Structure Prediction of G-Protein Coupled Receptors

G-protein coupled receptors (GPCRs) form a large family of proteins and are very important drug targets. They are membrane proteins, which makes computational prediction of their structure challenging. Homology modeling is further complicated by low sequence similarly of the GPCR superfamily.

In this dissertation, we analyze the conserved inter-helical contacts of recently solved crystal structures, and we develop a unified sequence-structural alignment of the GPCR superfamily. We use this method to align 817 human GPCRs, 399 of which are nonolfactory. This alignment can be used to generate high quality homology models for the 817 GPCRs.

To refine the provided GPCR homology models we developed the Trihelix sampling method. We use a multi-scale approach to simplify the problem by treating the transmembrane helices as rigid bodies. In contrast to Monte Carlo structure prediction methods, the Trihelix method does a complete local sampling using discretized coordinates for the transmembrane helices. We validate the method on existing structures and apply it to predict the structure of the lactate receptor, HCAR1. For this receptor, we also build extracellular loops by taking into account constraints from three disulfide bonds. Docking of lactate and 3,5-dihydroxybenzoic acid shows likely involvement of three Arg residues on different transmembrane helices in binding a single ligand molecule.

Protein structure prediction relies on accurate force fields. We next present an effort to improve the quality of charge assignment for large atomic models. In particular, we introduce the formalism of the polarizable charge equilibration scheme (PQEQ) and we describe its implementation in the molecular simulation package Lammps. PQEQ allows fast on the fly charge assignment even for reactive force fields.

Fiber-optic integration and efficient detection schemes for optomechanical resonators

With the advent of the laser in the year 1960, the field of optics experienced a renaissance from what was considered to be a dull, solved subject to an active area of development, with applications and discoveries which are yet to be exhausted 55 years later. Light is now nearly ubiquitous not only in cutting-edge research in physics, chemistry, and biology, but also in modern technology and infrastructure. One quality of light, that of the imparted radiation pressure force upon reflection from an object, has attracted intense interest from researchers seeking to precisely monitor and control the motional degrees of freedom of an object using light. These optomechanical interactions have inspired myriad proposals, ranging from quantum memories and transducers in quantum information networks to precision metrology of classical forces. Alongside advances in micro- and nano-fabrication, the burgeoning field of optomechanics has yielded a class of highly engineered systems designed to produce strong interactions between light and motion.

Optomechanical crystals are one such system in which the patterning of periodic holes in thin dielectric films traps both light and sound waves to a micro-scale volume. These devices feature strong radiation pressure coupling between high-quality optical cavity modes and internal nanomechanical resonances. Whether for applications in the quantum or classical domain, the utility of optomechanical crystals hinges on the degree to which light radiating from the device, having interacted with mechanical motion, can be collected and detected in an experimental apparatus consisting of conventional optical components such as lenses and optical fibers. While several efficient methods of optical coupling exist to meet this task, most are unsuitable for the cryogenic or vacuum integration required for many applications. The first portion of this dissertation will detail the development of robust and efficient methods of optically coupling optomechanical resonators to optical fibers, with an emphasis on fabrication processes and optical characterization.

I will then proceed to describe a few experiments enabled by the fiber couplers. The first studies the performance of an optomechanical resonator as a precise sensor for continuous position measurement. The sensitivity of the measurement, limited by the detection efficiency of intracavity photons, is compared to the standard quantum limit imposed by the quantum properties of the laser probe light. The added noise of the measurement is seen to fall within a factor of 3 of the standard quantum limit, representing an order of magnitude improvement over previous experiments utilizing optomechanical crystals, and matching the performance of similar measurements in the microwave domain.

The next experiment uses single photon counting to detect individual phonon emission and absorption events within the nanomechanical oscillator. The scattering of laser light from mechanical motion produces correlated photon-phonon pairs, and detection of the emitted photon corresponds to an effective phonon counting scheme. In the process of scattering, the coherence properties of the mechanical oscillation are mapped onto the reflected light. Intensity interferometry of the reflected light then allows measurement of the temporal coherence of the acoustic field. These correlations are measured for a range of experimental conditions, including the optomechanical amplification of the mechanics to a self-oscillation regime, and comparisons are drawn to a laser system for phonons. Finally, prospects for using phonon counting and intensity interferometry to produce non-classical mechanical states are detailed following recent proposals in literature.

Light-matter Interactions in Semiconductors and Metals: From Nitride Optoelectronics to Quantum Plasmonics

This thesis puts forth a theory-directed approach coupled with spectroscopy aimed at the discovery and understanding of light-matter interactions in semiconductors and metals.

The first part of the thesis presents the discovery and development of Zn-IV nitride materials.The commercial prominence in the optoelectronics industry of tunable semiconductor alloy materials based on nitride semiconductor devices, specifically InGaN, motivates the search for earth-abundant alternatives for use in efficient, high-quality optoelectronic devices. II-IV-N2 compounds, which are closely related to the wurtzite-structured III-N semiconductors, have similar electronic and optical properties to InGaN namely direct band gaps, high quantum efficiencies and large optical absorption coefficients. The choice of different group II and group IV elements provides chemical diversity that can be exploited to tune the structural and electronic properties through the series of alloys. The first theoretical and experimental investigation of the ZnSnxGe1−xN2 series as a replacement for III-nitrides is discussed here.

The second half of the thesis shows ab−initio calculations for surface plasmons and plasmonic hot carrier dynamics. Surface plasmons, electromagnetic modes confined to the surface of a conductor-dielectric interface, have sparked renewed interest because of their quantum nature and their broad range of applications. The decay of surface plasmons is usually a detriment in the field of plasmonics, but the possibility to capture the energy normally lost to heat would open new opportunities in photon sensors, energy conversion devices and switching. A theoretical understanding of plasmon-driven hot carrier generation and relaxation dynamics in the ultrafast regime is presented here. Additionally calculations for plasmon-mediated upconversion as well as an energy-dependent transport model for these non-equilibrium carriers are shown.

Finally, this thesis gives an outlook on the potential of non-equilibrium phenomena in metals and semiconductors for future light-based technologies.

Connectivity and Function of the Primate Insula

The insula is a mammalian cortical structure that has been implicated in a wide range of low- and high-level functions governing one’s sensory, emotional, and cognitive experiences. One particular role of this region is considered to be processing of olfactory stimuli. The ability to detect and evaluate odors has significant effects on an organism’s eating behavior and survival and, in case of humans, on complex decision making. Despite such importance of this function, the mechanism in which olfactory information is processed in the insula has not been thoroughly studied. Moreover, due to the structure’s close spatial relationship with the neighboring claustrum, it is not entirely clear whether the connectivity and olfactory functions attributed to the insula are truly those of the insula, rather than of the claustrum. My graduate work, consisting of two studies, seeks to help fill these gaps. In the first, the structural connectivity patterns of the insula and the claustrum in a non-human primate brain is assayed using an ultra-high-quality diffusion magnetic resonance image, and the results suggest dissociation of connectivity — and hence function — between the two structures. In the second study, a functional neuroimaging experiment investigates the insular activity during odor evaluation tasks in humans, and uncovers a potential spatial organization within the anterior portion of the insula for processing different aspects of odor characteristics.

I. The 3.7 Å crystal structure of horse heart ferricytochrome C. II. The application of the Karle-Hauptman tangent formula to protein phasing

I. The 3.7 Å Crystal Structure of Horse Heart Ferricytochrome C.

The crystal structure of horse heart ferricytochrome c has been determined to a resolution of 3.7 Å using the multiple isomorphous replacement technique. Two isomorphous derivatives were used in the analysis, leading to a map with a mean figure of merit of 0.458. The quality of the resulting map was extremely high, even though the derivative data did not appear to be of high quality.

Although it was impossible to fit the known amino acid sequence to the calculated structure in an unambiguous way, many important features of the molecule could still be determined from the 3.7 Å electron density map. Among these was the fact that cytochrome c contains little or no α-helix. The polypeptide chain appears to be wound about the heme group in such a way as to form a loosely packed hydrophobic core in the molecule.

The heme group is located in a cleft on the molecule with one edge exposed to the solvent. The fifth coordinating ligand is His 18 and the sixth coordinating ligand is probably neither His 26 nor His 33.

The high resolution analysis of cytochrome c is now in progress and should be completed within the next year.

II. The Application of the Karle-Hauptman Tangent Formula to Protein Phasing.

The Karle-Hauptman tangent formula has been shown to be applicable to the refinement of previously determined protein phases. Tests were made with both the cytochrome c data from Part I and a theoretical structure based on the myoglobin molecule. The refinement process was found to be highly dependent upon the manner in which the tangent formula was applied. Iterative procedures did not work well, at least at low resolution.

The tangent formula worked very well in selecting the true phase from the two possible phase choices resulting from a single isomorphous replacement phase analysis. The only restriction on this application is that the heavy atoms form a non-centric cluster in the unit cell.

Pages 156 through 284 in this Thesis consist of previously published papers relating to the above two sections. References to these papers can be found on page 155.