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Modulating Biophysical Properties of Insulin with Non-Canonical Mutagenesis at Position B28
Get PDFNon-canonical amino acids are tools for altering the chemical and physical properties of proteins, providing a facile strategy to engineer proteins with novel properties, especially where canonical amino acid mutagenesis has exhausted nearly all avenues for further optimization. Insulin, for example, is one of the most widely studied therapeutic proteins; however, non-canonical insulin engineering is a subfield that has been largely unexplored. To this end, my thesis research has focused on the use of non-canonical amino acids to understand and engineer the biophysical properties of insulin.
Protein structure and function are sensitive to the smallest of changes; even small differences such as a single atom substitution or change in stereo-orientation can cause large, unsuspected, and unpredictable global changes. Chapters 2 to 4 describe substitutions at the 4th position of proline at position 28 of insulin’s B chain (ProB28) in a manner analogous to the structure-activity-relationships that are widely used in medicinal chemistry. Canonical mutagenesis at ProB28 has led to the discovery of rapid-acting insulins (RAIs): a class of therapeutic insulins with enhanced pharmacokinetic properties. Therefore, we chose to incorporate proline analogs with substitutions such as hydroxyl and fluoro groups, and different ring compositions to assess their effects on the biophysical properties (i.e. stability, dissociation rates and oligomerizations states) of insulin. Chapter 2 describes the discovery of a hydroxyinsulin variant with faster hexamer dissociation rates and enhanced stability compared to wild-type insulin in vitro. We find crystallographic evidence of a novel hydrogen bond in the insulin dimer interface which we hypothesize stabilizes the insulin dimer state. To complement the findings in chapter 2, chapters 3 and 4 detail an investigation of the importance of hydrophobic and nonpolar interactions for modulating the biophysical properties of insulin.
Establishing structure-activity-relationships for insulin will create new opportunities for further engineering using non-canonical amino acids. In chapter 5, we describe progress towards a general, simple screening method to discover new aminoacyl-tRNA synthetases for the incorporation of non-canonical amino acids in E. coli. Implementing such a high-throughput screening system will allow scientists to perform medicinal chemistry on proteins and discover new or improved therapeutics to help manage human diseases.</p
Compact Microscope System for Biomedical Applications
Get PDFDemands for an imaging system which has high space-bandwidth product (SBP) are increasing in modern biomedical research as the amount of information to be dealt with is increasing. However, conventional microscopy has a limited SBP of about 10 mega pixels, and as such if a user wants an image in high resolution, the field of view (FOV) of the image is reduced, or if a wide FOV is necessary, the user needs to give up the resolution of image. A common way of overcoming this SBP limit in the conventional microscopy is to use mechanical moving stages and scan through wide sample area, however, it is time consuming to image large area using a high numerical aperture (NA) objective lens. This thesis presents compact imaging systems based on Fourier ptychographic microscopy for biomedical applications which are able to increase SBP without having any mechanical moving parts: one imaging system for an incubator embedded imaging system to be used in in-vitro cell culture monitoring, and the other for a high throughput 96 well plate imaging system for fast drug screening.</p
Genome Activation and Regulation of Signaling in the Rapidly Dividing Drosophila Embryo
Get PDFEmbryonic development of the fruit fly Drosophila melanogaster is unique among model organisms and animals in general, as rapid and syncytial nuclear divisions characterize the early stages before cell membranes form. These nuclear divisions occur every eight to fifteen minutes, culminating with a 45-minute cell cycle where cell membranes form and the 6000 nuclei become 6000 cells before the embryo undergoes gastrulation. At the beginning of development, maternally deposited transcripts define the major axes of the embryo and control all processes that occur. As the syncytial nuclear cycles slow and nuclei migrate to the periphery of the embryo, maternal transcripts are degraded and the zygotic genome is first activated. The rapid pace of nuclear divisions concurrent with the activation of the zygotic genome presents unique challenges to the developing embryo, as the constraints imposed by mitosis limit the ability to transcribe new genes. This switch of control, the Maternal to Zygotic Transition, has been the subject of studies at the molecular and genetic level for almost 30 years. Here, we use new tools and approaches to study the developing embryo at a time scale not previously achieved. We show how the gene regulatory network along the dorsal-ventral axis, including entire signaling pathways, is activated using time point intervals of 10 minutes. Using mutants, we show the contribution of individual genes in the process of development and the resulting changes in expression levels for the entire network. Finally, we examine the transcription of long genes during the rapid syncytial nuclear cycles, when time constraints limit the ability to transcribe the entire gene. We show how an RNA binding protein regulates the truncation of the transcripts into short isoforms with novel coding sequences, and how these short gene products code for functional proteins that regulate the spatiotemporal activation of key signaling pathways in the embryo.</p
Structure and Function of the Mycobacterial Mechanosensitive Channel of Large Conductance, MscL
Get PDFMscL is a ubiquitous channel found in bacterial membranes. It provides a protective response to osmotic downshock by opening and closing in response to tension in the membrane. A number of studies have aimed to develop a mechanism for the gating of MscL in E. coli, but structural details describing the process have remained elusive. A few structures of non-conducting states of MscL have been solved using X-ray crystallography, Mycobacterium tuberculosis (Mt) MscL and Staphylococcus aureus (Sa) MscL with a C-terminal domain truncation. In addition, the structure of the E. coli (Ec) MscL C-terminal cytoplasmic domain has been solved.
The goals of the studies presented in this thesis are as follows: (i) capturing a C-terminal domain truncation of MtMscL using X-ray crystallography, and (ii) analyzing the functional regulation of MscL channels in mycobacteria. To achieve the latter goal, we generated a knockout of the mscL gene in a fast-growing mycobacteria species, Mycobacterium smegmatis. This strain was used to analyze the role of MscL in the cell during antibiotic entry. Structural studies of MtMscL are focused on identifying the role of the C-terminal domain by studying a channel with a truncation at the C-terminal domain. The motivation for this goal comes from the structure of SaMscL, which showed that truncation of the C-terminal domain resulted in crystallizing the protein as a tetramer, an alternative oligomeric state to the pentameric state observed for the MtMscL structure. Studies on an MtMscL C-terminal domain truncation aimed to further establish that correlation. This protein was overexpressed in E. coli BL21 DE3 mscL-, purified, and crystallized by sitting drop vapor diffusion. Native crystals diffracted to 6.5 Å, and heavy atom derivative crystals diffracted to 5.8 Å. The structure of the MtMscL C-terminal truncation has been solved, and is presented in this thesis. Our studies on the structure show that the pentameric state of the channel remains intact upon truncation of the C-terminal domain. To analyze the function of our mutant, we utilized patch clamp electrophysiology studies using our expression strain as the giant spheroplast platform. The findings from the electrophysiology studies indicate that MtMscL C-terminal domain truncation results in a channel that has gating tension requirements similar to EcMscL, whereas full-length MtMscL has much higher gating tension requirements than our construct. In addition, the role of MscL in mycobacterial antibiotic susceptibility is being tested in Mycobacterium smegmatis. We have created a strain of M. smegmatis with the mscL gene knocked out, MC2155 mscL- and we have observed that upon deletion of mscL an increase in tolerance to spectinomycin is observed in our knockout strain.</p
Optical Focusing and Imaging through Scattering Media
Get PDFOptical techniques, which have been widely used in various fields including bio-medicine, remote sensing, astronomy, and industrial production, play an important role in modern life. Optical focusing and imaging, which correspond to the basic methods of utilizing light, are key to the implementation of optical techniques. In free space or a nearly transparent medium, optical imaging and focusing can be easily realized by using conventional optical elements, such as lenses and mirrors, due to the ballistic propagation of light in these media. However, in scattering media like biological tissue and fog, refractive index inhomogeneities cause diffusive propagation of light that increases with depth, which restricts the use of optical methods in thick, scattering media. Generally speaking, scattering media poses three challenges to optical focusing and imaging: wavefront aberrations, glare, and decorrelation. Wavefront aberrations can randomize light traveling through a scattering medium, disrupt the formation of focus, and break the conjugate relation in imaging. Glare caused by backscattering will largely impair the visibility of imaging, and decorrelation in dynamic media requires systems that counter the effect of scattering to operate faster than the decorrelation time. In this thesis, we explored solutions to the problem of scattering from different aspects. We presented Time Reversal by Analysis of Changing wavefronts from Kinetic targets (TRACK) technique to realize noninvasive optical focusing through a scattering medium. We showed that by taking the difference between time-varying scattering fields caused by a moving object and applying optical phase conjugation, light can be focused back to the location previously occupied by the object. To tackle the decorrelation of living tissue, we built up a fast digital optical phase conjugation (DOPC) system based on FPGA and DMD, which has a response time of 5.3 ms and was the fastest DOPC system in the world before 2017. We demonstrated that the system is fast enough to focus light through 2.3mm-thick living mouse skin. As for glare, inspired by noise canceling headphones, we invented an optical analogue termed coherence gated negation (CGN) technique. CGN can optically cancel out the glare in an active illumination imaging scenario to realize imaging through scattering media, like fog. In the experiment, we suppressed the glare by an order of magnitude and allowed improved imaging of a weak target. Finally, we demonstrated a method to image a moving target through scattering media noninvasively. Its principle roots are in the speckle-correlation-based imaging (SCI) invented by Ori Katz. We improved the technique and extended its application to bright field imaging of a moving target.</p
Studies in Palladium-Catalyzed Allylic Alkylation: Enantioselective Total Syntheses of Structurally Diverse Alkaloids
Get PDFPresented herein are three projects, all unified by the use of palladium-catalyzed, enantioconvergent, decarboxylative allylic alkylations to synthesize stereochemically rich, nitrogen-containing small molecules. The ubiquity of nitrogen in biologically active natural products and pharmaceutical ingredients necessitates perpetual exploration and development of relevant small molecules. Highly robust palladium-catalyzed allylic alkylation reactions of non-stabilized enolates enable the construction of sterically encumbered all-carbon quaternary and tetrasubstituted tertiary stereocenters present within such targets.
The successful development of a novel substrate class for palladium-catalyzed allylic alkylation, namely dihydropyrido[1,2-a]indolones (DHPIs), has enabled divergent syntheses of multiple monoterpene indole alkaloids. By setting the C20 quaternary stereocenter present within these alkaloids at an early stage in the synthesis, the remaining stereocenters can be forged with exquisite levels of control. Critical to the success of this work was the identification of highly tunable and predictable cyclizations between an indole and a C2-tethered iminium moiety. Regiodivergent cyclizations were used to complete the first catalytic enantioselective total synthesis of (–)-goniomitine, along with efficient formal syntheses of (+)-aspidospermidine and (–)-quebrachamine. Stereodivergent cyclization strategies were then employed in total syntheses of (+)-limaspermidine and (+)-kopsihainanine A. Synthetic efforts toward the highly caged Kopsia alkaloids (–)-kopsinine, (–)-kopsinilam, and (–)-kopsifoline G are also discussed.
Lastly, the synthesis of challenging alpha-quaternary Mannich-type products was accomplished through a simple, elegant inversion of strategy. The chiral building blocks made available by this technology bear significant potential in the realm of medicinal chemistry. Furthermore, this work enabled rapid total syntheses of (–)-isonitramine and (+)-sibirinine.</p
A Toolbox for Exoplanet Exploration
Get PDFIn this thesis, I develop a new suite of tools to address two questions in exoplanet science: how common are Earth-mass planets in the habitable zones of Solar-type stars, and can we detect signs of life on other worlds?
Answering the first question requires a method for detecting Earth-Sun analogs. Currently, the radial velocity (RV) method of exoplanet detection is one of the most successful tools for probing inner planetary systems. However, degeneracy between a spectrometer's wavelength calibration and the astrophysical RV shift has limited the sensitivity of today's instruments. In my thesis, I address a method for breaking this degeneracy: by combining a traditional spectrometer design with a dynamic interferometer, a fringe pattern is generated at the image plane that is highly sensitive to changes in the radial velocity of the target star. I augmented previous theoretical studies of the method, creating an end-to-end simulation to 1) introduce and recover wavelength calibration errors, and 2) investigate the effects of interferometer position errors on the RV precision. My simulation showed that using this kind of interferometric system, a 5-m class telescope could detect an Earth-Sun analog.
Addressing the occurrence rate of Earth twins also requires an understanding of planet formation in multiple star systems, which encompass half of all Solar-type stars. Gravitational interactions between binary components separated by 10-100 astronomical units are predicted to truncate the outer edges of their respective disks, possibly reducing the disks' lifetimes. Consequently, the pool of material and the amount of time available for planet formation may be smaller than in single star systems. The stars' rotational periods provide a fossil record of these events: star-disk magnetic interactions initially prevent a contracting pre-main sequence star from spinning up, and hence a star with a shorter-lived disk is expected to be spinning more quickly when it reaches the zero age main sequence. In order to conduct a large-scale multiplicity survey to investigate the relationship between stellar rotation and binary system properties (e.g. their separations and mass ratios), I contributed to the commissioning of Robo-AO, a robotic laser guide star adaptive optics system, at the Kitt Peak 2.1-m. After the instrument's installation, I wrote a data pipeline to optimize the system's sensitivity to close stellar companions via reference star differential imaging. I then characterized Robo-AO's performance during its first year of operations. Finally, I used Robo-AO to search for binaries among the 759 stars in the Pleiades with rotational periods measured using the photometric data of the re-purposed Kepler telescope, K2.
Detecting signs of life on other worlds will require detailed characterization of rocky exoplanet atmospheres. Polarimetry has long been proposed as a means of probing these atmospheres, but current instruments lack the sensitivity to detect the starlight reflected and polarized by such small, close-in planets. However, the latest generation of high contrast imaging instruments (e.g. GPI and SPHERE) may be able to detect the polarization of thermal emission by young gas giants due to scattering by aerosols in their atmospheres. Observational constraints on the details of clouds physics imposed by polarized emission will improve our understanding of the planets' compositions, and hence their formation histories. For the case of the brown dwarf HD19467 B orbiting a nearby Sun-like star, I demonstrated that the Gemini Planet Imager can detect linear polarizations on the order predicted for these cloudy exoplanets. My current pilot programs can produce the first detections of polarized exoplanet emission, while also building expertise for reflected starlight polarimetry with future observatories.</p
Nanophotonic Resonators for Optical Quantum Memories based on Rare-Earth-Doped Materials
Get PDFThe growing interest in optical quantum systems has led to the exploration of multiple platforms. Though pioneering experiments were performed in trapped atom and trapped ion systems, solid state systems show promise of being scalable and robust. Rare earth dopants in crystalline hosts are an appealing option because they possess a rich spectrum of energy levels that result from a partially filled electron orbital. While level structure varies across the period, all elements possess crystal field splittings corresponding to near infra-red or optical frequencies, as well as Zeeman and often hyperfine levels separated by radio frequency and microwave frequencies. These levels demonstrate long excited-state lifetimes and coherence times and have been used in diverse applications, including demonstrating storage of a photonic state, converting of optical to microwave photons, and manipulating a single ion as a single qubit. The ions' weak interaction with their environment results in low coupling to optical fields, which had previously required measurements with macroscopically large ensembles of ions. Coupling the ions to an optical cavity enables the use of a smaller ensemble, which is required for the development of the aforementioned technologies in an on-chip scalable architecture.
This thesis contains recent progress towards fabricating optical micro and nanocavities coupled to ensembles of erbium ions, mainly erbium in yttrium orthosilicate. In one design, focused ion beam milling was used to create a triangular nanobeam photonic crystal cavity in a bulk erbium-doped substrate. A second design leveraged the fabrication capabilities of silicon photonics, defining amorphous silicon ring resonators using electron beam lithography and dry etching. These devices coupled evanescently to erbium ions below the ring, in the bulk substrate. Simulation, design, fabrication, and characterization of both resonators are discussed. Coupling between the ions and the resonator is demonstrated for each, and capabilities offered by these devices are described. Preliminary work implementing coherent control of erbium ions is presented. Lastly, alternative substrates are evaluated for possible future solid-state erbium systems.</p
Biophysical Characterization of an ABC L-methionine Transporter
Get PDFThe ATP-binding cassette (ABC) superfamily is pivotal to a number of important biochemical processes and ubiquitous in all kingdoms of life. Previous studies of ABC transporters have been heavily focused on the structural determination of the different intermediates of the transport cycle. In order to characterize the mechanism of an E. coli L-methionine transporter, which is an ABC importer, we first collated previously reported structural information on the conformational states of several well characterized ABC importers and associated binding proteins, and identified four major conformations (i.e., pre-T, outward, post-T, and inward state). We stabilized these intermediates using appropriate mutations, substrates, and nucleotides. We then studied the kinetics and thermodynamics of the formation of these states using surface plasmon resonance (BiaCore, GE Healthcare) and MicroScale Thermophoresis (NanoTemper). We developed a quantitative model that details the kinetic and molecular mechanism of E. coli MetNI. Towards this goal, we extended the Two-State, alternating access model to include other intermediates that are crucial to transport and are using this to provide a temporal understanding of transport. While this model is developed to describe the behavior of the Lmethionine MetNI importer, it may also have predictive power for other ABC Type I importers, since the NBD’s response for coupling transport to ATP-binding and hydrolysis are highly conserved in this family.</p
I. Tin Silsesquioxanes as Analogs for the Open and Closed Sites in Tin-Containing Zeotype Beta and II. Enantiomerically Enriched, Polycrystalline Molecular Sieves
Get PDFThe use of biomass as a resource to produce value-added products has garnered significant interest as a means of reducing reliance on fossil fuels. This task is complicated by the complex, highly functionalized nature of abundant biomass derivatives, such as glucose. Tin-containing zeolite Beta (Sn-Beta) has been investigated as a catalyst for isomerizing aldohexoses into ketohexoses through a Lewis acid mediated hydride shift (1,2-intramolecular hydride shift, 1,2-HS). Recent studies on the reactivities of Lewis base-doped and alkali-exchanged Sn-Beta samples have conclusively demonstrated that the open tin site performs the glucose isomerization reaction. With Lewis base doped Sn-Beta, glucose conversion is almost completely eliminated and product selectivity is shifted predominantly to mannose, formed through a 1,2-intramolecular carbon shift (1,2-CS). To understand the structure-activity relationships between the conditions of the active sites in the zeolite, three molecular models (tin silsesquioxanes) of the tin sites in the zeolite are synthesized. Two tin silsesquioxanes that contain an octahedral tin site with and without an adjacent silanol group are prepared and used as catalysts for the reaction of glucose. The catalyst that contains the adjacent silanol group selectively forms fructose through a 1,2-HS while the catalyst without the silanol group yields mannose through a 1,2-CS. These results provide further evidence for the nature of the active sites in Sn-Beta. A methyl-ligated tin silsesquioxane is experimentally and theoretically examined to examine possible reactivities at the closed site. This compound is an active glucose conversion catalyst that selectively produces mannose, although the rates of reaction are far below those obtained from Sn-Beta. Additionally, a hybrid quantum mechanical/molecular mechanics model is constructed, and the complete catalytic cycle is computationally examined via considering ring-opening, three distinct pathways for each hydride- and carbon-shift reaction, and ring-closing. The combined experimental and computational results suggest that there could be reaction pathways that involve Si-O-Sn cleavage that give much slower reaction rates than the open tin site in Sn-Beta.
Zeolite and zeolite-like molecular sieves are being used in a large number of applications such as adsorption and catalysis. Achievement of the long-standing goal of creating a chiral, polycrystalline molecular sieve with bulk enantioenrichment would enable these materials to perform enantioselective functions. In part II of this thesis, the synthesis of enantiomerically enriched samples of a molecular sieve is reported. Enantiopure organic structure directing agents (OSDAs) are designed with the assistance of computational methods, and used to synthesize enantioenriched, polycrystalline molecular sieve samples of either enantiomer. Computational results correctly predicted which enantiomer is obtained, and enantiomeric enrichment is proven by high-resolution transmission electron microscopy. The enantioenriched and racemic samples of the molecular sieves are tested as adsorbents and heterogeneous catalysts. The enantioenriched molecular sieves show enantioselectivity for the ring opening reaction of epoxides and enantioselective adsorption of 2-butanol (R enantiomer of the molecular sieve shows opposite and approximately equal enantioselectivity from the S enantiomer of the molecular sieve, while the racemic sample of the molecular sieve shows no enantioselectivity).</p