Biochemistry, Biophysics, and Structural Biology Commons^™

Atpase Regulation In The Maltose Transporter, Alister D. Gould

Electronic Thesis and Dissertation Repository

This thesis investigates the mechanism of activity-coupling in the maltose transporter of Escherichia coli (MalFGK₂); the way ATP hydrolysis is prevented in the absence of maltose, and then enabled to drive maltose transport. Like other ATP binding cassette importers, MalFGK₂ requires substrate to be presented by a peripheral substrate-binding protein, in this case the maltose binding protein (MBP). MBP predominantly adopts an ‘open’ resting state, but undergoes a rotation of its two domains to a ‘closed’ state after maltose binding. In the closed state MBP is able to activate MalFGK₂ to stimulate ATP hydrolysis and maltose …

Structure And Dynamics Of The Membrane Protein Bacteriorhodopsin Studied By Mass Spectrometry, Yan Pan

Electronic Thesis and Dissertation Repository

Membrane proteins continue to represent a major challenge for most analytical techniques. Using bacteriorhodopsin (BR) as model system, this work aims to develop mass spectrometry (MS)-based approaches for exploring the structure, dynamics and folding of membrane proteins.

As the first step, BR in its native lipid environment was exposed to hydroxyl radicals, which were produced by laser photolysis of hydrogen peroxide. It was found that the resulting methionine (Met) labeling pattern was consistent with the known BR structure. This finding demonstrates that laser-induced oxidative Met labeling can provide structural information on membrane proteins. In subsequent experiments, the effects of different …

Structural Characterization Of Hip2 Enzyme Interactions In Ubiquitination, Benjamin W. Cook

Electronic Thesis and Dissertation Repository

The ubiquitin proteolysis pathway utilizes three enzymes, an E1 activating enzyme, an E2 conjugating enzyme and an E3 ligating enzyme, to respectively activate, transfer and ligate ubiquitin (Ub) onto a substrate protein. The creation of a K48-linked poly-Ub chain on a substrate will target this protein to be degraded by the 26S proteosome. E2 conjugating enzymes are central proteins in this pathway and interact with the E1 and E3 enzymes to perform Ub transfer. The mechanism by which Ub molecules are interconnected remains poorly understood. The E2 enzymes HIP2 and Ubc1 have been shown to create poly-Ub chains in the …

Structure-Function Analysis Of Enzymes Of The Polyisoprenyl-Phosphate Hexose-1-Phosphate Transferase Family, Kinnari B. Patel

Electronic Thesis and Dissertation Repository

Enzymes of the polyisoprenyl-phosphate hexose-1-phosphate transferase (PHPT) family are integral membrane proteins that initiate the synthesis of glycans by catalyzing the transfer of a hexose-1-phosphate sugar from UDP-hexose to the lipid carrier undecaprenyl phosphate (Und-P). These glycans such as O antigen and exopolysaccharide (EPS) provide bacteria with protection and adaptation to the environment and host immune factors. The role of PHPT proteins in initiation and the absence of any eukaryotic homologues make them an attractive target for novel antimicrobials; however study of these proteins is difficult due to the presence of multiple transmembrane helices. A requirement of the C-terminal domain …

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 …

Mitochondrial Metabolic Suppression And Reactive Oxygen Species Production During Hypometabolism In Mammals, Jason Cl Brown

Electronic Thesis and Dissertation Repository

During hibernation, daily torpor, and fasting, mammals reduce metabolic rate (MR) up to 99%, 95%, and 30%, respectively, compared to resting levels. Mitochondrial metabolic suppression likely contributes to this MR reduction, and the first objective of this study was to determine the relative contributions of active, regulated inhibition and passive thermal effects as body temperature (T_b) falls, to mitochondrial metabolic suppression, and to examine the mechanisms involved using top-down elasticity analysis and novel statistical approach. The second objective of this study was to determine how mitochondrial metabolic suppression affects mitochondrial reactive oxygen species (ROS) production, a topic which …

Regulation Of G Protein Signaling By Goloco Motif Containing Proteins, Peishen Zhao

Electronic Thesis and Dissertation Repository

Signal transduction via heterotrimeric G proteins in response to transmembrane G protein-coupled receptors plays a central aspect in how cells integrate extracellular stimuli and produce biological responses. In addition to receptor-mediated activation of heterotrimeric G proteins, during the last few decades, accessory proteins have been found to regulate G protein activity via different mechanisms. Several proteins have been identified that contain multiple G protein regulatory domains. Using various molecular and biochemical approaches, we have characterized the effects of two such proteins, G18 and RGS14, on G protein activity. Both proteins contain a second G protein binding domain in addition to …

Dissecting The Molecular Role Of Distinct Binding Interfaces On The Retinoblastoma Tumor Suppressor In Growth Control And Tumorigenesis., Matthew J. Cecchini

Electronic Thesis and Dissertation Repository

The retinoblastoma tumor suppressor protein (pRB) functions to maintain proliferative control and act as a barrier to tumorigenesis. pRB is capable of regulating E2F transcription factors to mediate control of proliferation through transcriptional regulation of S-phase target gene expression. In addition, pRB can stabilize the CDK inhibitor p27 through an interaction with two ubiquitin ligase complexes. Further, pRB is capable of forming a unique interaction with E2F1 termed the ‘specific’ interaction that is capable of blocking E2F1 induced apoptosis. These functions of pRB are mediated by distinct binding interfaces and their contributions to the overall functionality of pRB are not …

Transactivation By Human Adenovirus Early Region 1a-Conserved Region Three, Jailal Ng Ablack

Electronic Thesis and Dissertation Repository

One of the critical functions of human adenovirus (hAd) early region 1A (E1A) protein is to activate transcription of the early viral genes. The largest isoform of E1A contains a unique region termed conserved region 3 (CR3), which includes a Cysteine-4 (C4) zinc finger domain. This region activates viral gene expression by interacting with and recruiting cellular transcription machinery to the regulatory regions of early viral genes. Although this process has been studied at length with hAd type 5 E1A, far less is known about how the E1A proteins from other hAd types activate transcription. There are dramatic differences in …

Two Rotary Motors In F-Atp Synthase Are Elastically Coupled By A Flexible Rotor And A Stiff Stator Stalk., André Wächter, Yumin Bi, Stanley D Dunn, Brian D Cain, Hendrik Sielaff, Frank Wintermann, Siegfried Engelbrecht, Wolfgang Junge

Biochemistry Publications

ATP is synthesized by ATP synthase (F(O)F(1)-ATPase). Its rotary electromotor (F(O)) translocates protons (in some organisms sodium cations) and generates torque to drive the rotary chemical generator (F(1)). Elastic power transmission between F(O) and F(1) is essential for smoothing the cooperation of these stepping motors, thereby increasing their kinetic efficiency. A particularly compliant elastic domain is located on the central rotor (c(10-15)/ε/γ), right between the two sites of torque generation and consumption. The hinge on the active lever on subunit β adds further compliance. It is under contention whether or not the peripheral stalk (and the "stator" as a whole) …

The Hiv-1 Tat Protein And Adverse Drug Reactions: A Model System Utilizing Jurkat T Cells And Sulphamethoxazole-Hydroxylamine, Kaothara Adeyanju

Electronic Thesis and Dissertation Repository

In 2009 approximately 2.6 million people became infected with the Human Immunodeficiency Virus (HIV). In addition to the estimated 33.3 million people currently living with the virus, this makes HIV/ AIDS an epidemic of unprecedented scale in modern times. Treatment of HIV infection requires antiretroviral agents as well as a number of other drugs such as antimicrobials. Hypersensitivity adverse drug reactions (ADRs) to a variety of drugs are common in HIV-infected individuals, but the antimicrobial Sulphamethoxazole remains a major culprit. Hypersensitivity ADRs cause significant morbidity, with the skin and liver most commonly affected and are among the top causes of …

Decreased Stability And Increased Formation Of Soluble Aggregates By Immature Superoxide Dismutase Do Not Account For Disease Severity In Als., Kenrick A Vassall, Helen R Stubbs, Heather A Primmer, Ming Sze Tong, Sarah M Sullivan, Ryan Sobering, Saipraveen Srinivasan, Lee-Ann K Briere, Stanley D Dunn, Wilfredo Colón, Elizabeth M Meiering

Biochemistry Publications

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic …