Biology Commons^™

The Neuronal Calcium Sensor Ncs-1 Regulates The Phosphorylation State And Activity Of The Gα Chaperone And Gef Ric-8a, Daniel Muñoz-Reyes, Levi J. Mcclelland, Sandra Arroyo-Urea, Sonia Sánchez-Yepes, Juan Sabín, Sara Pérez-Suárez, Margarita Menendez, Alicia Mansilla, Javier García-Nafría, Stephen Sprang, Maria Jose Sanchez-Barrena

Biological Sciences Faculty Publications

The neuronal calcium sensor 1 (NCS-1), an EF-hand Ca binding protein, and Ric-8A coregulate synapse number and probability of neurotransmitter release. Recently, the structures of Ric-8A bound to Gα have revealed how Ric-8A phosphorylation promotes Gα recognition and activity as a chaperone and guanine nucleotide exchange factor. However, the molecular mechanism by which NCS-1 regulates Ric-8A activity and its interaction with Gα subunits is not well understood. Given the interest in the NCS-1/Ric-8A complex as a therapeutic target in nervous system disorders, it is necessary to shed light on this molecular mechanism of action at atomic level. We have reconstituted …

Application Of Sulfur Sad To Small Crystals With A Large Asymmetric Unit And Anomalous Substructure, Tung Chung Mou, Baisen Zeng, Tzanko I. Doukov, Stephen R. Sprang

Biological Sciences Faculty Publications

The application of sulfur single-wavelength anomalous dispersion (S-SAD) to determine the crystal structures of macromolecules can be challenging if the asymmetric unit is large, the crystals are small, the size of the anomalously scattering sulfur structure is large and the resolution at which the anomalous signals can be accurately measured is modest. Here, as a study of such a case, approaches to the SAD phasing of orthorhombic Ric-8A crystals are described. The structure of Ric-8A was published with only a brief description of the phasing process [Zeng et al. (2019), Structure, 27, 1137-1141]. Here, alternative approaches to determining the 40-atom …

Evolutionary Dynamics And Structural Consequences Of De Novo Beneficial Mutations And Mutant Lineages Arising In A Constant Environment, Margie Kinnersley, Katja Schwartz, Dong Dong Yang, Gavin Sherlock, Frank Rosenzweig

Biological Sciences Faculty Publications

Background: Microbial evolution experiments can be used to study the tempo and dynamics of evolutionary change in asexual populations, founded from single clones and growing into large populations with multiple clonal lineages. High-throughput sequencing can be used to catalog de novo mutations as potential targets of selection, determine in which lineages they arise, and track the fates of those lineages. Here, we describe a long-term experimental evolution study to identify targets of selection and to determine when, where, and how often those targets are hit. Results: We experimentally evolved replicate Escherichia coli populations that originated from a mutator/nonsense suppressor ancestor …

The Structure Of The Cysteine-Rich Region From Human Histone-Lysine N-Methyltransferase Ehmt2 (G9a), Keshia M. Kerchner, Tung-Chung Mou, Yizhi Sun, Domniţa-Valeria Rusnac, Stephen R. Sprang, Klára Briknarová

Biological Sciences Faculty Publications

Euchromatic histone-lysine N-methyltransferase 1 (EHMT1; G9a-like protein; GLP) and euchromatic histone-lysine N-methyltransferase 2 (EHMT2; G9a) are protein lysine methyltransferases that regulate gene expression and are essential for development and the ability of organisms to change and adapt. In addition to ankyrin repeats and the catalytic SET domain, the EHMT proteins contain a unique cysteine-rich region (CRR) that mediates protein-protein interactions and recruitment of the methyltransferases to specific sites in chromatin. We have determined the structure of the CRR from human EHMT2 by X-ray crystallography and show that the CRR adopts an unusual compact fold with four bound zinc atoms. The …

Novel Small Rnas Expressed By Bartonella Bacilliformis Under Multiple Conditions Reveal Potential Mechanisms For Persistence In The Sand Fly Vector And Human Host, Shaun Wachter, Linda D. Hicks, Rahul Raghavan, Michael F. Minnick

Biological Sciences Faculty Publications

Bartonella bacilliformis, the etiological agent of Carrión's disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión's disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing …

Matrices (Re)Loaded: Durability, Viability, And Fermentative Capacity Of Yeast Encapsulated In Beads Of Different Composition During Long-Term Fed-Batch Culture, Jordan Gulli, Peter Yunker, Frank Rosenzweig

Biological Sciences Faculty Publications

Encapsulated microbes have been used for decades to produce commodities ranging from methyl ketone to beer. Encapsulated cells undergo limited replication, which enables them to more efficiently convert substrate to product than planktonic cells and which contributes to their stress resistance. To determine how encapsulated yeast supports long-term, repeated fed-batch ethanologenic fermentation, and whether different matrices influence that process, fermentation and indicators of matrix durability and cell viability were monitored in high-dextrose, fed-batch culture over 7 weeks. At most timepoints, ethanol yield (g/g) in encapsulated cultures exceeded that in planktonic cultures. And frequently, ethanol yield differed among the four matrices …

Human Vascular Endothelial Cells Express Epithelial Growth Factor In Response To Infection By Bartonella Bacilliformis, Linda D. Hicks, Michael F. Minnick

Biological Sciences Faculty Publications

Bartonella are Gram-negative bacterial pathogens that trigger pathological angiogenesis during infection of humans. Bartonella bacilliformis (Bb) is a neglected tropical agent endemic to South America, where it causes Carrión's disease. Little is known about Bb's virulence determinants or how the pathogen elicits hyperproliferation of the vasculature, culminating in Peruvian warts (verruga peruana) of the skin. In this study, we determined that active infection of human umbilical vein endothelial cells (HUVECs) by live Bb induced host cell secretion of epidermal growth factor (EGF) using ELISA. Killed bacteria or lysates of various Bb strains did not cause EGF production, suggesting that an …

Structure Of The G Protein Chaperone And Guanine Nucleotide Exchange Factor Ric-8a Bound To Gαi1, Levi J. Mcclelland, Kaiming Zhang, Tung-Chung Mou, Jake Johnston, Cindee Yates-Hansen, Shanshan Li, Celestine J. Thomas, Tzanko I. Doukov, Sarah Triest, Alexandre Wohlkonig, Gregory G. Tall, Jan Steyaert, Wah Chiu, Stephen R. Sprang

Biological Sciences Faculty Publications

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that …

Encapsulation Enhances Protoplast Fusant Stability, Jordan Gulli, Eugene Kroll, Frank Rosenzweig

Biological Sciences Faculty Publications

A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as …

Gene Duplication And Deletion, Not Horizontal Transfer, Drove Intra-Species Mosaicism Of Bartonella Henselae, Rachana Banerjee, Oshina Shine, Vyshakh Rajachandran, Govind Krishnadas, Michael F. Minnick, Sandip Paul, Sujay Chattopadhyay

Biological Sciences Faculty Publications

Bartonella henselae is a facultative intracellular pathogen that occurs worldwide and is responsible primarily for cat-scratch disease in young people and bacillary angiomatosis in immunocompromised patients. The principal source of genome-level diversity that contributes to B. henselae's host-adaptive features is thought to be horizontal gene transfer events. However, our analyses did not reveal the acquisition of horizontally-transferred islands in B. henselae after its divergence from other Bartonella. Rather, diversity in gene content and genome size was apparently acquired through two alternative mechanisms, including deletion and, more predominantly, duplication of genes. Interestingly, a majority of these events occurred in regions that …

A Csra-Binding, Trans-Acting Srna Of Coxiella Burnetii Is Necessary For Optimal Intracellular Growth And Vacuole Formation During Early Infection Of Host Cells, Shaun Wachter, Matteo Bonazzi, Kyle Shifflett, Abraham S. Moses, Rahul Raghavan, Michael F. Minnick

Biological Sciences Faculty Publications

Coxiella burnetii is an obligate intracellular gammaproteobacterium and zoonotic agent of Q fever. We previously identified 15 small noncoding RNAs (sRNAs) of C. burnetii. One of them, CbsR12 (Coxiella burnetii small RNA 12), is highly transcribed during axenic growth and becomes more prominent during infection of cultured mammalian cells. Secondary structure predictions of CbsR12 revealed four putative CsrA-binding sites in stem loops with consensus AGGA/ANGGA motifs. We subsequently determined that CbsR12 binds to recombinant C. burnetii CsrA-2, but not CsrA-1, proteins in vitro. Moreover, through a combination of in vitro and cell culture assays, we identified several in trans mRNA …

Diverse Conditions Support Near-Zero Growth In Yeast: Implications For The Study Of Cell Lifespan, Jordan Gulli, Emily Cook, Eugene Kroll, Adam Rosebrock, Amy Caudy, Frank Rosenzweig

Biological Sciences Faculty Publications

Baker’s yeast has a finite lifespan and ages in two ways: a mother cell can only divide so many times (its replicative lifespan), and a non-dividing cell can only live so long (its chronological lifespan). Wild and laboratory yeast strains exhibit natural variation for each type of lifespan, and the genetic basis for this variation has been generalized to other eukaryotes, including met-azoans. To date, yeast chronological lifespan has chiefly been studied in relation to the rate and mode of functional decline among non-dividing cells in nutrient-depleted batch culture. However, this culture method does not accurately capture two major classes …

Structure, Function, And Dynamics Of The Gα Binding Domain Of Ric-8a, Baisen Zeng, Tung-Chung Mou, Tzanko I. Doukov, Andrea Steiner, Wenxi Yu, Makaia Papasergi-Scott, Gregory G. Tall, Franz Hagn, Stephen R. Sprang

Biological Sciences Faculty Publications

Ric-8A is a 530-amino acid cytoplasmic molecular chaperone and guanine nucleotide exchange factor (GEF) for i, q, and 12/13 classes of heterortrimeric G protein alpha subunits (Gα). We report the 2.2-Å crystal structure of the Ric-8A Gα-binding domain with GEF activity, residues 1-452, and is phosphorylated at Ser435 and Thr440. Residues 1-429 adopt a superhelical fold comprised of Armadillo (ARM) and HEAT repeats, and the C terminus is disordered. One of the phosphorylated residues potentially binds to a basic cluster in an ARM motif. Amino acid sequence conservation and published hydrogen-deuterium exchange data indicate repeats 3 through 6 to be …

De Novo Origins Of Multicellularity In Response To Predation, Matthew D. Herron, Joshua M. Borin, Jacob C. Boswell, Jillian Walker, I. Chen Kimberly Chen, Charles A. Knox, Margrethe Boyd, Frank Rosenzweig, William C. Ratcliff

Biological Sciences Faculty Publications

The transition from unicellular to multicellular life was one of a few major events in the history of life that created new opportunities for more complex biological systems to evolve. Predation is hypothesized as one selective pressure that may have driven the evolution of multicellularity. Here we show that de novo origins of simple multicellularity can evolve in response to predation. We subjected outcrossed populations of the unicellular green alga Chlamydomonas reinhardtii to selection by the filter-feeding predator Paramecium tetraurelia. Two of five experimental populations evolved multicellular structures not observed in unselected control populations within ~750 asexual generations. Considerable variation …

Atypical Activation Of The G Protein Gα By The Oncogenic Mutation Q209p, Marcin Maziarz, Anthony Leyme, Arthur Marivin, Alex Luebbers, Prachi P. Patel, Zhe Chen, Stephen R. Sprang, Mikel Garcia-Marcos

Biological Sciences Faculty Publications

The causative role of G protein-coupled receptor (GPCR) pathway mutations in uveal melanoma (UM) has been well-established. Nearly all UMs bear an activating mutation in a GPCR pathway mediated by G proteins of the G family, driving tumor initiation and possibly metastatic progression. Thus, targeting this pathway holds therapeutic promise for managing UM. However, direct targeting of oncogenic Gα mutants, present in ∼90% of UMs, is complicated by the belief that these mutants structurally resemble active Gα WT. This notion is solidly founded on previous studies characterizing Gα mutants in which a conserved catalytic glutamine (Gln-209 in Gα) is replaced …

Genetics Of A De Novo Origin Of Undifferentiated Multicellularity, Matthew D. Herron, William C. Ratcliff, Jacob Boswell, Frank Rosenzweig

Biological Sciences Faculty Publications

The evolution of multicellularity was a major transition in evolution and set the stage for unprecedented increases in complexity, especially in land plants and animals. Here, we explore the genetics underlying a de novo origin of multicellularity in a microbial evolution experiment carried out on the green alga Chlamydomonas reinhardtii. We show that large-scale changes in gene expression underlie the transition to a multicellular life cycle. Among these, changes to genes involved in cell cycle and reproductive processes were overrepresented, as were changes to C. reinhardtii-specific and volvocine-specific genes. These results suggest that the genetic basis for the experimental evolution …

Streamlined Preparation Of Genomic Dna In Agarose Plugs For Pulsed-Field Gel Electrophoresis, Linda D. Hicks, Charlotte M. Van Der Graaf, Jacob Childress, Emily Cook, Karen Schmidt, Frank Rosenzweig, Eugene Kroll

Biological Sciences Faculty Publications

Genome analysis using pulsed-field gel electrophoresis (PFGE) has been used in applications ranging from typing bacterial strains to radiobiology to cancer research. While methods for running PFGE have been significantly improved since its invention, the method for preparing chromosomal DNA itself has remained essentially unchanged. This limits the applicability of PFGE, especially when analyses require many samples. We have streamlined sample preparation for routine applications of PFGE through the use of deep-well 48-well plates. Besides saving time, our protocol has the added advantage of reducing the volume of expensive reagents. Our improved protocol enables us to reduce throughput time and …

Analysis Of Motility In Multicellular Chlamydomonas Reinhardtii Evolved Under Predation, Margrethe Boyd, Frank Rosenzweig, Matthew D. Herron

Biological Sciences Faculty Publications

The advent of multicellularity was a watershed event in the history of life, yet the transition from unicellularity to multicellularity is not well understood. Multicellularity opens up opportunities for innovations in intercellular communication, cooperation, and specialization, which can provide selective advantages under certain ecological conditions. The unicellular alga Chlamydomonas reinhardtii has never had a multicellular ancestor yet it is closely related to the volvocine algae, a clade containing taxa that range from simple unicells to large, specialized multicellular colonies. Simple multicellular structures have been observed to evolve in C. reinhardtii in response to predation or to settling rate-based selection. Structures …

Deciphering The Origin, Evolution, And Physiological Function Of The Subtelomeric Arylalcohol Dehydrogenase Gene Family In The Yeast Saccharomyces Cerevisiae, Dong Dong Yang, Gustavo M. De Billerbeck, Jin Jing Zhang, Frank Rosenzweig, Jean Marie Francois

Biological Sciences Faculty Publications

Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). Yeast AAD genes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes, AAD4 and AAD14, encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeast AAD genes are undergoing pseudogenization. The 5' sequence of AAD15 has been deleted from the genome. Repair of an AAD3 missense mutation at the catalytically essential Tyr73 …

Identification Of Novel Mites (Miniature Inverted-Repeat Transposable Elements) In Coxiella Burnetii: Implications For Protein And Small Rna Evolution, Shaun Wachter, Rahul Raghavan, Jenny Wachter, Michael F. Minnick

Biological Sciences Faculty Publications

Background: Coxiella burnetii is a Gram-negative gammaproteobacterium and zoonotic agent of Q fever. C. burnetii’s genome contains an abundance of pseudogenes and numerous selfish genetic elements. MITEs (miniature invertedrepeat transposable elements) are non-autonomous transposons that occur in all domains of life and are thought to be insertion sequences (ISs) that have lost their transposase function. Like most transposable elements (TEs), MITEs are thought to play an active role in evolution by altering gene function and expression through insertion and deletion activities. However, information regarding bacterial MITEs is limited.

Results: We describe two MITE families discovered during research on small non-coding …

Crystal Structure Of The Major Quadruplex Formed In The Promoter Region Of The Human C-Myc Oncogene, Sascha Stump, Tung-Chung Mou, Stephen R. Sprang, Nicholas R. Natale, Howard D. Beall

Biological Sciences Faculty Publications

The c-MYC oncogene mediates multiple tumor cell survival pathways and is dysregulated or overexpressed in the majority of human cancers. The NHE III1 region of the c-MYC promoter forms a DNA quadruplex. Stabilization of this structure with small molecules has been shown to reduce expression of c-MYC, and targeting the c-MYC quadruplex has become an emerging strategy for development of antitumor compounds. Previous solution NMR studies of the c-MYC quadruplex have assigned the major conformer and topology of this important target, however, regions outside the G-quartet core were not as well-defined. Here, we report a high-resolution crystal structure (2.35 Å) …

Accommodating Mixed-Severity Fire To Restore And Maintain Ecosystem Integrity With A Focus On The Sierra Nevada Of California, Usa, Dominick A. Dellasala, Richard L. Hutto, Chad T. Hanson, Monica L. Bond, Timothy Ingalsbee, Dennis C. Odion, William L. Baker

Biological Sciences Faculty Publications

Existing fire policy encourages the maintenance of ecosystem integrity in fire management, yet this is difficult to implement on lands managed for competing economic, human safety, and air quality concerns. We discuss a fire management approach in the mid-elevations of the Sierra Nevada, California, USA, that may exemplify similar challenges in other fire-adapted regions of the western USA. We also discuss how managing for pyrodiversity through mixed-severity fires can promote ecosystem integrity in Sierran mixed conifer and ponderosa pine (Pinus ponderosa Laws) forests. To illustrate, we show how coarse-filter (landscape-level) and complementary fine-filter (species-level) approaches can enhance forest management …

Reply To Marques Et Al. (2017): How To Best Handle Potential Detectability Bias, Richard L. Hutto

Biological Sciences Faculty Publications

Marques et al. (2017) write in response to a communications paper in which I (Hutto 2016) question whether a model-based approach is the only way to deal with the detectability problem inherent in bird survey work. The model-based approach to dealing with potential detectability bias is widely viewed as the operational gold standard, and I welcome the comments from experts in this field. The authors did uncover some errors on my part, which I address below, but they also misrepresented my most important points, so I am glad to have the opportunity to respond here. [1st paragraph]

Analysis Of The Caenorhabditis Elegans Innate Immune Response To Coxiella Burnetii, James M. Battisti, Lance A. Watson, Myo T. Naung, Adam M. Drobish, Ekaterina Voronina, Michael F. Minnick

Biological Sciences Faculty Publications

The nematode Caenorhabditis elegans is well established as a system for characterization and discovery of molecular mechanisms mediating microbe-specific inducible innate immune responses to human pathogens. Coxiella burnetii is an obligate intracellular bacterium that causes a flu-like syndrome in humans (Q fever), as well as abortions in domesticated livestock, worldwide. Initially, when wild type C. elegans (N2 strain) was exposed to mCherry-expressing C. burnetii (CCB) a number of overt pathological manifestations resulted, including intestinal distension, deformed anal region and a decreased lifespan. However, nematodes fed autoclave-killed CCB did not exhibit these symptoms. Although vertebrates detect C. burnetii via TLRs, pathologies …

Stability Of Cross-Feeding Polymorphisms In Microbial Communities, Ivana Gudelj, Margie Kinnersley, Peter Rashkov, Karen Schmidt, Frank Rosenzweig

Biological Sciences Faculty Publications

Cross-feeding, a relationship wherein one organism consumes metabolites excreted by another, is a ubiquitous feature of natural and clinically-relevant microbial communities and could be a key factor promoting diversity in extreme and/or nutrient-poor environments. However, it remains unclear how readily cross-feeding interactions form, and therefore our ability to predict their emergence is limited. In this paper we developed a mathematical model parameterized using data from the biochemistry and ecology of an E. coli cross-feeding laboratory system. The model accurately captures short-term dynamics of the two competitors that have been observed empirically and we use it to systematically explore the stability …

Cytochrome C Can Form A Well-Defined Binding Pocket For Hydrocarbons, Levi J. Mcclelland, Harmen B. Steele, Frank G. Whitby, Tung-Chung Mou, David Holley, J B. Ross, Stephen R. Sprang, Bruce E. Bowler

Biological Sciences Faculty Publications

Cytochrome c can acquire peroxidase activity when it binds to cardiolipin in mitochondrial membranes. The resulting oxygenation of cardiolipin by cytochrome c provides an early signal for the onset of apoptosis. The structure of this enzyme-substrate complex is a matter of considerable debate. We present three structures at 1.7-2.0 Å resolution of a domain-swapped dimer of yeast iso-1-cytochrome c with the detergents, CYMAL-5, CYMAL-6, and ω-undecylenyl-β-d-maltopyranoside, bound in a channel that places the hydrocarbon moieties of these detergents next to the heme. The heme is poised for peroxidase activity with water bound in place of Met80, which serves as the …

Ric-8a, A G Protein Chaperone With Nucleotide Exchange Activity Induces Long-Range Secondary Structure Changes In Gα, Ravi Kant, Baisen Zeng, Celestine J. Thomas, Brian Bothner, Stephen R. Sprang

Biological Sciences Faculty Publications

Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein α subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Gα Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gαi1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Gα interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, …

Structural Basis For Negative Allosteric Modulation Of Glun2a-Containing Nmda Receptors, Feng Yi, Tung-Chung Mou, Katherine N. Dorsett, Robert A. Volkmann, Frank S. Menniti, Stephen R. Sprang, Kasper B. Hansen

Biological Sciences Faculty Publications

NMDA receptors mediate excitatory synaptic transmission and regulate synaptic plasticity in the central nervous system, but their dysregulation is also implicated in numerous brain disorders. Here, we describe GluN2A-selective negative allosteric modulators (NAMs) that inhibit NMDA receptors by stabilizing the apo state of the GluN1 ligand-binding domain (LBD), which is incapable of triggering channel gating. We describe structural determinants of NAM binding in crystal structures of the GluN1/2A LBD heterodimer, and analyses of NAM-bound LBD structures corresponding to active and inhibited receptor states reveal a molecular switch in the modulatory binding site that mediate the allosteric inhibition. NAM binding causes …

Nanosecond Dynamics Of Gαi1 Bound To Nucleotides Or Ric-8a, A Gα Chaperone With Gef Activity, Labe A. Black, Celestine J. Thomas, Gwendolyn N. Nix, Michelle C. Terwilliger, Stephen R. Sprang, J B. Ross

Biological Sciences Faculty Publications

Resistance to Inhibitors of Cholinesterase A (Ric-8A) is a 60-kDa cytosolic protein that has chaperone and guanine nucleotide exchange (GEF) activity toward heterotrimeric G protein α subunits of the i, q, and 12/13 classes, catalyzing the release of GDP from Gα and subsequent binding of GTP. In the absence of GTP or GTP analogs, and subsequent to GDP release, Gα forms a stable nucleotide-free complex with Ric-8A. In this study, time-resolved fluorescence anisotropy measurements were employed to detect local motions of Gαi1 labeled at selected sites with Alexa 488 (C5) fluorescent dye (Ax) in the GDP, GTPγS (collectively, GXP), and …

Activation Of G Proteins By Gtp And The Mechanism Of Gα-Catalyzed Gtp Hydrolysis, Stephen R. Sprang

Biological Sciences Faculty Publications

This review addresses the regulatory consequences of the binding of GTP to the alpha subunits (Gα) of heterotrimeric G proteins, the reaction mechanism of GTP hydrolysis catalyzed by Gα and the means by which GTPase activating proteins (GAPs) stimulate the GTPase activity of Gα. The high energy of GTP binding is used to restrain and stabilize the conformation of the Gα switch segments, particularly switch II, to afford stable complementary to the surfaces of Gα effectors, while excluding interaction with Gβγ, the regulatory binding partner of GDP-bound Gα. Upon GTP hydrolysis, the energy of these conformational restraints is dissipated and …