A Bioengineered Memory Storage Device Using Bacteriorhodopsin and Graphene

Bacteriorhodopsin (BR) is a photoactive protein, which has been studied as a memory storage device owing to its photochemical and thermal stability. BR photocycle comprises of two distinct stable binary states, bR (0) and Q (1) based on the wavelength of the applied radiation. However, such devices have a limited success due to low quantum yield of the Q state1. Many studies have used genetic and chemical modification as optimization strategies to increase the yield of the Q state compromising the overall photochemical stability of the BR1. Here we come up with a unique way of stabilizing the conformations of BR and thereby the BR and Q states of the protein through its adsorption onto graphene. We have used all-atom molecular dynamics (MD) simulations utilizing NAMD (Nanoscale Molecular Dynamics) and the CHARMM (Chemistry at HARvard Macromolecular Mechanics) force field to understand the interactive events at the interface of BR and a single layer graphene sheet. Based on the stable RMSD (Root Mean Square Deviation) and interactive energies such as Van-der-Waals and electrostatics, we propose that the adsorption of BR onto graphene can stabilize the photochemical behavior of BR. Furthermore, the switching between Cis and Trans conformations of the retinal based on the angular change of the dihedral demonstrates that such an adsorption is beneficial to preserve the binary states

The Extraction of Hops for Inhibition of Bacterial Growth

A poster discussing the extraction of hops for the unique bacterial properties that hops contain

Cloning of Putative Cobalamin Reductases of Thermosipho melanesiensis

Cobalamin, commonly known as Vitamin B12, is a vitamin that plays an essential role in keeping human nerve and blood cells healthy. It is also a cofactor for the synthesis of enzymes involved in citric acid cycle metabolism, DNA synthesis, and gene regulation. Only certain Bacteria and Archaea possess the required enzymes for Cobalamin biosynthesis. Eukaryotes cannot synthesize Cobalamin de novo, but obtain it in one of two ways: via gut microorganisms that synthesize Cobalamin, or via food sources. Humans use the latter method by consuming animal products. Our aim is to uncover the unknown gene identities of three reductase enzymes in Thermosipho melanesiensis that are suspected to be required for de novo Cobalamin synthesis. Previous research on protein comparison to Salmonella enterica has targeted three DNA sequences as possible reductase genes

Investigating the origins of B12 biosynthesis in the most ancient roots of the tree of life

Vitamin B12, also known as B12 or cobalamin, is a vital nutrient required across all branches of life, but the ability to synthesize this complex molecule de novo is limited to only a few archaea and bacteria. De novo synthesis begins with glutamate and utilizes over 30 gene products to produce an active cobalamin [1]. Previous studies suggest that of the available bacterial genomes, only half utilizing cobalamin can synthesize it [2]. The other half either take up complete cobalamin from the environment via an ABC transporter, or scavenge incomplete corrinoids (partial cobalamin molecules) as precursors to synthesize active cobalamin [3, 4]. The evolutionary histories and identities of multiple genes within these B12 pathways are unknown, leaving gaping holes in our understanding of the only source of biosynthesis of the vitamin so essential to human survival. Genes of particular interest to this investigator are those responsible for producing reductases that act upon the central cobalt atom of B12. Three reductases with unknown gene identities are located within the B12 biosynthetic pathway and it is the aim of this research to identify those genes responsible

UB Knightlines Spring 2015

The UB Knightlines newsletter for the Spring of 2015. This issue contains articles discussing Seed Money Grants, alumnus Milan Bull's work to save threatened bird species in Connecticut, alumna Amelia Amon's work combining solar power with design, UB professor Steve Jackowicz's work with pharmaceutical plants, professor Xingguo Xiong's work to use MEMS technology to track miniscule pollutants, UB's alumni at Welcome Back Weekend, UB alumnus Ger Duany's experience starring in a film with Reese Witherspoon, filmmaker Monica Lange speaking at "Necessary Voices", UB security officer Ralph Gonzalez and his bike patrol, UB students attending Heifer International's Global Gateway program, UB student Bao Lei winning the Chunhui Cup contest, BackCountry Jazz Billie Holiday Celebration Concerts sponsored by UB, UB named best online education in the nation by U.S. News, faculty news, books published by alums and faculty, a focus on alumnus Rindy Higgins, alumni news, the UB Taekwondo winning 12 medals at the U.S. Open Taekwondo Championship, the story of UB Basketball player Willie Williams III, the UB alumni Basketball game, the continued success of UB women's volleyball, UB Martial Arts major Edward Jeong to seventh at the World Taekwondo Poomsae Championships in Mexico, and other campus and sports news

GlxA is a new structural member of the radical copper oxidase family and is required for glycan deposition at hyphal tips and morphogenesis of <i>Streptomyces lividans</i>

Streptomyces lividans displays a distinct dependence on copper to fully initiate morphological development. Evidence has accumulated to implicate the participation of an extracytoplasmic cuproenzyme in morphogenesis. In the present study, we show that GlxA fulfils all criteria to be that cuproenzyme. GlxA is membrane associated and has an active site consisting of a mononuclear copper and a cross-linked Y-C cofactor. The domain organization of the tertiary structure defines GlxA as a new structural member of the mono-copper oxidase family, with copper co-ordination geometry similar to, but spectroscopically distinct from fungal galactose oxidase (Gox). EPR spectroscopy reveals that the oxidation of cupric GlxA generates a protein radical residing on the Y-C cross-link. A variety of canonical Gox substrates (including D-galactose) were tested but none were readily turned over by GlxA. A glxA null-mutant leads to loss of glycan accumulation at hyphal tips and consequently a drastically changed morphology both on solid substrates and in liquid-grown environments, a scenario similarly observed in the absence of the neighbouring glycan synthase CslA (cellulase synthase-like protein). In addition the glxA mutant has lost the stimulation of development by copper, supporting a model whereby the enzymatic action of GlxA on the glycan is required for development and morphology. From a biotechnology perspective, the open mycelium morphology observed with the glxA mutant in submerged culture has implications for use as an enzyme production host.</jats:p

Active site maturation and activity of the copper-radical oxidase GlxA is governed by a tryptophan residue

GlxA from Streptomyces lividans is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct member of the AA5 family in which the fungal galactose 6-oxidase (Gox) is the best-characterized. GlxA is a key cuproenzyme in the copper-dependent morphological development of S. lividans with a function that is linked to the processing of an extracytoplasmic glycan. The catalytic site in GlxA and Gox contain two distinct one-electron acceptors comprising the copper ion and a 3'-(S-cysteinyl) tyrosine. The latter is formed post-translationally through a covalent bond between a cysteine and a copper coordinating tyrosine ligand and houses a radical. In GlxA and Gox a second coordination sphere tryptophan residue (Trp288 in GlxA) is present, but the orientation of the indole ring differs between the two enzymes creating a marked difference in the ?-? stacking interaction of the benzyl ring with the 3'-(S-cysteinyl) tyrosine. Differences in the spectroscopic and enzymatic activity have been reported between GlxA and Gox with the indole orientation suggested as a reason. Here we report a series of in vivo and in vitro studies using the W288F and W288A variants of GlxA to assess the role of Trp288 on the morphology, maturation, spectroscopic and enzymatic properties. Our findings point towards a salient role for Trp288 in the kinetics of copper loading and maturation of GlxA, with its presence essential for stabilising the metalloradical site required for coupling catalytic activity and morphological development

Genes for the Major Structural Components of Thermotogales Species’ Togas Revealed by Proteomic and Evolutionary Analyses of OmpA and OmpB Homologs

The unifying structural characteristic of members of the bacterial order Thermotogales is their toga, an unusual cell envelope that includes a loose-fitting sheath around each cell. Only two toga-associated structural proteins have been purified and characterized in Thermotoga maritima: the anchor protein OmpA1 (or Ompα) and the porin OmpB (or Ompβ). The gene encoding OmpA1 (ompA1) was cloned and sequenced and later assigned to TM0477 in the genome sequence, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. We identified six porin candidates in the genome sequence of T. maritima. Of these candidates, only one, encoded by TM0476, has all the characteristics reported for OmpB and characteristics expected of a porin including predominant β-sheet structure, a carboxy terminus porin anchoring motif, and a porin-specific amino acid composition. We highly enriched a toga fraction of cells for OmpB by sucrose gradient centrifugation and hydroxyapatite chromatography and analyzed it by LC/MS/MS. We found that the only porin candidate that it contained was the TM0476 product. This cell fraction also had β-sheet character as determined by circular dichroism, consistent with its enrichment for OmpB. We conclude that TM0476 encodes OmpB. A phylogenetic analysis of OmpB found orthologs encoded in syntenic locations in the genomes of all but two Thermotogales species. Those without orthologs have putative isofunctional genes in their place. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one or two OmpA homologs. T. maritima has two OmpA homologs, encoded by ompA1 (TM0477) and ompA2 (TM1729), both of which were found in the toga protein-enriched cell extracts. These annotations of the genes encoding toga structural proteins will guide future examinations of the structure and function of this unusual lineage-defining cell sheath

Genes for the Major Structural Components of Thermotogales Species’ Togas Revealed by Proteomic and Evolutionary Analyses of OmpA and OmpB Homologs

The unifying structural characteristic of members of the bacterial order Thermotogales is their toga, an unusual cell envelope that includes a loose-fitting sheath around each cell. Only two toga-associated structural proteins have been purified and characterized in Thermotoga maritima: the anchor protein OmpA1 (or Ompα) and the porin OmpB (or Ompβ). The gene encoding OmpA1 (ompA1) was cloned and sequenced and later assigned to TM0477 in the genome sequence, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. We identified six porin candidates in the genome sequence of T. maritima. Of these candidates, only one, encoded by TM0476, has all the characteristics reported for OmpB and characteristics expected of a porin including predominant β-sheet structure, a carboxy terminus porin anchoring motif, and a porin-specific amino acid composition. We highly enriched a toga fraction of cells for OmpB by sucrose gradient centrifugation and hydroxyapatite chromatography and analyzed it by LC/MS/MS. We found that the only porin candidate that it contained was the TM0476 product. This cell fraction also had β-sheet character as determined by circular dichroism, consistent with its enrichment for OmpB. We conclude that TM0476 encodes OmpB. A phylogenetic analysis of OmpB found orthologs encoded in syntenic locations in the genomes of all but two Thermotogales species. Those without orthologs have putative isofunctional genes in their place. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one or two OmpA homologs. T. maritima has two OmpA homologs, encoded by ompA1 (TM0477) and ompA2 (TM1729), both of which were found in the toga protein-enriched cell extracts. These annotations of the genes encoding toga structural proteins will guide future examinations of the structure and function of this unusual lineage-defining cell sheath

Characteristics of <i>T. maritima</i> OmpB orthologs and putative syntenic analogs in Thermotogales species.

<p>Characteristics are defined in the text. <b>Legend:</b> +, protein is predicted to possesses the attribute; –, protein is predicted to not possess the attribute.</p><p>ORFs are from TM, T. maritima; TRQ2, Thermotoga species strain RQ2; Tpet, Thermotoga petrophila; Tnap, Thermotoga naphthophila; CTN, Thermotoga neapolitana; Tmel, Thermotoga melanesiensis; Fnod, Fervidobacterium nodosum; Tlet, Thermotoga lettingae; THA, Thermosipho africanus; Kole, Kosmotoga olearia; Pmob, Petrotoga mobilis; and Theba, Thermotogales bacterium mesG1.Ag.4.2 (Mesotoga prima). ORFs above the line are homologs of TM0476, those below the line are possible analogs of TM0476.</p>‡<p>Values of the percent β strand content were calculated utilizing the multisequence alignment programs STRAP and SCRATCH, respectively.</p