Tag: Biochemistry

Alkaline Synthesis of Amidines—Exploring a New Approach to Accessing a Pharmaceutically Relevant Functional Group

Presenter: Muhammad Khalifa

Mentor: Michael Haley

Oral Presentation

Major: Biochemistry

Aryl amidines have been used against a variety of diseases, most notably pneumocystis pneumonia. They continue to be relevant in the search for cures against malaria, Alzheimer’s disease, and myotonic dystrophy type
1 (DM1). Current methods of amidine synthesis feature harsh, acidic reaction conditions that limit possibilities for functionalization of the amidine group, a process of particular interest in developing small molecule therapeutics
for DM1. Preparation of amidines via basic conditions has been described, but not well studied. Here we outline a method of preparing N-substituted aryl amidines under alkaline conditions and examine the accessibility of series
of amidines to delineate important properties of this synthetic approach. Our results indicate that increasing the nucleophilicity of the amine and/or increasing the electrophilicity of the nitrile afford higher yields of the desired amidine. Results also show that alkaline synthesis is sufficiently chemoselective to form amidines in the presence of competing, nucleophilic aromatic substitution sites on the nitrile. Featuring reaction times at least 50% shorter, up to 40% greater yields, and better compatibility with a broad range of starting materials, our method of alkaline amidine synthesis makes accessible a host of new N-substituted amidines for study in a variety of diseases previously described.

Mapping Interactions Of Single-Stranded (Ss) DNA With the Ss-DNA Binding Protein (Gp32) of the T4 DNA Replication Complex at Specific Nucleotide Residue Positions

Presenter(s): Anson Dang − Biochemistry

Faculty Mentor(s): Pete von Hippel

Poster 19

Research Area: Natural Science, Biochemistry

Funding: Dreyfus Undergraduate Mentorships

The single-stranded (ss)DNA binding protein (gp32) of bacteriophage T4 plays a central role in regulating the functions and integration of the helicase, polymerase and primase components of the T4 DNA replication system. To understand how gp32 interacts with itself and with the other regulatory proteins and sub-assemblies of the T4 replication complex, we must first understand the structural details of how this protein binds to ssDNA lattices, both as isolated monomer subunits and as cooperatively bound gp32 clusters. We have explored these issues by monitoring differences in the fluorescence and circular dichroism (CD) spectra of site-specifically positioned monomers and dimer-pairs of 2-aminopurine (2-AP) probes located at various ssDNA positions within the binding site. In its cooperatively bound form gp32 spans 7 nucleotide residues per protein subunit, and by mapping spectral changes on binding to ssDNA lattices that are exact multiples of 7 residues in length we have been able to characterize interactions at defined positions within the gp32 binding cleft. We have extended these studies using acrylamide quenching and permanganate foot-printing assays to monitor degrees of base exposure at various lattice positions. Our results show that gp32 binds randomly at low concentrations, and then shifts toward preferential binding at the 5’-ends of the lattice as cooperatively bound gp32 clusters form at higher gp32 concentrations. Bases located near the middle of a gp32 binding site display lower solvent accessibility than those near the ends of the site. These differences in base ‘shielding’ may reflect deeper burial of the middle bases within the electropositive binding cleft, while bases at the ends may be made more accessible by fluctuations of the C- and N-terminal regulatory sub-domains of the protein. Insights into gp32- ssDNA interactions involved in controlling the functions of the T4 DNA replication complex that result from these studies will be discussed.

Ensembles link RNA thermodynamics and molecular evolution

Presenter(s): Daria Wonderlick—Biochemistry

Faculty Mentor(s): Mike Harms

Session 5: The Bonds that Make Us

Designing better biomolecules is a long-standing goal for biochemists . Doing so requires a rigorous understanding of how the sequence of a biomolecule determines its properties . Sequence changes, known as mutations, alter these properties and drive the natural evolutionary process . If we can accurately predict how mutations impact biomolecular properties, we can engineer novel biomolecules for applications in medicine, energy, and technology . Predicting a mutational effect is challenging, however, because the effect often depends on the presence of other mutations . Previous work in the Harms lab suggests that some of these mutational interactions emerge from a thermodynamic property of biomolecules—the ensemble . A biomolecule’s ensemble is the collection of interchanging structures it can adopt . A mutation may impact any structure in the ensemble, and its effect arises from perturbations to the relative populations of these structures . Mutations will have different effects depending on the degree to which other mutations have redistributed the ensemble . To mechanistically understand how the ensemble mediates mutational interactions, I am characterizing the effects of five mutations alone and in combination on a magnesium- and adenine-binding RNA molecule with a simple four-structure ensemble . By measuring the amount of a fluorescent adenine analog bound in the presence of varying magnesium concentrations, I can detect the effect of mutations on each of the four structures in this ensemble . The simplicity of this system will provide detailed mechanistic insight into the relationship between ensembles and mutations that can be used to improve the mutational predictions required for successful biomolecule design .

The role of the Chemoreceptor Zinc-Binding Domain in bacterial signal transduction

Presenter(s): Dan Tudorica—Biochemistry

Faculty Mentor(s): Arden Perkins

Session 3: The Substance of Us

Previous work presented at the undergraduate research symposium hypothesized that the chemoreceptor zinc-binding (CZB) domain acted to sense bleach in certain bacteria’s environment and correspondingly direct bacterial swimming patterns . This project presents an expanded view of the CZB domain as being responsible not just for informing bacterial swimming patterns in the presence of bleach, but also for regulating the formation and dispersal of bacterial biofilms . Through the use of genetically-modified bacteria and biofilm-quantification assays, we determined that bleach in the bacteria’s environment encourages the formation of biofilms . In addition, we find that modifying the active site of the CZB domain in such a way as to make the domain “always on” increases the amount of biofilm produced by the bacteria in a fashion largely insensitive to subsequent addition of bleach . This evidence suggests that the CZB domain, known to regulate bacterial swimming patterns, is also used by bacteria to modulate the amount of biofilm that they form . This work helps us understand the biochemistry of how bacteria, particularly gut-colonizing human pathogens, behave in order to survive and thrive in their environment, possibly setting the groundwork for future therapeutic interventions .

Sensors and Materials for In-field Aqueous Analysis of Nitrate and Other Ions

Presenter(s): Ian Torrence—Biochemistry

Faculty Mentor(s): Sean Fontenot

Session 2: Cells R Us

Chemically sensitive field effect transistor (ChemFET) development have been well studied as ion- sensing chemical sensors . These devices are attractive to other chemical sensors due to their low cost, low power consumption, small size, and their compatibility with electronics . By applying an ion- selective material, typically a polymer, on the ChemFET it is possible to create an interfacial potential difference between the environment and the gate-oxide of the FET . This ion-selective material can be designed to ensure the potential difference is dependent only on the activity of a target analyte. Currently, there is a need for a real-time chemical sensor to detect both nitrate and ammonium concentrations in soil, dubbed “total-N” content of the soil, as described by the NSF grand challenge for closing the nitrate cycle . This is primarily to combat fertilizer runoff caused by over fertilization of crops resulting in high concentration of nitrate in lakes, rivers, and streams . My research shows promising results for two ChemFETs that are sensitive and selective for ammonium and nitrate respectively which can be measured simultaneously for real time nitrate sensing in aqueous systems .

Rational Design of s-Indacene-cored Small Molecule Organic Semiconductors as a Paradigm to Tune Electronic Characteristics

Presenter(s): Eric Strand—Biology/Biochemistry

Faculty Mentor(s): Joshua Barker

Session: Prerecorded Poster Presentation

The Haley Lab is interested in the synthesis and characterization of organic hydrocarbon scaffolds which can be used as semiconductors . The family of indenofluorene hydrocarbons exhibit unique electronic properties such as antiaromaticity and diradical character, which contribute to their allure for scientists . Specifically, our studies into indenofluorenes have shown promise in regard to the ability of these molecules to serve as potential replacements for current inorganic counterparts within devices . Continuous fundamental studies into the electronic abilities of these molecules will help to elucidate the ideal characteristics of organic semiconductors, which is imperative for the feasible implementation of these molecules into devices . Our lab has developed highly modular synthetic routes toward many analogues of this parent scaffold, which can be further optimized through subtle synthetic tuning . Fusing a variety of aryl moieties onto the parent scaffold allows for a decrease in the HOMO-LUMO energy gap and subsequent improvement in electron mobility and conductivity . Our project initially focused on proving the diradical character in an analogue of indenoindenodibenzothiopene, and has successfully shown this by reacting the molecule through a known radical degradation pathway . This project is now focused on the optimization of previous synthetic routes such that further studies into these highly interesting molecules can be carried out . Our goal is to create a library of analogues with various electronic characteristics such that we may identify the most promising candidates for device implementation .

Effects of Feedback-Related Negativity on Excecutive Function and Development in Preschoolers

Presenter(s): Dakota Paulus—Biology, Biochemistry Minor

Co-Presenter(s): Nisha Sridhar, Katia Pramono

Faculty Mentor(s): Tyson Barker, Leticia Hayes

Session 5: The Wonders of the Brain

Executive function (EF) is a set of higher-order cognitive skills that support early learning and development . EF is highly influenced by environmental factors such as exposure to stress and social interaction . The prefrontal cortex (PFC) is one of the primary neural regions underlying EF . As the PFC develops during early childhood, the brain begins to lay the groundwork for more complex processing . One neural component that supports EF, feedback-related negativity (FRN), is measurable using electroencephalography (EEG), a device that measures the brain’s electrical activity . FRN is observed following both positive and negative feedback and is generated by the PFC . Although FRN is theorized to represent EF, little is known about the FRN development in early childhood: a period of critical EF development .

We predict that children’s FRN will be positively related to a behavioral measure of EF, which was collected during a previous study . Thus, we propose that FRN will reflect an early neural indicator
of EF . Previous research has used tasks without intermittent reinforcement making it difficult to maintain children’s attention . We will be using the Doors Game, which is a novel feedback-based task providing intermittent random reinforcement to children upon their selection between two doors . This task presents the reward immediately alongside feedback, thus it is more age-appropriate due to its ability to sustain their motivation . As feedback processing serves an important role in early childhood development and may serve as a novel indicator of EF, it is a promising area for research .

Characterizing the Conformational Fluctuations of DNA Under Physiological and Salt-Stabilized Conditions

Presenter(s): Maya Pande—Biochemistry, Political Science

Co-Presenter(s): Anabel Chang

Faculty Mentor(s): Andrew Marcus

Session: Prerecorded Poster Presentation

The Marcus Group conducts studies on the dynamics of macromolecules in biological environments . In our experiments, we used a variety of techniques to analyze the structure of DNA with the overall goal of better understanding the conformations it can take . Our studies were focused in two areas: (1) understanding the mechanisms of DNA breathing, and (2) conducting experiments on the stabilizing and destabilizing properties of salt solutions on DNA . Techniques included circular and linear dichroism, UV-Vis spectroscopy, and Förster Resonance Energy Transfer (FRET) . Determining the structure of DNA is crucial to understanding biochemical and molecular events essential for gene expression and DNA replication . For these processes to occur, various proteins must access ssDNA coding templates which are otherwise inaccessible due to complementary base pairing in dsDNA . Proteins rely on thermal fluctuations in the DNA double-stranded region at physiological temperatures known as DNA ‘breathing’ . Studies are ongoing, but thus far have led us to a better understanding of the energetic favorability of various conformations of DNA .

Characterization of sound-evoked responses of photo-identified auditory striatal neurons

Presenter(s): Matthew Nardoci—Biochemistry/Biology

Co-Presenter(s): Jewlyssa Pedregon

Faculty Mentor(s): Santiago Jaramillo

Session: Prerecorded Poster Presentation

The striatum plays a critical role in decision-making based on sensory input . Specifically, the posterior region of the striatum receives projections from auditory regions of both the cerebral cortex and the thalamus . This posterior region of the striatum contains several classes of neurons, but it is not known whether these distinct neuron classes respond uniquely to different sound stimuli . Specifically, the striatum contains two types of medium spiny neurons (MSNs) that form the direct and indirect pathways, suggesting that these MSNs play distinct roles . To test whether these two types of MSNs differ in their responses to sounds, we characterized evoked responses to basic acoustic stimuli such as pure tones and amplitude modulated white noise in naive mice . We discovered that the two populations of striatal neurons differ in the way they represent temporal modulations of sounds . This suggests that direct and indirect pathway neurons in the posterior striatum differently influence sound-driven decisions as they each process distinct sound features .

The Evolution of Coronaviruses: Cross-Species Transfers and Mechanisms of Infections

Presenter(s): Tristan McKibben—Biochemistry

Faculty Mentor(s): J. Josh Snodgrass

Session 5.5: McNair Scholars Presentations

The emergence of the SARS-CoV-2 virus and its accompanying disease, COVID-19, in late 2019 has had a global impact that will likely be felt for decades to come . As the number of infections and deaths around the world keep rising, there is a pressing need to better understand the virus and its origin . This research reviews the evolution of coronaviruses as well as mechanisms of cross-species transfers and infection . Researchers have identified that SARS-CoV-2 uses the same receptor for cell entry as SARS-CoV (the virus responsible for the SARS epidemic of 2002-2004), which may aid in combating SARS-CoV-2 . In comparing the mechanisms by which SARS-CoV entered the human population and proceeded to infect human hosts to what is currently known about SARS-CoV-2, it is possible to identify areas of research that potentially provide the most utility in the search for a vaccine . The present paper systematically reviews published literature on coronaviruses with the goal of identifying promising avenues for future research .