Biochemistry Commons^™

Advances In Structural And Functional Analysis Of Membrane Proteins By Electron Crystallography, Goragot Wisedchaisri, Steve Reichow, Tamir Gonen

Chemistry Faculty Publications and Presentations

Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane …

Exploring The Roles Of Nucleobase Desolvation And Shape Complementarity During The Misreplication Of O6-Methylguanine, Delia Chavarria, Andrea Ramos Serrano, Ichiro Hirao, Anthony J. Berdis

Chemistry Faculty Publications

O6-methylguanine is a miscoding DNA lesion arising from the alkylation of guanine. This report uses the bacteriophage T4 DNA polymerase as a model to probe the roles hydrogen-bonding interactions, shape/size, and nucleobase desolvation during the replication of this miscoding lesion. This was accomplished by using transient kinetic techniques to monitor the kinetic parameters for incorporating and extending natural and non-natural nucleotides. In general, the efficiency of nucleotide incorporation does not depend on the hydrogen-bonding potential of the incoming nucleotide. Instead, nucleobase hydrophobicity and shape complementarity appear to be the preeminent factors controlling nucleotide incorporation. In addition, shape complementarity plays a …

Rna Oxidation Adducts 8-Ohg And 8-Oha Change With Aβ42 Levels In Late-Stage Alzheimer's Disease, Adam M. Weidner, Melissa A. Bradley, Tina L. Beckett, Dana M. Niedowicz, Amy L.S. Dowling, Sergey V. Matveev, Harry Levine, Mark A. Lovell, M. Paul Murphy

Sanders-Brown Center on Aging Faculty Publications

While research supports amyloid-β (Aβ) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aβ. Using data from four disease-affected brain …

Beta-Lysine Discrimination By Lysyl-Trna Synthetase, Marla S. Gilreath, Hervé Roy, Tammy J. Bullwinkle, Assaf Katz, Michael Ibba, William Wiley Navarre

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P is modified with (R)‐β‐lysine by the lysyl‐tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate α‐ and β‐lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site guided by the PoxA structure. A233S LysRS behaved as wild type with α‐lysine, while the G469A and A233S/G469A variants decreased stable α‐lysyl‐adenylate formation. A233S LysRS recognized β‐lysine better than wildtype, suggesting a role for this residue in discriminating α‐ and β‐amino acids. Both enantiomers of β‐lysine were substrates for tRNA aminoacylation by LysRS, which, together with …

Follicular Dendritic Cells, Resting Cd4+ T Cells And Human Immunodeficiency Virus Expression, Changna Wang

Theses and Dissertations

Many events associated with Human Immunodeficiency Virus (HIV) infection/replication occur in and around the germinal centers (GCs) of secondary lymphoid tissues where follicular dendritic cells (FDCs) reside, suggesting that this microenvironment may contribute unique signaling that is important to viral progression. My research focused on characterizing signaling, both positive and negative, contributed by FDCs that affects HIV infection and replication. Specifically, I determined if FDC signals could induce the expression of latent HIV in T cells and if so, to characterize the signaling pathways involved. Moreover, I also examined the ability of FDCs to produce inhibitory signals that might block …

Stomatin-Like Protein 2 Binds Cardiolipin And Regulates Mitochondrial Biogenesis And Function., Darah Christie, Caitlin D Lemke, Isaac M Elias, Luan A Chau, Mark G Kirchhof, Bo Li, Eric H Ball, Stanley D Dunn, Grant M Hatch, Joaquín Madrenas

Biochemistry Publications

Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched …

Single Molecule Analysis Of The Arabidopsis Fra1 Kinesin Shows That It Is A Functional Motor Protein With Unusually High Processivity, Chuanmei Zhu, Ram Dixit

Biology Faculty Research

The Arabidopsis FRA1 kinesin contributes to the organization of cellulose microfibrils through an unknown mechanism. The cortical localization of this kinesin during interphase raises the possibility that it transports cell wall-related cargoes along cortical microtubules that either directly or indirectly influence cellulose microfibril patterning. To determine whether FRA1 is an authentic motor protein, we combined bulk biochemical assays and single molecule fluorescence imaging to analyze the motor properties of recombinant, GFP-tagged FRA1 containing the motor and coiled-coil domains (designated as FRA1(707)–GFP). We found that FRA1(707)–GFP binds to microtubules in an ATP-dependent manner and that its ATPase activity is dramatically stimulated …

The Worlds Of Splicing And Chromatin Collide, J. Adam Hall, Philippe T. Georgel

Biological Sciences Faculty Research

Both transcription and splicing take place in a nuclear environment which, at face value, may seem refractory to the efficiency afforded by the coupling of both processes. This environment, chromatin, was once viewed as only a passive packaging system for genetic material, with very little contribution to the variety of nuclear activities occurring within and around it. However, overwhelming evidence now points to the chromatin environment as being highly dynamic, and an active player in nuclear activities.

Mutant Alcohol Dehydrogenase Leads To Improved Ethanol Tolerance In Clostridium Thermocellum, Steven D. Brown, Adam M. Guss, Tatiana V. Karpinets, Jerry M. Parks

Dartmouth Scholarship

Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. …

The Trna Synthetase Paralog Poxa Modifies Elongation Factor-P With (R)-Β-Lysine, Hervé Roy, S. Betty Zou, Tammy J. Bullwinkle, Benjamin S. Wolfe, Marla S. Gilreath, Craig J. Forsyth, William Wiley Navarre, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with α-lysine at low efficiency. Cell-free extracts containing non–α-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of β-lysine, a substrate also predicted by genomic analyses. EF-P was efficiently functionally modified with (R)-β-lysine but not (S)-β-lysine or genetically encoded α-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases.

The Nucleotide Exchange Factor Ric-8a Is A Chaperone For The Conformationally Dynamic Nucleotide-Free State Of G Alpha I1, Celestine J. Thomas, Klara Briknarova, Jonathan K. Hilmer, Navid Movahed, Brian Bothner, John P. Sumida, Gregory G. Tall, Stephen R. Sprang

Chemistry and Biochemistry Faculty Publications

Heterotrimeric G protein alpha subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of G alpha, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class G alpha subunits. Ric-8A binds to G alpha.GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free G alpha i1 is stable, but dissociates upon binding of GTP to G alpha i1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release …

Aryl Acetate Phase Transfer Catalysis: Method And Computation Studies, Meisha A. Binkley

Theses and Dissertations

Brief explanation and history of cinchona based Phase Transfer Catalysis (PTC). Studied aryl acetates in PTC, encompassing napthoyl, 6-methoxy napthoyl, phenyl and protected 4-hydroxy phenyl acetates. Investigated means of controlling the selectivity of the PTC reaction by changing the electrophile size, the ether side group size or by addition of inorganic salts. Found that either small or aromatic electophiles increased enantioselectivity more than aliphatic electrophiles, and that increasing the size of ether protecting group also increased selectivity. Positive effects of salt addition included either decreasing reaction time or increasing enantiomeric excess. Applied findings towards the synthesis of S-equol. Computational experiments …

The Role Of A Transcription Factor In Regulating Rice Response To Drought Stress, Diana Ha, Liyuan A. Zhang, Jeffery Shen

Undergraduate Research Opportunities Program (UROP)

The current water shortage is a major concern in regard to our global climate change crisis. A decrease in the availability of water will have direct effects on the development of plants. Some crops, such as Oryza sativa, or commonly known as rice, requires an abundant amount of water for adequate growth. With the water shortage crisis, it will become extremely difficult to harvest such crops to meet the world’s food demand. However, many plants have evolved mechanisms for overcoming and tolerating stresses such as drought. My research focuses on studying the proteins involved with these mechanisms. The WRKY superfamily …

Ubiquitylation Of Proteins In The Frozen Wood Frog, Alexa Khan, Michael Ulrich, Kenneth Storey, Frank Van Breukelen

Undergraduate Research Opportunities Program (UROP)

Wood frogs (Rana sylvatica) are able to withstand freezing. Respiratory and cardiac activity ceases when frozen. Homeostatic functions like protein synthesis and degradation presumably must also be compromised. We investigated the fate of ubiquitin-dependent proteolysis in the freeze-thaw cycle and how that might gives clues to wood frog survival. We performed western blots for ubiquitin conjugates

Novel Thermophilic Cellulolytic Isolates Belonging To The Phylum Chloroflexi, Maryknoll Palisoc, Jessica K. Guy, Joseph P. Peacock, Duy C. Trinh, Jeremy A. Dodsworth, Brian P. Hedlund

Undergraduate Research Opportunities Program (UROP)

Current biofuel technologies utilize valuable foodstuffs, such as corn kernels and cane sugar, as sources of easily metabolized sugars. Microbes are used to ferment these sugars into bioethanol, a first-generation biofuel. However, in order to avoid diverting foodstuffs from the food supply, the development of second-generation biofuels technology is necessary. Second-generation biofuels are produced by converting structurally complex lignocellulosic biomass, such as agricultural and municipal wastes, to fermentable sugars or directly to biofuels.

The major technological hurdle limiting the mass production of second-generation biofuels is the difficulty in efficiently converting structurally complex lignocellulosic materials to fermentable sugars or directly to …

Inactivation Of Spo0a Gene Increases Stationary Phase Mutagenesis In Bacillus Subtilis, Denisse Reyes, Amanda Prisbrey, Holly Martin, Eduardo Robleto

Undergraduate Research Opportunities Program (UROP)

Stationary phase mutagenesis occurs when a population of cells acquires mutations conferring escape from nongrowing or stress conditions. This type of mutations is observed in nutritionally starved cells. Because the mutations occur after the onset of stress and in cells that are in non-replicative conditions, elucidating the underlying mechanisms contributes novel views to the process of evolution and apply to the formation of cancer in human cells and antibiotic resistance in microbial pathogens. Studies have shown that in Bacillus subtilis, the Mfd protein which is a transcription repair coupling factor is necessary for this phenomenon to occur. Here, we investigate …

Synthesis Of Chimeric Receptors Essential For Spore Germination, Christopher Yip, Christian Ross, Eduardo Robleto, Ernesto Abel-Santos

Undergraduate Research Opportunities Program (UROP)

Various species of bacteria have been reported to form an endospore, a metabolically dormant cell, during times of nutrient deficiencies and extreme stress. These said structures are outstandingly resistant to harsh chemicals, extreme temperatures, and can revert back to a metabolically active cell, through a process known as germination, when the necessary conditions are met. The rigid membrane of the endospore contains various germination (Ger) receptors which sense the external environment for necessary metabolites and germinants. Ger receptors are encoded by tricistronic operons that produce three distinct membrane proteins, the A, B, and C subunits. Although the function of the …

Investigating The Origin Of Coprolites From Three Great Basin Caves, Chelsey Vandrisse, Duane P. Moser, David Rhode

Undergraduate Research Opportunities Program (UROP)

The study of coprolites (mummified feces) is a relatively new endeavor, which enables investigations of the health and diet of ancient people and provides some of the oldest evidence to date for the human habitation in North America (2). In this project, 18 coprolites were examined from archeological digs at three Great Basin caves: the Bonneville Estates Rockshelter (UT), Hidden Cave (NV), and Top of the Terrace Rockshelter (UT). The main objectives were: 1) to verify human origin through the presence of mitochondrial DNA (mtDNA) and 2) assuming human origin, characterize intestinal microflora of Native Americans prior to European contact. …

The Role Of An Abc Transporter As A Steroid Antagonist In Drosophila, Gregory King, Andrew Andres

Undergraduate Research Opportunities Program (UROP)

Drosophila melanogaster are holometabolous insects that have several distinct life stages including larvae and a winged adult. The larval stage is mainly a time of feeding and growth, while the adult stage is optimized for sexual reproduction and dissemination. The larval stage can itself be divided into three time periods, or instars: 1st (L1), 2nd (L2), and 3rd (L3) (Figure 1). Larval growth – both between instars and beyond – depends on specific signaling pathways controlled by a cholesterol derived steroid, 20-hydroxyecdysone (20E). Although 20E is a systemic developmental signal, little is known about the molecular details of how different …

Cell Migration Dynamics After Alteration Of Cell-Cell Contacts In Fibrosarcoma And Glioblastoma Cell Lines, Hassan S. Rizvi, Ronald K. Gary

Undergraduate Research Opportunities Program (UROP)

Cell migration is a vital component of metastasis. In this study, our intent was to study cell migration by alteration of the Wnt/GSK-3 Pathway. Since BeSO4 is a known GSK-3 kinase inhibitor, we hypothesized that this agent would cause cell migration to decrease as a result of β-catenin stabilization. Two human cell lines, HT-1080 (fibrosarcoma) and A172 (glioblastoma), were used to observe migration levels in the presence and absence of BeSO4. Our results show that cell migration is diminished for cells that were pre-treated with BeSO4, in comparison to the untreated (control) cells.