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Instruments / Technical Area	Publication Details	Published
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
B21-High Throughput SAXS	A cholesterol analogue induces an oligomeric reorganization of VDAC Fraser G. Ferens , Trushar R. Patel , George Orriss , Deborah A. Court , Jörg Stetefeld Biophysical Journal DOI: 10.1016/j.bpj.2019.01.031 Diamond Proposal Number(s): [16028] Show Abstract ▼ Abstract: The oligomeric organization of the voltage-dependent anion-selective channel (VDAC) and its interactions with hexokinase play integral roles in mitochondrially mediated apoptotic signaling. Various small to large assemblies of VDAC are observed in mitochondrial outer membranes but they do not predominate in detergent solubilized VDAC samples. In this study, a cholesterol analogue, cholesteryl-hemisuccinate (CHS), was shown induce the formation of detergent soluble VDAC multimers. The various oligomeric states of VDAC induced by the addition of CHS were deciphered through an integrated biophysics approach using microscale thermophoresis, analytical ultracentrifugation and size exclusion chromatography-small angle X-ray scattering. Furthermore, CHS stabilizes the interaction between VDAC and hexokinase (Kd of 27 ± 6 μM), confirming the biological relevance of oligomers generated. Thus, sterols such as cholesterol in higher eukaryotes or ergosterol in fungi may regulate VDAC oligomeric state and may provide a potential target for the modulation of apoptotic signaling by effecting VDAC-VDAC and VDAC-hexokinase interactions. In addition, the integrated biophysical approach described provides a powerful platform for the study of membrane protein complexes in solution.	Feb 2019
B21-High Throughput SAXS	Structure and hydrodynamics of a DNA G-quadruplex with a cytosine bulge Markus Meier , Aniel Moya-torres , Natalie J. Krahn , Matthew D. Mcdougall , George L. Orriss , Ewan K. S. Mcrae , Evan P. Booy , Kevin Mceleney , Trushar R. Patel , Sean A. Mckenna , Jörg Stetefeld Nucleic Acids Research 34 DOI: 10.1093/nar/gky307 Diamond Proposal Number(s): [16028] Open Access Show Abstract ▼ Abstract: The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5′-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.	May 2018
B23-Circular Dichroism	Intrinsic disorder in the partitioning protein KorB persists after cooperative complex formation with operator DNA and KorA Eva Hyde , Phillip Callow , Karthik V. Rajasekar , Peter Timmins , Trushar R. Patel , Giuliano Siligardi , Rohanah Hussain , Scott White , Christopher M. Thomas , David J. Scott Biochemical Journal DOI: 10.1042/BCJ20170281 Diamond Proposal Number(s): [14894, 16440] Show Abstract ▼ Abstract: The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism, and small angle neutron scattering, we have studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered, with a mobile C-terminal domain and no changes in secondary structure but increases in the radius of gyration on complex formation. Comparison of wild type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.	Jul 2017

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