The clinical drug candidate anle138b binds in a cavity of lipidic α-synuclein fibrils

DOI

This dataset includes NMR spectra raw data generated in this study, Negative stain EM micrographs for αSyn fibrils in the presence of anle138b and mass spectrometry data for the synthesis of selectively labelled α-synuclein and 13C- and 15N-labelled anle138b, respectively. Solid-state NMR: 3D (H)CANH experiments on fibrils in the presence of anle138b were recorded on an 800 MHz Bruker Avance III HD spectrometer at a magnetic field of 18.8 T equipped with a 1.3 mm magic-angle spinning (MAS) HCN probe and MAS at 55 kHz. The temperature of the cooling gas was set to 250 K, resulting in an estimated sample temperature of 20 °C. 3D (H)CANH spectra on fibrils in the absence of anle138b were recorded on a 950 MHz Bruker Avance III HD spectrometer at a magnetic field of 22.3 T equipped with a 0.7 mm HCDN probe and MAS at 100 kHz. The temperature of the cooling gas was set to 250 K, resulting in an estimated sample temperature of 20 °C. 2D 13C13C-DARR spectra were acquired on an 850 MHz Avance III spectrometer with a 3.2-mm MAS HCN probe with mixing times of 200 ms at a magnetic field of 20.0 T and MAS at 17 kHz. The temperature of the cooling gas was set to 265 K, resulting in an estimated sample temperature of 20 °C. 3D (H)CANH spectra were acquired in three blocks of 32 h in the presence and four blocks of 19 h in the absence of anle138b. All 2D 13C13C DARR spectra were acquired in 34 blocks of 7.2 h in the presence and 25 equivalent blocks in the presence of anle138b. All related blocks were corrected for linear drift of the static magnetic field using an in-house program executed from the command line in Bruker Topspin 4.0.8. The drift corrected blocks were then averaged and processed as one spectrum. Spectra were analyzed using CcpNmr Analysis and NMRFAM-Sparky. Peak intensities were determined by integrating cross peaks using the Lorentzian fit algorithm in Sparky, not allowing peak center motion. Assignments on α-synuclein fibrils in the absence of anle138b had previously been reported. Chemical shift perturbations were calculated as the average of HN, NH, Cα and Cβ chemical shifts. Solution NMR: NOESY spectra were acquired with 72 scans using 256 increments in the indirect dimension and a relaxation delay of 2.3 s. The mixing time was set to 3 ms. Proton 90° flip pulses were 14.4 μs. Water suppression was achieved by presaturation at a power level of 100 Hz. Experiments were recorded on a Bruker 900-MHz spectrometer. Temperature during measurements was kept at 298 K. 2D datasets were processed in NMRPipe and analyzed in NMRFAM-Sparky. Spectral traces were fitted to a mixed Gaussian and Lorentzian function using the line shapes tool in Bruker Topspin 4.0.8. Preparation of isotope labelled protein α-Synuclein was recombinantly produced in E. coli strain BL21(DE3) as previously described. Uniformly 15N- and 13C,15N-labeled samples were expressed in minimal medium supplemented with 15NH4Cl and 13C6-D-glucose (Cambridge Isotope Laboratories and Sigma Aldrich). For production of amino acid specific forward-labeled protein (13C,15N-Leu/Tyr, 13C-Phe, 13C,15N-Ile), the labeled amino acids were added to the minimal medium one hour before induction of protein expression. For forward labeling with 13C- and 13C,15N-Gly, protein expression was per-formed with the glycine auxotrophic E. coli strain DL39 (DE3) GlyA following a published protocol. The protein was finally dialyzed against buffer (50 mM HEPES, 100 mM NaCl, pH 7.4) to obtain a 0.3 mM solution and the resulting solution was stored at -80 °C until use.

Identifier
DOI https://doi.org/10.17617/3.9C6TEW
Metadata Access https://edmond.mpg.de/api/datasets/export?exporter=dataverse_json&persistentId=doi:10.17617/3.9C6TEW
Provenance
Creator Antonschmidt, Leif; Dirk Matthes; Rıza Dervişoğlu; Benedikt Frieg; Christian Dienemann; Andrei Leonov; Evgeny Nimerovsky; Vrinda Sant; Sergey Ryazanov; Armin Giese; Gunnar Schröder; Stefan Becker; Bert L. de Groot; Christian Griesinger; Loren B. Andreas
Publisher Edmond
Publication Year 2022
OpenAccess true
Contact lean(at)mpinat.mpg.de
Representation
Language English
Resource Type Dataset
Version 1
Discipline Other