The Shape of the Periphery

Includes: Familiar Strangers; Recoil; (Despair)ity; The Shape of the Periphery; ImaginedBachelor of Art

Stratigraphy and Porosity Modeling of South-Central Illinois (USA) Chester (Upper Mississippian) Series Sandstones Using Petrel

Maximizing resource extraction from mature oilfields requires enhanced and secondary recovery techniques. The success of these methods relies on knowledge and understanding of the reservoir geology and hydraulics. At the Loudon Oilfield (Illinois, USA), enhanced oil recovery is being used to extend the production life of the reservoir. The suitability and placement of additional wells for oil recovery processes required three-dimensional (3D) facies and porosity modeling of the oilfield. The purpose of this work was to assess the ability of a porosity model to predict sandstone facies. The facies model for the Loudon field was generated using data obtained from digitized-wireline logs. The facies model provided sand thickness and insight in the geometries and interconnections of the producing formations. The porosity model identified zones of high porosity, and illustrated the discontinuous nature of the porosity zones within the oilfield. Comparison of facies and porosity models revealed strong correlation and similarity between the models

R4 and R12 subfamily RGS proteins: structures, functions, and emerging chemical biology

It is essential that cells respond to their extracellular environment with appropriate intracellular changes. Many environmental cues are received at the cell membrane, by a family of G-protein coupled receptors (GPCRs) and their heterotrimeric G-proteins, composed of Gα, Gβ and Gγ subunits. Upon binding of a hormone, neurotransmitter, tastant, or small molecule agonist at the membrane-bound GPCR, the receptor catalyzes the exchange of GDP for GTP on the heterotrimeric Gα subunit. This change results in the release of Gβγ from the Gα subunit. The dissociated Gα and Gβγ dimer can each signal to downstream effectors until the Gα hydrolyzes GTP, resulting in the reassociation of the Gαβγ heterotrimer. The duration of effector activation is therefore controlled by the duration of the Gα subunit in its GTP-bound state. The state of Gα as a GTP-bound protein is short-lived, however, given that the protein has an intrinsic ability to hydrolyze GTP to GDP and inorganic phosphate - an activity that can be greatly accelerated by Regulator of G-protein Signaling (RGS) proteins, which are known to act as GTPase-accelerating proteins (GAPs) for Gα subunits. The work described herein represents series of studies aimed at furthering our understanding of the molecular determinants of RGS protein/Gα interaction specificity, facilitating the identification of small molecule modulators of RGS protein activity, and understanding the biochemical function and physiological roles of RGS21. Toward the first aim, I performed mutagenesis on residues predicted to change the Gα specificity of RGS2 and extensively characterized these mutants using GTP hydrolysis assays and Gα interaction assays employing surface plasmon resonance and in vitro FRET. To comprehensively understand the role that each mutation was playing in allowing RGS2 to bind to a non-native Gα binding partner, I solved a crystal structure of a mutant RGS2 in complex with Gαi. Toward the second aim, facilitating the identification of small molecule modulators of RGS protein function, I used a variety of biophysical tools to determine the mechanism of action of the first commercially available RGS protein inhibitor - which was ultimately determined to be a non-specific, thiol-reactive compound. In order to identify new small molecule modulators of RGS protein function, I developed and validated a high-throughput screen for the RGS12/Gαi1 interaction. This screen was run against several compound libraries, both locally and at the NIH Chemical Genomics Center (NCGC); however, no hits were subsequently validated as in vivo inhibitors of the RGS12/Gαi1 interaction. Given these setbacks, we rethought how we were screening for RGS protein inhibitors and developed a completely novel, enzymatic-based assay that can be used for high-throughput screening. Toward the final aim, we confirmed the disputed report by von Buchholtz et al. that RGS21 is expressed only in chemosensory cells; however, we were also able to identify RGS21 transcripts in sensory digestive and pulmonary epithelia. Using biochemical methods, we demonstrated that RGS21 exhibits high affinity binding toward a variety of Gα substrates and that it can accelerate their GTP hydrolysis in vitro. We also present data that endogenous RGS21 expression serves to negatively regulate tastant receptor signal transduction in a cellular model of gustation

Molecular architecture of Gαo and the structural basis for RGS16-mediated deactivation

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an α subunit with intrinsic GTPase activity and a βγ heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the α subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Gα subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Gα subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein Gαo in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon Gαo binding, the determinants for GAP activity, and the structurally unique features of Gαo that likely distinguish it physiologically from other members of the larger Gαi family, affording insight to receptor, GAP and effector specificity

A P-loop Mutation in Gα Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis

Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gαi1(G42R) binding to GDP·AlF4− or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gβγ and GoLoco motifs in any nucleotide state. The corresponding Gαq(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants