Chemical-scale studies of G protein-coupled receptors and ligand-gated ion channels

This dissertation describes studies of G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) using unnatural amino acid mutagenesis to gain high precision insights into the function of these important membrane proteins.

Chapter 2 considers the functional role of highly conserved proline residues within the transmembrane helices of the D2 dopamine GPCR. Through mutagenesis employing unnatural α-hydroxy acids, proline analogs, and N-methyl amino acids, we find that lack of backbone hydrogen bond donor ability is important to proline function. At one proline site we additionally find that a substituent on the proline backbone N is important to receptor function.

In Chapter 3, side chain conformation is probed by mutagenesis of GPCRs and the muscle-type nAChR. Specific side chain rearrangements of highly conserved residues have been proposed to accompany activation of these receptors. These rearrangements were probed using conformationally-biased β-substituted analogs of Trp and Phe and unnatural stereoisomers of Thr and Ile. We also modeled the conformational bias of the unnatural Trp and Phe analogs employed.

Chapters 4 and 5 examine details of ligand binding to nAChRs. Chapter 4 describes a study investigating the importance of hydrogen bonds between ligands and the complementary face of muscle-type and α4β4 nAChRs. A hydrogen bond involving the agonist appears to be important for ligand binding in the muscle-type receptor but not the α4β4 receptor.

Chapter 5 describes a study characterizing the binding of varenicline, an actively prescribed smoking cessation therapeutic, to the α7 nAChR. Additionally, binding interactions to the complementary face of the α7 binding site were examined for a small panel of agonists. We identified side chains important for binding large agonists such as varenicline, but dispensable for binding the small agonist ACh.

Chapter 6 describes efforts to image nAChRs site-specifically modified with a fluorophore by unnatural amino acid mutagenesis. While progress was hampered by high levels of fluorescent background, improvements to sample preparation and alternative strategies for fluorophore incorporation are described.

Chapter 7 describes efforts toward a fluorescence assay for G protein association with a GPCR, with the ultimate goal of probing key protein-protein interactions along the G protein/receptor interface. A wide range of fluorescent protein fusions were generated, expressed in Xenopus oocytes, and evaluated for their ability to associate with each other.

Genetically engineered sensors of cell signaling

Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed FlaSh: a novel, genetically-encoded probe that can be used to measure trans-membrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive potassium channel so that voltage dependent rearrangements in the potassium channel induce changes in the fluorescence of GFP. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism non-invasively and targeted to specific developmental stages, brain regions, cell types, and sub-cellular compartments.

We also describe modifications to FlaSh that shift its color, kinetics, and dynamic range. We used multiple green fluorescent proteins to produce variants of the FlaSh sensor that generate ratiometric signal output via fluorescence resonance energy transfer (FRET). Finally, we describe initial work toward FlaSh variants that are sensitive to G-protein coupled receptor (GPCR) activation. These sensors can be used to design functional assays for receptor activation in living cells.

Fluorescence optofluidic microscopy and fluorescence microscopy based on the Talbot effect

Light microscopy has been one of the most common tools in biological research, because of its high resolution and non-invasive nature of the light. Due to its high sensitivity and specificity, fluorescence is one of the most important readout modes of light microscopy. This thesis presents two new fluorescence microscopic imaging techniques: fluorescence optofluidic microscopy and fluorescent Talbot microscopy. The designs of the two systems are fundamentally different from conventional microscopy, which makes compact and portable devices possible. The components of the devices are suitable for mass-production, making the microscopic imaging system more affordable for biological research and clinical diagnostics.

Fluorescence optofluidic microscopy (FOFM) is capable of imaging fluorescent samples in fluid media. The FOFM employs an array of Fresnel zone plates (FZP) to generate an array of focused light spots within a microfluidic channel. As a sample flows through the channel and across the array of focused light spots, a filter-coated CMOS sensor collects the fluorescence emissions. The collected data can then be processed to render a fluorescence microscopic image. The resolution, which is determined by the focused light spot size, is experimentally measured to be 0.65 μm.

Fluorescence Talbot microscopy (FTM) is a fluorescence chip-scale microscopy technique that enables large field-of-view (FOV) and high-resolution imaging. The FTM method utilizes the Talbot effect to project a grid of focused excitation light spots onto the sample. The sample is placed on a filter-coated CMOS sensor chip. The fluorescence emissions associated with each focal spot are collected by the sensor chip and are composed into a sparsely sampled fluorescence image. By raster scanning the Talbot focal spot grid across the sample and collecting a sequence of sparse images, a filled-in high-resolution fluorescence image can be reconstructed. In contrast to a conventional microscope, a collection efficiency, resolution, and FOV are not tied to each other for this technique. The FOV of FTM is directly scalable. Our FTM prototype has demonstrated a resolution of 1.2 μm, and the collection efficiency equivalent to a conventional microscope objective with a 0.70 N.A. The FOV is 3.9 mm × 3.5 mm, which is 100 times larger than that of a 20X/0.40 N.A. conventional microscope objective. Due to its large FOV, high collection efficiency, compactness, and its potential for integration with other on-chip devices, FTM is suitable for diverse applications, such as point-of-care diagnostics, large-scale functional screens, and long-term automated imaging.

Deep tissue fluorescence imaging with time-reversed light

Advances in optical techniques have enabled many breakthroughs in biology and medicine. However, light scattering by biological tissues remains a great obstacle, restricting the use of optical methods to thin ex vivo sections or superficial layers in vivo. In this thesis, we present two related methods that overcome the optical depth limit—digital time reversal of ultrasound encoded light (digital TRUE) and time reversal of variance-encoded light (TROVE). These two techniques share the same principle of using acousto-optic beacons for time reversal optical focusing within highly scattering media, like biological tissues. Ultrasound, unlike light, is not significantly scattered in soft biological tissues, allowing for ultrasound focusing. In addition, a fraction of the scattered optical wavefront that passes through the ultrasound focus gets frequency-shifted via the acousto-optic effect, essentially creating a virtual source of frequency-shifted light within the tissue. The scattered ultrasound-tagged wavefront can be selectively measured outside the tissue and time-reversed to converge at the location of the ultrasound focus, enabling optical focusing within deep tissues. In digital TRUE, we time reverse ultrasound-tagged light with an optoelectronic time reversal device (the digital optical phase conjugate mirror, DOPC). The use of the DOPC enables high optical gain, allowing for high intensity optical focusing and focal fluorescence imaging in thick tissues at a lateral resolution of 36 µm by 52 µm. The resolution of the TRUE approach is fundamentally limited to that of the wavelength of ultrasound. The ultrasound focus (~ tens of microns wide) usually contains hundreds to thousands of optical modes, such that the scattered wavefront measured is a linear combination of the contributions of all these optical modes. In TROVE, we make use of our ability to digitally record, analyze and manipulate the scattered wavefront to demix the contributions of these spatial modes using variance encoding. In essence, we encode each spatial mode inside the scattering sample with a unique variance, allowing us to computationally derive the time reversal wavefront that corresponds to a single optical mode. In doing so, we uncouple the system resolution from the size of the ultrasound focus, demonstrating optical focusing and imaging between highly diffusing samples at an unprecedented, speckle-scale lateral resolution of ~ 5 µm. Our methods open up the possibility of fully exploiting the prowess and versatility of biomedical optics in deep tissues.

Sulfur-Cycling in Methane-Rich Ecosystems: Uncovering Microbial Processes and Novel Niches

Microbial sulfur cycling communities were investigated in two methane-rich ecosystems, terrestrial mud volcanoes (TMVs) and marine methane seeps, in order to investigate niches and processes that would likely be central to the functioning of these crucial ecosystems. Terrestrial mud volcanoes represent geochemically diverse habitats with varying sulfur sources and yet sulfur-cycling in these environments remains largely unexplored. Here we characterized the sulfur-metabolizing microorganisms and activity in 4 TMVs in Azerbaijan, supporting the presence of active sulfur-oxidizing and sulfate-reducing guilds in all 4 TMVs across a range of physiochemical conditions, with diversity of these guilds being unique to each TMV. We also found evidence for the anaerobic oxidation of methane coupled to sulfate reduction, a process which we explored further in the more tractable marine methane seeps. Diverse associations between methanotrophic archaea (ANME) and sulfate-reducing bacterial groups (SRB) often co-occur in marine methane seeps, however the ecophysiology of these different symbiotic associations has not been examined. Using a combination of molecular, geochemical and fluorescence in situ hybridization coupled to nano-scale secondary ion mass spectrometry (FISH-NanoSIMS) analyses of in situ seep sediments and methane-amended sediment incubations from diverse locations, we show that the unexplained diversity in SRB associated with ANME cells can be at least partially explained by preferential nitrate utilization by one particular partner, the seepDBB. This discovery reveals that nitrate is likely an important factor in community structuring and diversity in marine methane seep ecosystems. The thesis concludes with a study of the dynamics between ANME and their associated SRB partners. We inhibited sulfate reduction and followed the metabolic processes of the community as well as the effect of ANME/SRB aggregate composition and growth on a cellular level by tracking 15N substrate incorporation into biomass using FISH-NanoSIMS. We revealed that while sulfate-reducing bacteria gradually disappeared over time in incubations with an SRB inhibitor, the ANME archaea persisted in the form of ANME-only aggregates, which are capable of little to no growth when sulfate reduction is inhibited. These data suggest ANME are not able to synthesize new proteins when sulfate reduction is inhibited.

Algorithmic challenges in green data centers

With data centers being the supporting infrastructure for a wide range of IT services, their efficiency has become a big concern to operators, as well as to society, for both economic and environmental reasons. The goal of this thesis is to design energy-efficient algorithms that reduce energy cost while minimizing compromise to service. We focus on the algorithmic challenges at different levels of energy optimization across the data center stack. The algorithmic challenge at the device level is to improve the energy efficiency of a single computational device via techniques such as job scheduling and speed scaling. We analyze the common speed scaling algorithms in both the worst-case model and stochastic model to answer some fundamental issues in the design of speed scaling algorithms. The algorithmic challenge at the local data center level is to dynamically allocate resources (e.g., servers) and to dispatch the workload in a data center. We develop an online algorithm to make a data center more power-proportional by dynamically adapting the number of active servers. The algorithmic challenge at the global data center level is to dispatch the workload across multiple data centers, considering the geographical diversity of electricity price, availability of renewable energy, and network propagation delay. We propose algorithms to jointly optimize routing and provisioning in an online manner. Motivated by the above online decision problems, we move on to study a general class of online problem named "smoothed online convex optimization", which seeks to minimize the sum of a sequence of convex functions when "smooth" solutions are preferred. This model allows us to bridge different research communities and help us get a more fundamental understanding of general online decision problems.

Bright-field and fluorescence chip-scale microscopy for biological imaging

Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.

Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.

Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.

Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

Molecular mechanism of sulfated carbohydrate recognition: structural and biochemical studies of the cysteine-rich domain of mannose receptor

Mannose receptor (MR) is widely expressed on macrophages, immature dendritic cells, and a variety of epithelial and endothelial cells. It is a 180 kD type I transmembrane receptor whose extracellular region consists of three parts: the amino-terminal cysteine-rich domain (Cys-MR); a fibronectin type II-like domain; and a series of eight tandem C-type lectin carbohydrate recognition domains (CRDs). Two portions of MR have distinct carbohydrate recognition properties: Cys-MR recognizes sulfated carbohydrates and the tandem CRD region binds terminal mannose, fucose, and N-acetyl-glucosamine (GlcNAc). The dual carbohydrate binding specificity allows MR to interact with sulfated and nonsulfated polysaccharide chains, and thereby facilitating the involvement of MR in immunological and physiological processes. The immunological functions of MR include antigen capturing (through binding non-sulfated carbohydrates) and antigen targeting (through binding sulfated carbohydrates), and the physiological roles include rapid clearance of circulatory luteinizing hormone (LH), which bears polysaccharide chains terminating with sulfated and non-sulfated carbohydrates.

We have crystallized and determined the X-ray structures of unliganded Cys-MR (2.0 Å) and Cys-MR complexed with different ligands, including Hepes (1.7 Å), 4SO_4-N-Acetylgalactosamine (4SO_4-GalNAc; 2.2 Å), 3SO_4-Lewis^x (2.2 Å), 3S04-Lewis^a (1.9 Å), and 6SO_4-GalNAc (2.5 Å). The overall structure of Cys-MR consists of 12 anti-parallel β-strands arranged in three lobes with approximate three fold internal symmetry. The structure contains three disulfide bonds, formed by the six cysteines in the Cys-MR sequence. The ligand-binding site is located in a neutral pocket within the third lobe, in which the sulfate group of ligand is buried. Our results show that optimal binding is achieved by a carbohydrate ligand with a sulfate group that anchors the ligand by forming numerous hydrogen bonds and a sugar ring that makes ring-stacking interactions with Trpll7 of CysMR. Using a fluorescence-based assay, we characterized the binding affinities between CysMR and its ligands, and rationalized the derived affinities based upon the crystal structures. These studies reveal the mechanism of sulfated carbohydrate recognition by Cys-MR and facilitate our understanding of the role of Cys-MR in MR recognition of its ligands.

Innovations of wide-field optical-sectioning fluorescence microscopy : toward high-speed volumetric bio-imaging with simplicity

Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere.

The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths.

Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often result in certain degrees of photo-damages because of the high focal intensity at the scanning point. In order to overcome such an issue, several wide-field optical-sectioning techniques have been proposed and demonstrated, although not without introducing new limitations and compromises such as low signal-to-background ratios and reduced axial resolutions. As a result, single-point-scanning optical-sectioning techniques remain the most widely used instrumentations for volumetric imaging of living biological systems to date.

In order to develop wide-field optical-sectioning techniques that has equivalent optical performance as single-point-scanning ones, this thesis first introduces the mechanisms and limitations of existing wide-field optical-sectioning techniques, and then brings in our innovations that aim to overcome these limitations. We demonstrate, theoretically and experimentally, that our proposed wide-field optical-sectioning techniques can achieve diffraction-limited optical sectioning, low out-of-focus excitation and high-frame-rate imaging in living biological systems. In addition to such imaging capabilities, our proposed techniques can be instrumentally simple and economic, and are straightforward for implementation on conventional wide-field microscopes. These advantages together show the potential of our innovations to be widely used for high-speed, volumetric fluorescence imaging of living biological systems.

Fluorescence microscopy of nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels abundantly expressed in the central nervous system. Changes in the assembly and trafficking of nAChRs are pertinent to disease states including nicotine dependence, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), and Parkinson’s disease (PD). Here we investigate the application of high resolution fluorescence techniques for the study of nAChR assembly and trafficking. We also describe the construction and validation of a fluorescent α5 subunit and subsequent experiments to elucidate the cellular mechanisms through which α5 subunits are expressed, assembled into mature receptors, and trafficked to the cell surface. The effects of a known single nucleotide polymorphism (D398N) in the intracellular loop of α5 are also examined.

Additionally, this report describes the development of a combined total internal reflection fluorescence (TIRF) and lifetime imaging (FLIM) technique and the first application of this methodology for elucidation of stochiometric composition of nAChRs. Many distinct subunit combinations can form functional receptors. Receptor composition and stoichiometry confers unique biophysical and pharmacological properties to each receptor sub-type. Understanding the nature of assembly and expression of each receptor subtype yields important information about the molecular processes that may underlie the mechanisms through which nAChR contribute to disease and addiction states.

Exciplexes in fluorescence quenching of aromatic hydrocarbons by tertiary aliphatic amines

Strong quenching of the fluorescence of aromatic hydrocarbons by tertiary aliphatic amines has been observed in solution at room temperature. Accompanying the fluorescence quenching of aromatic hydrocarbons, an anomalous emission is observed. This new emission is very broad, structureless and red-shifted from the original hydrocarbon fluorescence.

Kinetic studies indicate that this anomalous emission is due to an exciplex formed by an aromatic hydrocarbon molecule in its lowest excited singlet state with an amine molecule. The fluorescence quenching of the aromatic hydrocarbons is due to the depopulation of excited hydrocarbon molecules by the formation of exciplexes, with subsequent de-excitation of exciplexes by either radiative or non-radiative processes.

Analysis of rate constants shows the electron-transfer nature of the exciplex. Through the study of the effects on the frequencies of exciplex emissions of substituents on the hydrocarbons, it is concluded that partial electron transfer from the amine molecule to the aromatic hydrocarbon molecule in its lowest excited singlet state occurs in the formation of exciplex. Solvent effects on the exciplex emission frequencies further demonstrate the polar nature of the exciplex.

A model based on this electron-transfer nature of exciplex is proposed and proves satisfactory in interpreting the exciplex emission phenomenon in the fluorescence quenching of aromatic hydrocarbons by tertiary aliphatic amines.

On concentrated loads and Green's functions in elastostatics

This investigation is concerned with the notion of concentrated loads in classical elastostatics and related issues. Following a limit treatment of problems involving concentrated internal and surface loads, the orders of the ensuing displacements and stress singularities, as well as the stress resultants of the latter, are determined. These conclusions are taken as a basis for an alternative direct formulation of concentrated-load problems, the completeness of which is established through an appropriate uniqueness theorem. In addition, the present work supplies a reciprocal theorem and an integral representation-theorem applicable to singular problems of the type under consideration. Finally, in the course of the analysis presented here, the theory of Green's functions in elastostatics is extended.

Fast Lattice Green's Function Methods for Viscous Incompressible Flows on Unbounded Domains

In this thesis, a collection of novel numerical techniques culminating in a fast, parallel method for the direct numerical simulation of incompressible viscous flows around surfaces immersed in unbounded fluid domains is presented. At the core of all these techniques is the use of the fundamental solutions, or lattice Green’s functions, of discrete operators to solve inhomogeneous elliptic difference equations arising in the discretization of the three-dimensional incompressible Navier-Stokes equations on unbounded regular grids. In addition to automatically enforcing the natural free-space boundary conditions, these new lattice Green’s function techniques facilitate the implementation of robust staggered-Cartesian-grid flow solvers with efficient nodal distributions and fast multipole methods. The provable conservation and stability properties of the appropriately combined discretization and solution techniques ensure robust numerical solutions. Numerical experiments on thin vortex rings, low-aspect-ratio flat plates, and spheres are used verify the accuracy, physical fidelity, and computational efficiency of the present formulations.