| 1. |
- Drago, Victoria N., et al.
(author)
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Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction
- 2022
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In: npj Microgravity. - : Springer Science and Business Media LLC. - 2373-8065. ; 8:1
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Journal article (peer-reviewed)abstract
- Biologically active vitamin B6-derivative pyridoxal 5′-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. Such comprehension requires atomic-level structural studies of PLP-dependent enzymes. Neutron diffraction affords the ability to directly observe hydrogen positions and therefore assign protonation states to the PLP cofactor and key active site residues. The low fluxes of neutron beamlines require large crystals (≥0.5 mm3). Tryptophan synthase (TS), a Fold Type II PLP-dependent enzyme, crystallizes in unit gravity with inclusions and high mosaicity, resulting in poor diffraction. Microgravity offers the opportunity to grow large, well-ordered crystals by reducing gravity-driven convection currents that impede crystal growth. We developed the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device, to grow neutron diffraction-quality crystals of perdeuterated TS in microgravity. Here, we present the design of the TCB and its implementation on Center for Advancement of Science in Space (CASIS) supported International Space Station (ISS) Missions Protein Crystal Growth (PCG)-8 and PCG-15. The TCB demonstrated the ability to improve X-ray diffraction and mosaicity on PCG-8. In comparison to ground control crystals of the same size, microgravity-grown crystals from PCG-15 produced higher quality neutron diffraction data. Neutron diffraction data to a resolution of 2.1 Å has been collected using microgravity-grown perdeuterated TS crystals from PCG-15.
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| 2. |
- Drago, Victoria N., et al.
(author)
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Neutron diffraction from a microgravity-grown crystal reveals the active site hydrogens of the internal aldimine form of tryptophan synthase
- 2024
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In: Cell Reports Physical Science. - 2666-3864. ; 5:2
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Journal article (peer-reviewed)abstract
- Pyridoxal 5′-phosphate (PLP), the biologically active form of vitamin B6, is an essential cofactor in many biosynthetic pathways. The emergence of PLP-dependent enzymes as drug targets and biocatalysts, such as tryptophan synthase (TS), has underlined the demand to understand PLP-dependent catalysis and reaction specificity. The ability of neutron diffraction to resolve the positions of hydrogen atoms makes it an ideal technique to understand how the electrostatic environment and selective protonation of PLP regulates PLP-dependent activities. Facilitated by microgravity crystallization of TS with the Toledo Crystallization Box, we report the 2.1 Å joint X-ray/neutron (XN) structure of TS with PLP in the internal aldimine form. Positions of hydrogens were directly determined in both the α- and β-active sites, including PLP cofactor. The joint XN structure thus provides insight into the selective protonation of the internal aldimine and the electrostatic environment of TS necessary to understand the overall catalytic mechanism.
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| 3. |
- Fisher, Zoe, et al.
(author)
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Biological Structures
- 2017
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In: Experimental Methods in the Physical Sciences. - 1079-4042. ; 49, s. 1-75
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Book chapter (peer-reviewed)abstract
- Neutron scattering methods are excellent for probing the detailed structure of biological systems, which rely on the intricate interplay of a large number of molecules from proteins and nucleic acids to lipids, hormones, and metabolites. With recent instrument developments and emergence of both new neutron sources and techniques, many biological systems that are not yet amenable to characterization by neutron scattering will become accessible in the near future, which will allow new experiments to be developed with a range of biologically relevant samples, offering new insights in life science. In this chapter, we will describe neutron methods for biological structure characterization on different length scales from atomic resolution to macromolecular length scales-up to micrometers. The dynamics of biological molecules are described by Seydel in Chapter 2 of this thematic volume.
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| 4. |
- Kovalevsky, Andrey, et al.
(author)
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"To Be or Not to Be" Protonated : Atomic Details of Human Carbonic Anhydrase-Clinical Drug Complexes by Neutron Crystallography and Simulation
- 2018
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In: Structure. - : Elsevier BV. - 0969-2126. ; 26:3, s. 3-390
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Journal article (peer-reviewed)abstract
- Human carbonic anhydrases (hCAs) play various roles in cells, and have been drug targets for decades. Sequence similarities of hCA isoforms necessitate designing specific inhibitors, which requires detailed structural information for hCA-inhibitor complexes. We present room temperature neutron structures of hCA II in complex with three clinical drugs that provide in-depth analysis of drug binding, including protonation states of the inhibitors, hydration water structure, and direct visualization of hydrogen-bonding networks in the enzyme's active site. All sulfonamide inhibitors studied bind to the Zn metal center in the deprotonated, anionic, form. Other chemical groups of the drugs can remain neutral or be protonated when bound to hCA II. MD simulations have shown that flexible functional groups of the inhibitors may alter their conformations at room temperature and occupy different sub-sites. This study offers insights into the design of specific drugs to target cancer-related hCA isoform IX. Kovalevsky et al. used macromolecular neutron crystallography and molecular dynamics simulations to obtain a detailed picture of clinical inhibitors binding to human carbonic anhydrase II. The study visualized hydrogen atom positions, revealing protonation/deprotonation events and intricate hydrogen-bonding networks, providing insights for drug design.
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| 5. |
- Wan, Qun, et al.
(author)
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Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography.
- 2015
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In: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 112:40, s. 12384-12389
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Journal article (peer-reviewed)abstract
- Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.
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