No description available.

<div>The family Flaviviridae includes important genera such as flavivirus and hepacivirus which comprise significant human pathogens that affect hundreds of millions annually. The understanding of these viruses, the viral life cycle, and pathogenicity is vital when it comes to developing therapeutics. Flavivirus virions undergo major conformational rearrangements during the life cycle, including the assembly and maturation steps. In order to create a reagent to investigate these processes, luminescent reporter viruses have been constructed. Luminescent reporter tags have yet to be incorporated into the structural proteins of dengue virus (DENV) without significantly affecting replication or infectivity and successful tagging would allow for targeted studies examining access to specific structural epitopes. Engineering tags in DENV structural proteins is particularly difficult because most reporter tags involve large insertions which may create steric hindrance and inhibit proper protein folding. However, the reporter system described here, developed by Promega, is much smaller than a full-size luciferase protein. It involves an eleven amino acid subunit (HiBiT) tagged to a viral protein that creates measurable luminescence when incubated with the larger subunit (LgBiT). Using the structure of the virion as a guide, the HiBiT reporter tag was incorporated into the structural region of the DENV genome including sites in capsid (C) as well as the glycoproteins membrane (M) and envelope (E). Resulting recombinant viruses were characterized and tag sites within the C protein membrane anchor as well as the transmembrane domain of M protein were found to tolerate HiBiT insertion and produce infectious particles. The recombinant virus possessing HiBiT in C protein was found to be stable over three rounds of serial passaging while virus containing the M protein tag site was found to be unstable. HiBiT activity of the capsid tagged virus was also found to directly correlate with purified infectious particles, suggesting the capsid membrane anchor may remain associated with the virus even after polyprotein processing. Additionally, insert composition was found to be a key determinant for the production of infectious virus. The lessons learned from engineering HiBiT in the DENV system were then applied to hepatitis C virus (HCV). </div><div>The highly lipophilic and pleiomorphic nature of HCV has made structural studies particularly difficult. However, by constructing multi-tagged reporter viruses containing both HiBiT and various purification tags, researchers will save time and resources in preparation for structural studies which are vital for vaccine development. In this study, HiBiT was incorporated into sites within HCV previously shown to tolerate tags of various sizes. Different insert compositions were engineered within the genome and the construct containing both FLAG and HiBiT tags within the N-terminus of E2 yielded highly infectious and quantifiable, luminescent virus. The recombinant HCV containing FLAG and HiBiT displayed similar peak titer as compared to WT while also demonstrating HiBiT activity. Furthermore, the FLAG peptide was found to be partially surface exposed and capable of being used for virus purification purposes. The multi-tagged reporter virus characterized in this study provides a robust platform for quantification and purification of HCV, two facets of research that are critical for the determination of viral structure via cryo-EM and other imaging techniques. The findings from both the DENV and HCV studies provide a robust foundation for future tagging of viruses within the family Flaviviridae and offer insight on the structural proteins that compose the virion.</div>

<p>Flaviviruses are enveloped, positive-strand RNA viruses that are spread by mosquitoes and ticks and can cause serious disease in humans. Flavivirus virions undergo extensive structural changes during their life cycle, including during maturation and fusion. Flaviviruses are initially assembled at the endoplasmic reticulum in a non-infectious, immature state, and then traffic to the trans-Golgi network, where a pH drop triggers a structural rearrangement of glycoproteins prM and E on the virus surface from 60 trimers to 90 dimers. A host protease, furin, then cleaves prM which makes the transition irreversible. Upon exiting the host cell, pr disassociates from the virus and the infectious, mature virus is able to enter a new cell. <br></p><p><br></p> <p> </p> <p>In Chapter 1, an overview of flaviviruses is presented, including a brief history of their discovery and interaction with humans, followed by what is known about their life cycle and the maturation process. The structure of a mature flavivirus is then described, including the symmetrical arrangement of glycoproteins on the virion surface, the lipid membrane, and the nucleocapsid core, followed by an introduction of the structural proteins that assemble into the virion. The structure of the immature flavivirus is then described. The chapter concludes with a description of the dynamics and heterogeneity observed for flaviviruses.</p><p><br></p> <p> </p> <p>The conformational rearrangements that occur during flavivirus maturation remain unclear. The structures of immature and mature flaviviruses determined with cryo-electron microscopy (cryo-EM) demonstrated that flaviviruses are icosahedral particles with 180 copies of glycoproteins on their surface. Icosahedral viruses typically have a quasi-equivalent arrangement of glycoproteins, but flaviviruses lack quasi-equivalence and instead the three subunits within an asymmetric unit occupy different chemical environments. Although the subunits are the same proteins, the unique environment of each subunit can be exploited for tracking subunits during conformational rearrangements. For example, the unique labeling of a subunit can be used to identify it in the immature and mature virion.</p><p><br></p> <p> </p> <p>In Chapter 2, the maturation process was studied by developing tools to differentially label protein subunits and trap potential intermediates of maturation. The tools included heavy-atom compounds and antibody Fabs, which were used to probe Kunjin virus (KUNV), an Australian subtype of West Nile virus (WNV). One heavy-atom compound, potassium tetranitroplatinate(II), was found to derivatize immature KUNV, likely at sites on both E and prM. Higher-resolution studies will be required to determine if the compound differentially labeled the three subunits. The other tool developed was the E16 Fab. E16 Fab, originally isolated from a mouse immunized with WNV E and found to bind to two out of three subunits on mature WNV, was used to differentially label subunits in immature KUNV. Based on poor epitope accessibility on immature KUNV, E16 Fab was hypothesized to trap an intermediate state of maturation. In the cryo-EM reconstruction of E16 Fab bound to immature KUNV it was found that the virion had localized distorted density and apparent non-uniform binding of the E16 Fab. Based on this result it was proposed that flaviviruses had imperfect icosahedral symmetry. <br></p><p><br></p> <p> </p> <p>The structural asymmetry of immature and mature flaviviruses was investigated in Chapter 3. Icosahedral symmetry has always been imposed during cryo-EM reconstructions of flaviviruses, as it led to stable convergence of orientations. When reconstructions of immature KUNV and ZIKV were performed without imposing symmetry, the reconstructions showed that the flaviviruses had an eccentric nucleocapsid core, which was positioned closer to the membrane at one pole. At the opposite pole, the glycoprotein and inner leaflet densities were weak and distorted. Furthermore, there were protrusions from the core that contacted the transmembrane helices of the glycoproteins. In the asymmetric reconstruction of mature KUNV, the core was positioned concentric with the glycoprotein shell, in contrast to the immature virion, indicating that maturation alters the interactions between the core and the glycoproteins. The asymmetric reconstructions suggested that there is variable contact between the core and glycoproteins during assembly, which may be due to membrane curvature restrictions in the budding process. </p> <p> </p> <p><br></p><p>In Chapter 4, extracellular vesicles (EVs) that were released during dengue virus (DENV) infection were characterized by mass spectrometry. EVs may play a significant role in flavivirus infection, as they have been shown to transport both viral proteins and infectious RNA. EVs likely represent alternative modes of virus transmission and aid in immune evasion. However, previous studies on EVs are controversial because EVs are potential contaminated during assays by co-purifying virions and other particulates. The identification of EV biomarkers would greatly reduce contamination because biomarkers would enable isolation of pure EVs by affinity purification. Therefore, a strategy was developed to isolate EVs and profile them with proteomics. The four proteins cystatin-A, filamin B, fibrinogen beta chain, and endothelin converting enzyme 1 were found to be statistically enriched in the DENV sample and represent potential EV biomarkers. </p> <p> </p>

<p>Cryogenic transmission electron microscopy (cryoEM) has become an increasingly common tool for determining structures of proteins and protein complex at near atomic resolution. We seek to determine the structure of p97 by cryoEM using an affinity capture approach that employs a family of novel synthetic lipids bearing water soluble PEG units and known high affinity inhibitor molecules at the distal end of the polymer. A library of inhibitor modified affinity lipopolymers of 5000 KD PEG molecular weights were synthesized. The inhibitor modified lipid coated grids were used to capture p97. The reconstruction of p97 revealed the structure at dimeric state at 3.64 Å and monomeric state at 4.33 Å. A PEG unit composed of 20000 KD molecular weight based polyrotaxane containing NTA ligand as affinity tag has been synthesized, used to concentrate 6x-his tagged p97 on TEM which also enabled to see all 3D orientation of the target particles and an initial model of 10.64 Å resolution of p97 structure was resolved. </p>

No description available.

Understanding the structure-function relationship of proteins such as G protein-coupled receptors (GPCRs) requires detailed information on their three-dimensional (3D) structure and dynamics. Recent progress in the structural elucidation of receptor-G protein or -arrestin complexes yields valuable starting points to studying their function and dynamics. I investigated the dynamic process of β2 adrenergic receptor-G protein recognition and binding using molecular dynamics (MD) simulations. MD simulation is a well-established technique to monitor the time-resolved motions of proteins and is used to investigate the dynamics of GPCRs. In order to start MD simulations, the structures that are often incompletely resolved have to be further completed. Finally, the results from computational analysis have to be subjected to experimental validation. To promote broad applicability of that workflow, I automated critical steps such as modeling of missing segments or interactive analysis and visualization of the results of MD simulations. Web-tools allow researchers besides different methodological expertise to apply unfamiliar techniques on their biological systems of interest without the need to download software or databases. To facilitate usage, I included visualization allowing intuitive understanding and analysis. We developed the NGL molecular web-viewer, which accesses hardware-accelerated graphics through WebGL. This and other recent developments, reviewed by Mwalongo and others, opened up new possibilities for web molecular graphics and render- ing techniques, superseding plugin- and Java-based viewers and making them comparable with desktop tools, such as PyMOL, VMD, or UCSF Chimera. Here, I implemented and applied tools for interactive modeling of missing segments into single-particle cryo-electron microscopy (cryo-EM) density maps, for interactive analysis of structure-functional dynamics, and more general to promote interdisciplinary research. To allow non-expert users access to bioinformatics methods and biophysical techniques, all tools were generated as web-services allowing interactive analysis and visualization.:Introduction 1 GPCRs as a prime example in structural and pharmaceutical biology 2 Modeling missing segments into cryo-EM density maps 3 Research goal I: Web service for modeling of missing segments into cryo-EM density maps 4 From static to dynamics to investigate GPCR function 5 The complexity of sharing MD simulations 6 Research goal II: Interactive visual sharing of MD simulations Manuscripts 1 FragFit: a web-application for interactive modeling of protein segments into cryo-EM density maps 2 MDsrv: viewing and sharing molecular dynamics simulations on the web. 3 Bringing Molecular Dynamics Simulation Data into View Summary 1 Modeling of missing fragments into cryo-EM density maps 2 InteractiveanalysisofMDsimulationsontheweb . . . . . . References Appendix A Additional Manuscripts 1 SL2: an interactive webtool for modeling of missing segments in proteins 2 GPCR-SSFE 2.0-a fragment-based molecular modeling web tool for Class A G-protein coupled receptors. 3 A fragment based method for modeling of protein segments into cryo-EM density maps 4 Structural Insights into the Process of GPCR-G Protein Complex Formation 5 Sharing Data from Molecular Simulations Appendix B Darstellung des eigenen Beitrags Selbständigkeitserklärung Curriculumvitae & Publikationen Danksagung

<p><i>Enterovirus</i> (EV), a genus within the <i>Picornaviridae </i>family, contains icosahedral positive-stranded RNA viruses linked to different human and mammalian diseases with a variety of symptoms ranging from the common cold to central nervous system infection. An important member within this genus is EV-D68. Unlike many enteroviruses that use the gastrointestinal tract as the transmission and propagation route, EV-D68 infects the respiratory tract and causes respiratory illness, especially in children. Severe infections of EV-D68 also lead to acute flaccid myelitis (AFM), a polio-like neurological disease. Especially in recent years, EV-D68 has been on a global upswing. However, no antiviral interventions against EV-D68 infection have been developed to date. Antibodies neutralizing EV-D68 have significant vaccine and therapeutic potentials. Here, the structures of the immune complex between EV-D68 and the Fab molecules of EV-D68 human monoclonal antibodies have been reconstructed using cryo-electron microscopy (cryo-EM). These structures show two Fab binding loci on the virion surface as well as the essential amino acids involved in binding. In addition to antibodies, a drug candidate against EV-D68 has been investigated in this work as an antiviral strategy. It is likely that this drug blocks viral entry through binding in the hydrophobic pocket underneath the viral protein 1, the largest structural protein of EV-D68. Furthermore, the morphogenesis of EV-D94, another causative virus of polio-like disease, which is closely related to EV-D68 with 85% sequence identity, has been investigated using cryo-EM. Compared to EV-D68, the shape of the canyon and the loops containing the immunogenic recognition sites are different in EV-D94. The structures of each of the three stages of EV-D94 particles (the full native virion, the uncoating intermediate, and the empty virion) were identified and delineate the viral uncoating process. These findings reveal useful knowledge and new insights to develop treatments against human EVs. </p><p></p>

No description available.

G protein signaling depends on the ability of the individual subunits of the G protein heterotrimer to assemble into a functional complex. Formation of the G protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings the Gβ and Gγ subunits together. This system includes the cytosolic chaperonin containing TCP-1 (CCT) and a co-chaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1-Gβ-CCT complexes, were isolated and their structures determined by cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. These structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ β-propeller, disrupting interactions with CCT and releasing a PhLP1-Gβ dimer for assembly with Gγ. We also investigated the role of CCT and PhLP1 in folding and assembling mTOR complexes, which regulate cell growth through phosphorylation. We found that the β-propeller protein mLST8 and one of its binding partners called raptor, which is a large protein in which one domain forms a β-propeller, both bind to CCT. PhLP1 forms a ternary complex with mLST8 and CCT and may play a co-chaperone role. Depletion of PhLP1 or CCT reduces assembly of mTOR complexes in the cell. Collectively, this report reveals diversity in the contributions of CCT to the formation of protein complexes in signaling pathways and presents a molecular mechanism of Gβ folding by CCT and PhLP1.

<p dir="ltr">This thesis explores the intricate mechanisms governing paracellular permeability, a process vital for maintaining homeostasis and barrier integrity in epithelial and endothelial tissues. Central to this exploration are tight junctions (TJs), multi-protein complexes that regulate substance movement between cells. The thesis begins with a comprehensive literature review synthesizing current research on how bacterial and viral proteins can enhance paracellular permeability or modulate TJs, highlighting their significance in both scientific and medical fields.</p><p dir="ltr">A focal point of the thesis is the detailed study of the <i>Listeria</i> adhesion protein (LAP), an epithelial TJ modulator (TJM). LAP’s interaction with the epithelial receptor, Hsp60, facilitates the translocation of <i>Listeria monocytogenes </i>across the epithelial barrier, presenting a novel approach for drug delivery. Addressing the limitations of current TJMs, such as poor bioavailability and toxic side effects, the thesis provides an in-depth analysis of LAP as an alternative biologics’ delivery vehicle. Utilizing a multidisciplinary research approach, the study reveals the cryo-EM structure of LAP at 2.9 Å resolution, elucidating its multimeric formation and interaction with Hsp60 through electrostatic forces, as supported by computational modeling.</p><p dir="ltr">The functional efficacy of LAP is further established through experiments demonstrating its capability to transport drug analogs across epithelial cell monolayers comparably to established TJMs. Moreover, <i>in vivo</i> studies using a mouse oral gavage model show that LAP significantly enhances the blood levels of peptide drugs without triggering inflammatory responses or harming tight junction architecture. This thesis ultimately positions the LAP-Hsp60 complex as a promising TJM candidate, offering an innovative means for enhancing oral drug delivery and maintaining epithelial barrier integrity.</p>