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13 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
B21-High Throughput SAXS	Human DDX17 unwinds rift valley fever virus non-coding RNAs Corey R. Nelson , Tyler Mrozowich , Sean M. Park , Simmone D’souza , Amy Henrickson , Justin R. J. Vigar , Hans-joachim Wieden , Raymond J. Owens , Borries Demeler , Trushar R. Patel International Journal Of Molecular Sciences 22 DOI: 10.3390/ijms22010054 Diamond Proposal Number(s): [22113] Open Access Show Abstract ▼ Abstract: Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5′ non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17135–555) and RVFV non-coding RNAs, IGR and 5’ NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17135–555, IGR, and 5’ NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5’ NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17135–555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17135–555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17135–555 interacting with and unwinding RVFV non-coding regions.	Jan 2021
B21-High Throughput SAXS	Molecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replicase expression Ankoor Patel , Emmely E. Treffers , Markus Meier , Trushar R. Patel , Jörg Stetefeld , Eric J. Snijder , Brian L. Mark Journal Of Biological Chemistry 295, 17904-17921 DOI: 10.1074/jbc.RA120.016105 Diamond Proposal Number(s): [22113] Open Access Show Abstract ▼ Abstract: Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV non-structural protein 1β (nsp1β) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1β and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1β and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1β:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1β within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1β and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1β and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.	Dec 2020
B21-High Throughput SAXS	Structural and hydrodynamic characterization of dimeric human oligoadenylate synthetase 2 Amit Koul , Darren Gemmill , Nikhat Lubna , Markus Meier , Natalie Krahn , Evan P. Booy , Jörg Stetefeld , Trushar R. Patel , Sean A. Mckenna Biophysical Journal DOI: 10.1016/j.bpj.2020.04.025 Diamond Proposal Number(s): [16028, 22113] Open Access Show Abstract ▼ Abstract: Oligoadenylate synthetases (OASs) are a family of interferon-inducible enzymes that require double-stranded RNA (dsRNA) as a cofactor. Upon binding dsRNA, OAS undergoes a conformational change and is activated to polymerize ATP into 2′-5′-oligoadenylate chains. The OAS family consists of several isozymes, with unique domain organizations to potentially interact with dsRNA of variable length, providing diversity in viral RNA recognition. In addition, oligomerization of OAS isozymes, potentially OAS1 and OAS2, is hypothesized to be important for 2′-5′-oligoadenylate chain building. In this study, we present the solution conformation of dimeric human OAS2 using an integrated approach involving small-angle x-ray scattering, analytical ultracentrifugation, and dynamic light scattering techniques. We also demonstrate OAS2 dimerization using immunoprecipitation approaches in human cells. Whereas mutation of a key active-site aspartic acid residue prevents OAS2 activity, a C-terminal mutation previously hypothesized to disrupt OAS self-association had only a minor effect on OAS2 activity. Finally, we also present the solution structure of OAS1 monomer and dimer, comparing their hydrodynamic properties with OAS2. In summary, our work presents the first, to our knowledge, dimeric structural models of OAS2 that enhance our understanding of the oligomerization and catalytic function of OAS enzymes.	May 2020
B21-High Throughput SAXS	Solution structure and oligomeric state of the E. coli glycerol facilitator Mary Hernando , George Orris , Jacqueline Perodeau , Shixing Lei , Fraser G. Ferens , Trushar R. Patel , Jörg Stetefeld , Andrew J. Nieuwkoop , Joe D. O'neil Biochimica Et Biophysica Acta (bba) - Biomembranes DOI: 10.1016/j.bbamem.2020.183191 Diamond Proposal Number(s): [16028] Show Abstract ▼ Abstract: Protein dynamics at atomic resolution can provide deep insights into the biological activities of proteins and enzymes but they can also make structure and dynamics studies challenging. Despite their well-known biological and pharmaceutical importance, integral membrane protein structure and dynamics studies lag behind those of water-soluble proteins mainly owing to solubility problems that result upon their removal from the membrane. Escherichia coli glycerol facilitator (GF) is a member of the aquaglyceroporin family that allows for the highly selective passive diffusion of its substrate glycerol across the inner membrane of the bacterium. Previous molecular dynamics simulations and hydrogen-deuterium exchange studies suggested that protein dynamics play an important role in the passage of glycerol through the protein pore. With the aim of studying GF dynamics by solution and solid-state nuclear magnetic resonance (NMR) spectroscopy we optimized the expression of isotope-labelled GF and explored various solubilizing agents including detergents, osmolytes, amphipols, random heteropolymers, lipid nanodiscs, bicelles and other buffer additives to optimize the solubility and polydispersity of the protein. The GF protein is most stable and soluble in lauryl maltose neopentyl glycol (LMNG), where it exists in a tetramer-octamer equilibrium. The solution structures of the GF tetramer and octamer were determined by negative-stain transmission electron microscopy (TEM), size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) and solid-state magic-angle spinning NMR spectroscopy. Although NMR sample preparation still needs optimization for full structure and dynamics studies, negative stain TEM and SEC-SAXS revealed low-resolution structures of the detergent-solubilized tetramer and octamer particles. The non-native octamer appears to form from the association of the cytoplasmic faces of two tetramers, the interaction apparently mediated by their disordered N- and C-termini. This information may be useful in future studies directed at reducing the heterogeneity and self-association of the protein.	Jan 2020
B21-High Throughput SAXS	Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution Doo Nam Kim , Bernhard C. Thiel , Tyler Mrozowich , Scott P. Hennelly , Ivo L. Hofacker , Trushar R. Patel , Karissa Y. Sanbonmatsu Nature Communications 11 DOI: 10.1038/s41467-019-13942-4 Diamond Proposal Number(s): [16028] Open Access Show Abstract ▼ Abstract: Long non-coding RNAs (lncRNAs) constitute a significant fraction of the transcriptome, playing important roles in development and disease. However, our understanding of structure-function relationships for this emerging class of RNAs has been limited to secondary structures. Here, we report the 3-D atomistic structural study of epigenetic lncRNA, Braveheart (Bvht), and its complex with CNBP (Cellular Nucleic acid Binding Protein). Using small angle X-ray scattering (SAXS), we elucidate the ensemble of Bvht RNA conformations in solution, revealing that Bvht lncRNA has a well-defined, albeit flexible 3-D structure that is remodeled upon CNBP binding. Our study suggests that CNBP binding requires multiple domains of Bvht and the RHT/AGIL RNA motif. We show that RHT/AGIL, previously shown to interact with CNBP, contains a highly flexible loop surrounded by more ordered helices. As one of the largest RNA-only 3-D studies, the work lays the foundation for future structural studies of lncRNA-protein complexes.	Jan 2020
B21-High Throughput SAXS	Nanoscale structure determination of Murray Valley encephalitis and Powassan virus non-coding RNAs Tyler Mrozowich , Amy Henrickson , Borries Demeler , Trushar Patel Viruses 12 DOI: 10.3390/v12020190 Diamond Proposal Number(s): [22113] Open Access Show Abstract ▼ Abstract: Viral infections are responsible for numerous deaths worldwide. Flaviviruses, which contain RNA as their genetic material, are one of the most pathogenic families of viruses. There is an increasing amount of evidence suggesting that their 5’ and 3’ non-coding terminal regions are critical for their survival. Information on their structural features is essential to gain detailed insights into their functions and interactions with host proteins. In this study, the 5’ and 3’ terminal regions of Murray Valley encephalitis virus and Powassan virus were examined using biophysical and computational modeling methods. First, we used size exclusion chromatography and analytical ultracentrifuge methods to investigate the purity of in-vitro transcribed RNAs. Next, we employed small-angle X-ray scattering techniques to study solution conformation and low-resolution structures of these RNAs, which suggest that the 3’ terminal regions are highly extended as compared to the 5’ terminal regions for both viruses. Using computational modeling tools, we reconstructed 3-dimensional structures of each RNA fragment and compared them with derived small-angle X-ray scattering low-resolution structures. This approach allowed us to reinforce that the 5’ terminal regions adopt more dynamic structures compared to the mainly double-stranded structures of the 3’ terminal regions.	Jan 2020
B21-High Throughput SAXS	Experimental determination of second virial coefficients by small-angle X-ray scattering: a problem revisited Tyler Mrozowich , Donald J. Winzor , David J. Scott , Trushar R. Patel European Biophysics Journal 346 DOI: 10.1007/s00249-019-01404-0 Diamond Proposal Number(s): [16028] Show Abstract ▼ Abstract: This investigation examines the validity of employing single-solute theory to interpret SAXS measurements on buffered protein solutions—the current practice despite the necessity to regard the buffer components as additional non-scattering solutes rather than as part of the solvent. The present study of bovine serum albumin in phosphate-buffered saline supplemented with 20–100 g/L sucrose as small cosolute has certainly verified the prediction that the experimentally obtained second virial coefficient should contain protein–cosolute contributions. Nevertheless, the second virial coefficient determined for protein solutions supplemented with high cosolute concentrations on the basis of single-solute theory remains a valid means for identifying conditions conducive to protein crystallization, because the return of a slightly negative second virial coefficient based on single-solute theory 𝐴app2 still establishes the existence of slightly associative interactions between protein molecules, irrespective of the molecular source–protein self-interactions and/or protein–cosolute contributions.	Oct 2019
B21-High Throughput SAXS	Asparagine-84, a regulatory allosteric site residue, helps maintain the quaternary structure of Campylobacter jejuni dihydrodipicolinate synthase Mohadeseh Majdi Yazdi , Sagar Saran , Tyler Mrozowich , Cheyanne Lehnert , Trushar R. Patel , David A. R. Sanders , David R. J. Palmer Journal Of Structural Biology DOI: 10.1016/j.jsb.2019.107409 Diamond Proposal Number(s): [16028] Show Abstract ▼ Abstract: Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.	Oct 2019
B21-High Throughput SAXS	Solution structure and hydrodynamics of C. elegans UNC-6: a nematode paralogue of the vertebrate key axon guidance protein Netrin-1 Natalie Krahn , Markus Meier , Raphael Reuten , Manuel Koch , Joerg Stetefeld , Trushar R. Patel Biophysical Journal DOI: 10.1016/j.bpj.2019.04.033 Diamond Proposal Number(s): [16028] Show Abstract ▼ Abstract: UNC-6 (UNCoordinated-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1 it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40 specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution X-ray scattering experiments. Our biophysical analysis of UNC-6 ΔC solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins -1 and -4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix.	May 2019
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Crystal structure of the TreS-Pep2 complex, initiating α-glucan synthesis in the GlgE pathway of mycobacteria Ali A. Kermani , Rana Roy , Chai Gopalasingam , Klaudia I. Kocurek , Trushar R. Patel , Luke J. Alderwick , Gurdyal S. Besra , Klaus Fütterer Journal Of Biological Chemistry DOI: 10.1074/jbc.RA118.004297 Diamond Proposal Number(s): [6388, 8359, 10369] Open Access Show Abstract ▼ Abstract: A growing body of evidence implicates the mycobacterial capsule - the outermost layer of the mycobacterial cell envelope - in modulation of the host immune response and virulence of mycobacteria. Mycobacteria synthesize the dominant capsule component, α(1→4)-linked glucan, via three interconnected, and potentially redundant metabolic pathways. Here, we report the crystal structure of the Mycobacterium smegmatis TreS-Pep2 complex, containing trehalose synthase (TreS) and maltokinase (Pep2), which convert trehalose to maltose-1-phosphate as part of the TreS-Pep2-GlgE pathway. The structure, at 3.6 Å resolution, revealed that a diamond-shaped TreS tetramer forms the core of the complex, and that pairs of Pep2 monomers bind to opposite apices of the tetramer in a 4+4 configuration. However, for the M. smegmatis orthologues, results from isothermal titration calorimetry and analytical ultracentrifugation experiments indicated that the prevalent stoichiometry in solution is 4 TreS + 2 Pep2 protomers. The observed discrepancy between the crystallised complex and the behaviour in the solution state may be explained by the relatively weak affinity of Pep2 for TreS (Kd 3.5 μM at mildly acidic pH) and crystal packing favouring the 4+4 complex. Proximity of the ATP binding site in Pep2 to the complex interface provides a rational basis for rate enhancement of Pep2 upon binding to TreS, but the complex structure appears to rule out substrate channeling between the active sites of TreS and Pep2. Our findings provide a structural model for the trehalose synthase-maltokinase complex in M. smegmatis that offers critical insights into capsule assembly.	Mar 2019

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