Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography
| Autor: | Henry N. Chapman, Katarina Doerner, Andrea M. Katz, Valerio Mariani, Adrian P. Mancuso, Weijun Xu, Anton Barty, Kara A. Zielinski, Jose L. Olmos, Luca Gelisio, Petra Fromme, Grant Mills, Tokushi Sato, Rebecca Jernigan, Oleksandr Yefanov, George D. Calvey, Mohammad Vakili, Mitchell D. Miller, Alireza Sadri, Henry Kirkwood, Ishwor Poudyal, Richard Bean, Tek Narsingh Malla, Jay-How Yang, John C. H. Spence, Peter Schwander, Jayanath Koliyadu, Lois Pollack, A. Tolstikova, Abbas Ourmazd, Suraj Pandey, Saša Bajt, Garrett Nelson, Faisal Hammad Mekky Koua, Matthias Frank, George N. Phillips, Salah Awel, Marius Schmidt, Jose M. Martin-Garcia, Marco Kloos |
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| Přispěvatelé: | National Science Foundation (US), Department of Energy (US), National Institutes of Health (US), Lawrence Livermore National Laboratory, German Research Foundation, European Commission, European Research Council |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
serial femtosecond crystallography
antibiotic resistance Antibiotic resistance β-lactamases 02 engineering and technology Substrate diffusion in crystals Biochemistry substrate diffusion in crystals Atomic Crystal beta-lactamases enzyme mechanisms Particle and Plasma Physics enzyme kinetics General Materials Science Serial femtosecond crystallography Diffusion (business) mix-and-inject serial crystallography 0303 health sciences Crystallography Chemistry Drug discovery Resolution (electron density) Ceftriaxone Mix-and-inject serial crystallography protein structure determination 021001 nanoscience & nanotechnology Ligand (biochemistry) megahertz pulse-repetition rate Condensed Matter Physics Research Papers 3. Good health Sulbactam QD901-999 X-ray crystallography Enzyme mechanisms 0210 nano-technology Physical Chemistry (incl. Structural) sulbactam Catalysis drug discovery 03 medical and health sciences Rare Diseases Tuberculosis Nuclear ddc:530 Enzyme kinetics 030304 developmental biology irreversible inhibition Megahertz pulse-repetition rate Substrate (chemistry) Molecular General Chemistry Protein structure determination ceftriaxone Good Health and Well Being European X-ray Free-Electron Laser Irreversible inhibition |
| Zdroj: | IUCrJ 8(6), 878-895 (2021). doi:10.1107/S2052252521008125 IUCrJ, Vol 8, Iss 6, Pp 878-895 (2021) IUCrJ, vol 8, iss Pt 6 Digital.CSIC. Repositorio Institucional del CSIC instname IUCrJ |
| Popis: | 18 pags, 11 figs, 5 tabs Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research. This work was supported by the National Science Foundation Science and Technology Center 'BioXFEL' through award STC-1231306, and in part by the US Department of Energy, Office of Science, Basic Energy Sciences under contract DESC0002164 (AO, algorithm design and development) and by the National Science Foundation under contract Nos. 1551489 (AO, underlying analytical models) and DBI-2029533 (AO, functional conformations). This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 1450681 to JLO. The work was also supported by funds from the National Institutes of Health grant R01 GM117342-0404. Funding and support are also acknowledged from the National Institutes of Health grant R01 GM095583, from the Biodesign Center for Applied Structural Discovery at ASU, from National Science Foundation award No. 1565180 and the US Department of Energy through Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344. KAZ was supported by the Cornell Molecular Biophysics Training Program (NIH T32-GM008267). This work was also supported by the Cluster of Excellence 'CUI: Advanced Imaging of Matter' of the Deutsche Forschungsgemeinschaft (DFG), EXC 2056, project ID 390715994. CFEL is supported by the Gottfried Wilhelm Leibniz Program of the DFG, the 'X-probe' project funded by the European Union 2020 Research and Innovation Program under Marie Sklodowska-Curie grant agreement 637295, the European Research Council, 'Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS)', ERC-2013-SyG 609920, and the Human Frontiers Science Program grant RGP0010 2017. This work is also supported by the AXSIS project funded by the European Research Council under the European Union Seventh Framework Program (FP/2007-2013)/ERC Grant Agreement No. 609920. |
| Databáze: | OpenAIRE |
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