they have significant implications for the environment and global warming as well as for human health as more acidic aerosols are increasingly harmful to lung health. Past research has demonstrated that enzymes such as methionine aminopeptidase are also active in these aerosols, which removes a terminal methionine from a protein chain. Therefore, this project seeks to better understand the chemistry of biologically relevant molecules in sea spray aerosols. Specifically, the amino acids methionine, glycine, and aspartic acid, which have all been detected in aerosols but have not been thoroughly investigated at salt concentrations higher than 1M, were examined in high salt and both high and low pH conditions. Results suggest that the buffering capacity of these amino acids is greatly reduced in the presence of high salt concentrations (analogous to those found in SSA). Characterizing this phenomenon could help reveal whether these amino acids are available to react in SSA. Additionally, calculating the pKa values of these amino acid solutions, and how they may change, could shed light on the general behavior of enzymes in aerosols. Ultimately, the results of this project can inform our understanding of SSA chemistry and biology and, in turn, how these particles impact our environment and health.
The direct and indirect effects of aerosols represent the single largest uncertainty associated with radiative forcing and climate modeling. Some of this uncertainty is related to the impact of aerosols on cloud formation. Within this broad topic, our work is related to the uncertainty attributed to ice nucleating particles (INPs), a unique subset of aerosols which induce heterogeneous freezing at temperatures above which super-cooled water freezes at in the atmosphere (-38˚C). Of the multiple freezing pathways, immersion freezing is of particular interest due to its role in the formation of mixed phase clouds. This work probes the low temperature interactions of micron-size water droplets in the presence and absence of INPs. In particular, micro-Raman spectroscopy is used to gain insights into the structure of super-cooled liquid and the temperature at which these droplets freeze. The contributions of fully hydrogen bonded water (FHW) and partially hydrogen bonded water (PHW) were determined by deconvoluting the OH region of Raman spectra across a range of temperatures. From these measurements the change in enthalpy, entropy, and Gibbs free energy for the transition from PHW to FHW at the ice nucleation onset temperature were calculated and found to correlate with the ice nucleating ability of the systems investigated -Snomax, Lipopolysaccharide, Laminarin, and NaCl. This work suggests a correlation between the liquid water structure and ice nucleation onset temperature and a potential method to predict ice nucleation behavior in the atmosphere and cloud formation.
O-alkyl hydroxylamines are attractive synthetic targets due to their varied applications as aminating agents and imide precursors. They have traditionally been prepared through nucleophilic substitution reactions of primary or secondary alcohols using the Mitsunobu protocol. Mitsunobu reactions require hazardous azodicarboxylates and produce toxic phosphine oxides. A novel Mitsunobu catalyst containing pentavalent phosphorus was reported in 2019. The catalyst is redox-neutral and its sole byproduct is water, making it a green alternative to the traditional Mitsunobu reagents. Here, we apply the catalyst to a variety of primary and secondary alcohols with N-hydroxyphthalimide to synthesize O-alkyl hydroxylamines. The protocol was successful for sterically-hindered secondary alcohols including Cholestenol and 5’-protected nucleosides.
Representational Fluency in Undergraduate Organic Chemistry: A 3D Printed Chemists’ Triangle
Students in organic chemistry struggle to master the reading and drawing of molecular structures using bond line formalism. Drawings are typically messy, with inaccurate bond angles and lengths which can hinder a full understanding of the 3D structure and conformations. Renowned organic chemist, Louis Fieser, developed a stencil, published in 1967 and produced by the Aldrich Chemical Company, as a tool to enable the illustration of accurate and consistent organic structure diagrams. The Chemists’ Triangle, as the stencil is known, has since been replaced by computer graphic programs like ChemDraw and are no longer commercially available. However, stencils can still be useful in the classroom where students must rapidly draw structures by hand. Thus, we developed a modern, 3D printable version the Chemist’s Triangle to examine the potential benefits for organic chemistry students. The stencil can be printed on a consumer grade 3D printer in ~30 min using less than $0.14 of PLA.
Developing Amyloid-Selective Fluorescent Probes for Aiding in Diagnosis of Neurodegenerative Diseases
Alexander Lee Shiao, Rachel Ehrlich, and Jerry Yang
Alzheimer’s Disease (AD) and Parkinson’s Disease (PD) are neurodegenerative diseases that causes severe loss of memory and physical ability. Despite these diseases affecting millions in the US annually, no cure exists. Recent treatments slow the progression of AD and PD, but clinical detection is often too late as it relies on pronounced symptoms. The misfolding of proteins β-amyloid (Aβ) in AD and alpha synuclein (αS) in PD into fibrils is ubiquitous with neurodegenerative diseases and precedes clinical symptoms by several years. Therefore, targeting these amyloids has become a recent area of research for early diagnosis of neurodegenerative diseases. The Yang lab has previously developed the fluorescent probe aryl cyano amide (ARCAM) that can bind to Aβ both in solution and in postmortem brain tissue with a significant fluorescent response. The fluorescence enhancement of these probes has shown to be dependent on the rotatability of single bonds found within the π conjugations. These probes show immense clinical potential as they can serve as reliable and low-cost methods of detection of neurodegenerative diseases prior to clinical symptoms appearing. In this poster presentation, I will describe the ongoing research on exploring fluorescent molecules that can discriminate between Aβ and αS aggregates. New probes based on the ARCAM scaffold were designed by introducing steric hindrance that can affect rotation of single bonds and can improve fluorescence response upon binding to amyloids. This presentation will compare the synthesis and characterization of these new probes for binding to amyloid aggregates both in solution and in tissue.
Optimization of ring-opening metathesis polymerization (ROMP) under physiologically relevant conditions
Ring opening metathesis polymerization (ROMP) is widely considered an excellent living polymerization technique that proceeds rapidly under ambient conditions and is highly functional group tolerant when performed in organic solvents. However, achieving the same level of success in aqueous media has proved to be challenging, often requiring an organic co-solvent or a very low pH to obtain fast initiation and high monomer conversion. The ability to efficiently conduct ROMP under neutral pH aqueous conditions would mark an important step towards utilizing aqueous ROMP with acid-sensitive functional groups or within a biological setting. Herein we describe our efforts to optimize ROMP in an aqueous environment under neutral pH conditions. Specifically, we found that the presence of excess chloride in solution as well as relatively small changes in pH near physiological conditions have a profound effect on
molecular weight control, polymerization rate and overall monomer conversion. Additionally, we have applied our optimized conditions to polymerize a broad scope of water-soluble monomers and used this methodology to produce nanostructures via ring opening metathesis polymerization induced self-assembly (ROMPISA) under neutral pH aqueous conditions.
The purpose of this project is to integrate catalytic nanomaterials into fabrics in order to create protective clothing for people in combat. In this presentation, the development of catalytic silicon-copper nanostructures to degrade chemical nerve agents will be discussed. Various fabrics are also soaked in porous silicon nanoparticles and were subjected to wash and abrasion tests to evaluate lifespan and durability of the material. Two copper precursors, tetraamminecopper(II) sulfate monohydrate (TACu2+) and bis(ethylenediamine)copper(II) hydroxide (BECu2+), are loaded into porous silicon nanoparticles and measured for their ability to hydrolyze dimethyl p-nitrophenyl phosphate (DMNP), a nerve agent simulant, using UV-Vis Spectroscopy. Hydrolyzed and amino functionalized copper-silicon systems are also investigated. All of the copper-silicon nanostructures will be compared to the benchmark, a free tetramethylethylenediamine (TMEDA) catalyst (J. Am. Chem. Soc. 2000, 122, 5399-5400).