Patel, Trushar

Permanent URI for this collection

https://opus.uleth.ca/handle/10133/5244

Browse

Browsing Patel, Trushar by Subject "Analytical ultracentrifugation"

Now showing 1 - 6 of 6
  • Thumbnail Image
    Item
    Crystal structure of the TreS:Pep2 complex, initiating α-glucan synthesis in the GlgE pathway of mycobacteria
    (American Society for Biochemistry and Molecular Biology, 2019) Kermani, Ali A.; Roy, Rana; Gopalasingam, Chai; Kocurek, Klaudia I.; Patel, Trushar R.; Alderwick, Luke J.; Besra, Gurdyal S.; Fütterer, Klaus
    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 converts 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 crystallized complex and the behavior 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 favoring 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.
  • Thumbnail Image
    Item
    Investigating RNA-RNA interactions through computational and biophysical analysis
    (Oxford University Press, 2023) Mrozowich, Tyler; Park, Sean M.; Waldl, Maria; Henrickson, Amy; Tersteeg, Scott; Nelson, Corey R.; De Klerk, Anneke; Demeler, Borries; Hofacker, Ivo L.; Wolfinger, MIchael T.; Patel, Trushar R.
    Numerous viruses utilize essential long-range RNA–RNA genome interactions, specifically flaviviruses. Using Japanese encephalitis virus (JEV) as a model system, we computationally predicted and then biophysically validated and characterized its long-range RNA–RNA genomic interaction. Using multiple RNA computation assessment programs, we determine the primary RNA–RNA interacting site among JEV isolates and numerous related viruses. Following in vitro transcription of RNA, we provide, for the first time, characterization of an RNA–RNA interaction using size-exclusion chromatography coupled with multi-angle light scattering and analytical ultracentrifugation. Next, we demonstrate that the 5′ and 3′ terminal regions of JEV interact with nM affinity using microscale thermophoresis, and this affinity is significantly reduced when the conserved cyclization sequence is not present. Furthermore, we perform computational kinetic analyses validating the cyclization sequence as the primary driver of this RNA–RNA interaction. Finally, we examined the 3D structure of the interaction using small-angle X-ray scattering, revealing a flexible yet stable interaction. This pathway can be adapted and utilized to study various viral and human long-non-coding RNA–RNA interactions and determine their binding affinities, a critical pharmacological property of designing potential therapeutics.
  • Thumbnail Image
    Item
    Nanoscale structure determination of Murray Valley encephalitis and Powassan virus non-coding RNAs
    (MDPI, 2020) Mrozowich, Tyler; Henrickson, Amy; Demeler, Borries; Patel, Trushar R.
    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.
  • Thumbnail Image
    Item
    Proceedings of the 25th Analytical Ultracentrifugation workshops and symposium
    (Springer, 2023) Demeler, Borries; Gilbert, Robert; Patel, Trushar R.
    The 25th International Analytical Ultracentrifugation (AUC) Workshops and Symposium (AUC2022) took place at the University of Lethbridge in Lethbridge, Canada, in July 2022. In total, 104 attendees (Attendance Profile: 104 attendees, 69 in-person, 35 remote. Brazil 1, Canada 24, China 1, Czech Republic 2, Finland 1, France 3, Germany 22, India 3, Italy 1, Japan 4, Spain 1, Switzerland 3, Taiwan 1, United Kingdom 5, United States 32) participated in the event and presented the latest advances in the field. While the primary focus of the conference was to showcase the applications of AUC in chemical, life sciences, and nanoparticle disciplines, several presentations also integrated complementary methods, such as isothermal titration calorimetry, microscale thermophoresis, light scattering (static and dynamic), small-angle X-ray scattering, X-ray crystallography, and cryo-electron microscopy. Additionally, the delegates gained valuable hands-on experience from 20 workshops covering a broad range of applications, experimental designs and systems, and the latest software innovations in solution biophysics. The AUC2022 special volume highlights the sustained innovation, utility and relevance of AUC and related solution biophysical methods across various disciplines, including biochemistry, structural biology, synthetic polymer chemistry, carbohydrate chemistry, protein and nucleic acid characterization, nano-science, and macromolecular interactions.
  • Thumbnail Image
    Item
    Structural studies of RNA-protein complexes: a hybrid approach involving hydrodynamics, scattering, and computational methods
    (Elsevier, 2017) Patel, Trushar R.; Chojnowski, Grzegorz; Astha; Koul, Amit; McKenna, Sean A.; Bujnicki, Janusz M.
    The diverse functional cellular roles played by ribonucleic acids (RNA) have emphasized the need to develop rapid and accurate methodologies to elucidate the relationship between the structure and function of RNA. Structural biology tools such as X-ray crystallography and Nuclear Magnetic Resonance are highly useful methods to obtain atomic-level resolution models of macromolecules. However, both methods have sample, time, and technical limitations that prevent their application to a number of macromolecules of interest. An emerging alternative to high-resolution structural techniques is to employ a hybrid approach that combines low-resolution shape information about macromolecules and their complexes from experimental hydrodynamic (e.g. analytical ultracentrifugation) and solution scattering measurements (e.g., solution X-ray or neutron scattering), with computational modeling to obtain atomic-level models. While promising, scattering methods rely on aggregation-free, monodispersed preparations and therefore the careful development of a quality control pipeline is fundamental to an unbiased and reliable structural determination. This review article describes hydrodynamic techniques that are highly valuable for homogeneity studies, scattering techniques useful to study the low-resolution shape, and strategies for computational modeling to obtain high-resolution 3D structural models of RNAs, proteins, and RNA-protein complexes.
  • Thumbnail Image
    Item
    Structural studies of RNA-protein complexes: a hybrid approach involving hydrodynamics, scattering, and computational methods
    (Elsevier, 2017) Patel, Trushar R.; Chojnowski, Grzegorz; Astha; Koul, Amit; McKenna, Sean A.; Bujnicki, Janusz M.
    The diverse functional cellular roles played by ribonucleic acids (RNA) have emphasized the need to develop rapid and accurate methodologies to elucidate the relationship between the structure and function of RNA. Structural biology tools such as X-ray crystallography and Nuclear Magnetic Resonance are highly useful methods to obtain atomic-level resolution models of macromolecules. However, both methods have sample, time, and technical limitations that prevent their application to a number of macromolecules of interest. An emerging alternative to high-resolution structural techniques is to employ a hybrid approach that combines low-resolution shape information about macromolecules and their complexes from experimental hydrodynamic (e.g. analytical ultracentrifugation) and solution scattering measurements (e.g., solution X-ray or neutron scattering), with computational modeling to obtain atomic-level models. While promising, scattering methods rely on aggregation-free, monodispersed preparations and therefore the careful development of a quality control pipeline is fundamental to an unbiased and reliable structural determination. This review article describes hydrodynamic techniques that are highly valuable for homogeneity studies, scattering techniques useful to study the low-resolution shape, and strategies for computational modeling to obtain high-resolution 3D structural models of RNAs, proteins, and RNA-protein complexes.