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  • Undergraduate Poster Abstracts
  • Biochemistry/Biophysics

    FRI-G9 DISTINCT BIOPHYSICAL PROPERTIES IMPARTED BY H2A VARIANTS ON CHROMATIN COMPACTION

    • Israel Saucedo Gonzalez ;
    • Ahmad Nabhan ;
    • Francisco Guerrero ;
    • Diana Chu ;
    • Geeta Narlikar ;

    FRI-G9

    DISTINCT BIOPHYSICAL PROPERTIES IMPARTED BY H2A VARIANTS ON CHROMATIN COMPACTION

    Israel Saucedo Gonzalez1, Ahmad Nabhan1, Francisco Guerrero1, Diana Chu1, Geeta Narlikar2.

    1San Francisco State University, San Francisco, CA, 2University of California, San Francisco, San Francisco, CA.

    An organism’s ability to regulate gene expression and chromatin compaction is important to maintain proper function, genomic integrity, and development. Organisms accomplish this by incorporating small, basic proteins called histones into their genome, allowing the formation of transcriptionally active and silenced regions. Additionally, histone variants that differ in protein sequence from canonical histones regulate various chromatin processes. For example HTZ-1, an evolutionarily conserved H2A variant, localizes to the promoter region of developmental genes where it may poise genes for expression. Similarly, HTAS-1, a sperm-specific H2A variant, localizes to condensing chromatin during sperm development. How H2A histone variants function in distinct chromatin processes remains elusive. We hypothesize that variations in amino acid sequence at specific domains confer structural differences and are responsible for their function in distinct processes. We show HTZ-1 and HTAS-1 confer stability to the nucleosome when compared to canonical H2A, and that the C-terminal domain of HTZ-1 is responsible for the increased nucleosomal stability. To understand the impact of H2A variants incorporation in chromatin compaction, we are assembling arrays of nucleosomes in vitro. This will reveal the biophysical properties imparted by HTZ-1 and HTAS-1 on the compaction of larger chromatin structures. The results from this study will elucidate how structural features of H2A variants correlate with their function in active and repressive gene expression. This will provide mechanistic insight into the compaction of the genome, a process crucial for proper gene expression and survival of the organism.

    THU-G8 MAPPING THE SEQUENCE FITNESS LANDSCAPE OF CLASS C GPCRS FOR DETERMING CRITICAL REGIONS FOR ACTIVE AGONIST BINDING

    • Jeremiah Heredia ;
    • Erik Procko ;

    THU-G8

    MAPPING THE SEQUENCE FITNESS LANDSCAPE OF CLASS C GPCRS FOR DETERMING CRITICAL REGIONS FOR ACTIVE AGONIST BINDING

    Jeremiah Heredia, Erik Procko.

    University of Illinois at Urbana Champaign, Urbana, IL.

    G‐protein coupled receptors (GPCRs) are the largest class of membrane proteins and are major drug targets, yet most GPCR structures remain unsolved. One unusual subgroup is the class C GPCR proteins, which includes members for the recognition of neurotransmitters and sweet and savory tasting substances. Class-C GPCRs are dimeric and characterized by an extracellular hydrophilic ligand binding domain (LBD), an extracellular cysteine‐rich domain, and a C‐terminal 7 transmembrane domain. Receptor activation requires ligand interactions with the LBD, but it is unknown how ligand binding stabilizes a conformational change many angstroms away in the membrane‐spanning region. Our lab is researching how small molecule ligands are recognized and how ligand binding induces downstream signaling in class-C GPCRs. We have developed a method for comprehensively mapping the sequence‐fitness landscape of a GPCR. All possible single amino acid substitutions are combined in a library that is sorted for expression and high affinity ligand interactions. Deep sequencing pre‐ and post‐sort allows calculation of the phenotypic fitness of all sequence variants in a single experiment, and residues that are conserved for function are revealed. Our initial efforts have focused on simple GPCRs that recognize chemokines, and we hypothesize that planned sequence‐fitness landscape mapping of multidomain class-C GPCRs will determine regions critical for adopting their active, agonist‐bound conformation. The development of these saturation mutagenesis libraries could also be applied for the characterization of other receptor families and for engineering transmembrane proteins with new or improved properties.

    FRI-G10 REMODELING AN EXCLUSIVE TYPE III PROTEIN ARGININE METHYLTRANSFERASE

    • Tamar Caceres ;
    • Joan Hevel ;

    FRI-G10

    REMODELING AN EXCLUSIVE TYPE III PROTEIN ARGININE METHYLTRANSFERASE

    Tamar Caceres, Joan Hevel.

    Utah State University, Logan, UT.

    Proteins that are arginine methylated are involved in a number of different cellular processes including transcriptional regulation, RNA metabolism, and DNA damage repair. They are also important in human diseases, especially in cardiovascular disease and cancer. In cells, arginine methylation is carried out by the family of protein arginine methyltransferases (PRMTs). The PRMTs catalyze 3 types of arginine methylation products: monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine. Each type of methylated arginine can have a distinct biological function; therefore, there is great interest in understanding how product specificity is achieved within the PRMT family. Despite the great amount of information available regarding PRMTs, the biochemistry of these enzymes is not completely understood. Protein arginine methyltransferase 7 is unique within the family as it is the only exclusive type III methyltransferase known to date, meaning that it can only monomethylate substrates. So far, the structural and mechanistic basis for the inability of PRMT7 to dimethylate peptide substrates is unknown. Our hypothesis is that steric hindrances in the active site prevent the PRMT7 enzymes from adding a second methyl group, thus, restricting TbPRMT7 to monomethylation. To test this hypothesis, we initiated mutational studies on Trypanosomal PRMT7, followed by methylation and product analysis assays. As a result, we have been able to remodel a type III methyltransferase into a mixed type I, II, and III, that is, an enzyme that can now perform dimethylation. By remodeling the PRMT7 enzyme, we have a better understanding for how product specificity is regulated by PRMTs.

    FRI-G8 INVESTIGATION OF A POSSIBLE ENZYMATIC SWITCH IN XYLAN BACKBONE SYNTHESIS

    • John Tran ;
    • Jacob Jensen ;
    • Curtis Wilkerson ;

    FRI-G8

    INVESTIGATION OF A POSSIBLE ENZYMATIC SWITCH IN XYLAN BACKBONE SYNTHESIS

    John Tran, Jacob Jensen, Curtis Wilkerson.

    Michigan State University, East Lansing, MI.

    Xylans are an important group of plant cell wall hemicelluloses that are abundant in all higher plants. Xylans are key components of the cell walls that enable plants to grow upright by providing the mechanical strength and are important in allowing vessel elements to withstand the negative pressure created by transpiration. Although much is known about the structure of xylan, which is characterized by a linear backbone composed of (1→4)-linked β-D-xylosyl residues with side-chain modifications, less is known about synthesis and elongation of the xylan backbone. Recent studies by us and others have demonstrated xylan:xylosyltransferase activity in vitro by heterologous expressed protein of IRX10 and IRX10-like. Between the 2, IRX10 showed a lower level of activity than IRX10-like, yet the 2 proteins are 86% identical at the amino acid sequence level. We find this difference in activity intriguing and propose that it likely involves a regulatory mechanism controlling the enzymatic activity of IRX10. Revealing such a mechanism would allow for a better understanding of xylan formation and perhaps explain the role of a number of other proteins which genetic evidence suggest are involved in xylan backbone synthesis. As a first step in our investigations, we wish to identify regions of IRX10 or IRX10-like responsible for the different levels of enzymatic activity by conducting a series of domain-swap experiments between IRX10 and IRX10-like and expressing them in S. cerevisiae.

    THU-G10 REDOX CONTROL OF PROTEIN ARGININE METHYLTRANSFERASE 1 ACTIVITY

    • Yalemi Morales ;
    • Joan Hevel ;

    THU-G10

    REDOX CONTROL OF PROTEIN ARGININE METHYLTRANSFERASE 1 ACTIVITY

    Yalemi Morales, Joan Hevel.

    Utah State University, Logan, UT.

    Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase (DDAH). Reports have shown that DDAH activity is down regulated and PRMT1 protein expression is up regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H2O2 concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis, but rather demonstrates that enzymatic activity can be regulated in a redox-sensitive manner.

    THU-G11 FUNCTIONAL CHARACTERIZATION OF MYOSIN MUTATIONS LINKED TO EARLY-ONSET CARDIOMYOPATHY

    • Carlos Vera ;
    • Hector Rodriguez ;
    • Leslie Leinwand ;

    THU-G11

    FUNCTIONAL CHARACTERIZATION OF MYOSIN MUTATIONS LINKED TO EARLY-ONSET CARDIOMYOPATHY

    Carlos Vera1, Hector Rodriguez2, Leslie Leinwand.

    1University of Colorado Boulder, Boulder, CO, 2Myokardia, Inc, South San Francisco, CA, 3BioFrontiers Institute, University of Colorado Boulder, Boulder, CO.

    Hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young athletes and has an incidence of 1 in 500 people. Dilated cardiomyopathy (DCM) has a prevalence of 1 in 2,000 people. Both diseases can be inherited, with HCM being autosomal dominant, and DCM having different genotypic profiles. There are over 400 mutations in cardiac myosin (MYH7) that have been linked to both diseases. Also noteworthy, early-onset HCM and DCM patients typically have a worse prognosis than adult-onset patients. The only intervention for these pediatric patients is a heart transplant. Here we describe the kinetic analysis of several cardiac myosin mutations that have been identified as unique to early-onset patients. One set of mutations has been identified in pediatric HCM and the other set is DCM. Steady-state characterization of ATP hydrolysis for both mutant sets using an NADH coupled ATPase assay is reported. Additionally, transient kinetic analysis for several mutants was conducted illustrating differences in the cross-bridge kinetics between HCM and DCM mutants, namely ATP-induced dissociation, ADP release, and the binding constants of actin and the different nucleotide states. These data suggest that the motor activity is different among mutants and may provide insight into the mechanistic defects that can lead to both diseases.

    THU-G9 BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF THE FREQUENCY-INTERACTING RNA HELICASE FROM NEUROSPORA CRASSA

    • Jacqueline Johnson ;
    • Sean Johnson ;

    THU-G9

    BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF THE FREQUENCY-INTERACTING RNA HELICASE FROM NEUROSPORA CRASSA

    Jacqueline Johnson, Sean Johnson.

    Utah State University, Logan, UT.

    The frequency-interacting RNA helicase (FRH) is a 124 kDa Ski2-like helicase from the fungal organism Neurospora crassa. Sequence alignments indicate that FRH is a homolog of S. cerevisiae Mtr4 (55% sequence identity; 73% sequence similarity), another Ski2-like helicase that plays a central role in RNA processing and degradation pathways. The high similarity between these 2 proteins suggests they share common structural and functional characteristics. In contrast to Mtr4, however, FRH plays an important role in regulating circadian rhythms in N. crassa, a role that does not appear to require helicase activity. To better characterize FRH and to understand the relationship between FRH and Mtr4, we have initiated biochemical and structural studies of FRH. Here, we present the crystal structure of FRH at 3.5 Å resolution. FRH retains the 5-domain architecture observed in Mtr4. Surprisingly, FRH forms a dimer in the crystal, in contrast to the monomeric Mtr4 structure. This dimeric form is accommodated by significant structural rearrangements in the arch domain. While the biological significance of a dimeric structure is unclear, biochemical crosslinking experiments indicate that FRH can form a dimer in solution. Ongoing efforts are focused on further characterizing the functional relevance of this unique structure.