Amino Acids, Peptides, and Proteins Commons^™

Association Between Archaeal Prolyl- And Leucyl-Trna Synthetases Enhances TrnaPro Aminoacylation, Mette Praetorius-Ibba, Theresa E. Rogers, Rachel Samson, Zvi Kelman, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetase-containing complexes have been identified in different eukaryotes, and their existence has also been suggested in some Archaea. To investigate interactions involving aminoacyl-tRNA synthetases in Archaea, we undertook a yeast two-hybrid screen for interactions between Methanothermobacter thermautotrophicus proteins using prolyl-tRNA synthetase (ProRS) as the bait. Interacting proteins identified included components of methanogenesis, protein-modifying factors, and leucyl-tRNA synthetase (LeuRS). The association of ProRS with LeuRS was confirmed in vitro by native gel electrophoresis and size exclusion chromatography. Determination of the steady-state kinetics of tRNA^Pro charging showed that the catalytic efficiency (k_cat/K_m) of ProRS increased 5-fold …

Quality Control During Aminoacyl-Trna Synthesis, M. Praetorius-Ibba, S. Ataide, C. Hausmann, J. Levengood, J. Ling, S. Wang, H. Roy, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

The fidelity of translation is determined at two major points: the accuracy of aminoacyl-tRNA selection by the ribosomes and synthesis of cognate amino acid/tRNA pairs by aminoacyl-tRNA synthetases (aaRSs) in the course of the aminoacylation reaction. The most important point in aminoacylation is the accurate recognition of cognate substrates coupled with discrimination of non-cognates. While this is generally accomplished by a single enzyme, we have recently found that discrimination against lysine analogues requires the existence of two unrelated lysyl-tRNA synthetases. For other amino acids, initial recognition is not sufficiently accurate with errors being corrected by an intrinsic editing activity. Recent …

Photoreactive Bicyclic Amino Acids As Substrates For Mutant Escherichia Coli Phenylalanyl-Trna Synthetases, Thomas Bentin, Ramin Hamzavi, Jahan Salomonsson, Hervé Roy, Michael Ibba, Peter E. Nielsen

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Unnatural amino acids carrying reactive groups that can be selectively activated under non-invasive biologically benign conditions are of interest in protein engineering as biological tools for the analysis of protein-protein and protein-nucleic acids interactions. The double ring system phenylalanine analogues benzofuranylalanine and benzotriazolylalanine were synthesized, and their photolability was tested by UV irradiation at 254, 320, and 365 nm. Although both showed photo reactivity, benzofuranylalanine appeared as the most promising compound because this amino acid was activated by UVA (long wavelength) irradiation. These amino acids were also tested for in vitro charging of tRNA^Phe and for protein mutagenesis via …

Divergence In Non-Cognate Amino Acid Recognition Between Class I And Class Ii Lysyl-Trna Synthetases, Jeffrey D. Levengood, Sandro F. Ataide, Hervé Roy, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Lysine insertion during coded protein synthesis requires lysyl-tRNA^Lys, which is synthesized by lysyl-tRNA synthetase (LysRS). Two unrelated forms of LysRS are known: LysRS2, which is found in eukaryotes, most bacteria, and a few archaea, and LysRS1, which is found in most archaea and a few bacteria. To compare amino acid recognition between the two forms of LysRS, the effects of l-lysine analogues on aminoacylation were investigated. Both enzymes showed stereospecificity toward the l-enantiomer of lysine and discriminated against noncognate amino acids with different R-groups (arginine, ornithine). Lysine analogues containing substitutions at other positions were generally most effective as …

Aminoacyl-Trnas: Setting The Limits Of The Genetic Code, Michael Ibba, Dieter Söll

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNAs (aa-tRNAs) are simple molecules with a single purpose—to serve as substrates for translation. They consist of mature tRNAs to which an amino acid has been esterified at the 3′-end. The 20 different types of aa-tRNA are made by the 20 different aminoacyl-tRNA synthetases (aaRSs, of which there are two classes), one for each amino acid of the genetic code (Ibba and Söll 2000). This would be fine if it were not for the fact that such a straightforward textbook scenario is not true in a single known living organism. aa-tRNAs lie at the heart of gene expression; they interpret …

Direct And Indirect Excitation Of Laterodorsal Tegmental Neurons By Hypocretin/Orexin Peptides: Implications For Wakefulness And Narcolepsy, Sophie Burlet, Christopher Tyler, Christopher S. Leonard

NYMC Faculty Publications

Compelling evidence links the recently discovered hypothalamic peptides Hypocretin/Orexin (Hcrt/Orx) to rapid eye movement sleep (REM) control and the sleep disorder narcolepsy, yet how they influence sleep-related systems is not well understood. We investigated the action of Hcrt/Orx on mesopontine cholinergic (MPCh) neurons of the laterodorsal tegmental nucleus (LDT), a target group whose function is altered in canine narcolepsy and appears pivotal for normal REM and wakefulness. Extracellular recordings from mouse brainstem slices revealed that Hcrt/Orx evoked prolonged firing of LDT neurons. Whole-cell recordings revealed that Hcrt/Orx had actions on both presynaptic neurons and at postsynaptic sites. Hcrt/Orx produced an …

Impaired Fast-Spiking, Suppressed Cortical Inhibition, And Increased Susceptibility To Seizures In Mice Lacking Kv3.2 K+ Channel Proteins, David Lau, Eleazar Vega-Saenz De Miera, Diego Contreras, Alan Chow, Richard Paylor, Christopher S. Leonard, Bernardo Rudy

NYMC Faculty Publications

Voltage-gated K(+) channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to -10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one …

Expression And Requirement Of Epithelial Fatty Acid- Binding Protein In Neuronal Axon Growth, Gregory William Allen

Loma Linda University Electronic Theses, Dissertations & Projects

Epithelial fatty acid-binding protein (E-FABP) is expressed in the dorsal root ganglia following sciatic nerve injury and in migrating and differentiating neurons during neuronal development. It was hypothesized that E-FABP expression is required for the robust outgrowth of axons from developing and regenerating neurons. To test this hypothesis, E-FABP expression in both PC12 cells and primary retinal neurons was examined. In PC12 cells, NGF induces E-FABP mRNA and protein during the period of neurite outgrowth, and E-FABP localizes to the perinuclear cytoplasm, nucleus, and growth cone. Furthermore, E-FABP-deficient cell lines exposed to NGF were less differentiated and had shorter neurites …

Regulation Of Connexin43 By Phosphorylation, Maithili M. Shah

Loma Linda University Electronic Theses, Dissertations & Projects

Gap junctions play a crucial role in embryogenesis, especially of the heart where connexin43 is thought to be important for its formation as well as synchronized contraction. Several reports have shown that connexin43 protein is phosphorylated on serine residues in vitro and in vivo.

Studies performed in our laboratory have demonstrated that cell-to-cell communication in cells expressing connexin43 can be controlled rapidly and reversibly by microinjection of active protein kinases or phosphatases that target serine or threonine residues. Phosphorylation by cAMP-dependent protein kinase (PKA) seems to favor channel opening, whereas the effect of protein kinase C (PKC) on channel gating …

Archaeal Aminoacyl-Trna Synthesis: Unique Determinants Of A Universal Genetic Code?, Michael Ibba, A. W. Curnow, J. Bono, P. A. Rosa, C. R. Woese, D. Söll

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

No abstract provided.

The Role Of Gap Junctions In Congenital Diseases Of The Heart, Scott Henry Britz-Cunningham

Loma Linda University Electronic Theses, Dissertations & Projects

Background. Gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. Connexin43, the major protein of gap junctions in the heart, is targeted by several protein kinases that regulate myocardial cell-cell coupling. We hypothesized that mutations altering sites critical to this regulation would lead to functional or developmental abnormalities of the heart.

Methods. Connexin43 DNA from 25 normal subjects and 30 children with a variety of congenital heart diseases was amplified by the polymerase chain reaction and sequenced. Mutant DNA was expressed in cell culture and examined for its effect …

Cellular Responses In Escherichia Coli To Lethal And Sublethal Doses Of Ozone, Indira Ruth Komanapalli

Loma Linda University Electronic Theses, Dissertations & Projects

Ozone is a major component of photochemical smog. High levels of this pollutant, sufficient to affect human health are found in many urban areas worldwide. Though limited studies in humans are supported by extensive findings from animal experiments, a difficulty in interpreting the results of these experiments has lead to an ambiguity on the biochemical mechanism of ozone toxicity. To elucidate the mechanism by which ozone causes cell damage and eventual cell death we conducted a comprehensive study using Escherichia coli K-12 as a model.

Studies on the comparative inactivation of bacteriophage lambda (λ), Escherichia coli, and Candida albicans …

Laminin Potentiates Differentiation Of Pcc4uva Embryonal Carcinoma Into Neurons, T. M. Sweeney, Roy C. Ogle, C. D. Little

Medical Diagnostics & Translational Sciences Faculty Publications

The embryonal carcinoma PCC4uva differentiates into neurons in response to treatment with retinoic acid and dbcAMP. We used this in vitro model system to study the effects of laminin on early neural differentiation. Laminin substrata markedly potentiate neural differentiation of retinoic acid and dbcAMP-treated cultures. Only laminin induced more rapid neural cell body clustering, neurite growth and neurite fasciculation as compared to type IV collagen, type I collagen, and fibronectin substrata. Exogenous laminin substrata promoted greater cell attachment, cellular spreading and growth to confluence than type IV collagen, type I collagen, fibronectin and glass substrata. Laminin-induced effects were inhibited by …

An Analysis Of Protein-Ligand Interactions For Enzyme Ii-Mannitol From Escherichia Coli, Brent Lee Wood

Loma Linda University Electronic Theses, Dissertations & Projects

Enzyme II^mtl of Escherichia coli was purified from mtlA-overexpressing E. coli strain LJ1112 extraction of isolated membranes with deoxycholate, followed by hexyl agarose and ω-amino hexyl agarose chromatography in Lubrol PX. A 10-fold increase in total pure protein and 2.5-fold increase in specific activity over previously reported procedures was obtained. Tryptic fragments of Enzyme II^mtl were also purified and identified as the membrane and cytoplasmic domains by protein sequencing and SDS-PAGE. The fluorescence spectrum of Enzyme II^mtl suggested that two populations of tryptophan residues existed with emission maxima at 322 and 344 nm, corresponding to buried …