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@MISC{Gloeckner:272967,
      author       = {Gloeckner, Christian Johannes},
      title        = {{D}ataset: {S}upplemental data for '{I}ntramolecular
                      feedback regulation of the {LRRK}2 {R}oc {G} domain by a
                      {LRRK}2 kinase dependent mechanism' ({G}ilsbach et al.,
                      e{L}ife 2024, doi:10.7554/e{L}ife.91083), v2},
      publisher    = {Zenodo},
      reportid     = {DZNE-2024-01346},
      year         = {2024},
      abstract     = {Supportive data for the eLife version of record. (1) Data
                      used for the Michaelis Menten Kinetics. HPLC-based assay.
                      Steady-state kinetic measurements of LRRK2-mediated GTP
                      hydrolysis were performed as previously described (Ahmadian
                      et al., 1997). Briefly, 0.1 µM of full-length LRRK2 was
                      incubated with different amounts of GTP (0, 25, 75, 150,
                      250, 500, 1000, 2000, 3000 and 5000 µM) and production of
                      GDP was monitored by reversed phase C18 HPLC. To this end,
                      the samples (10 µl) were directly injected on a
                      reversed-phase C18 column (pre-column: Hypersil Gold, 3µm
                      particle size, 4.6x10mm; main column: Hypersil Gold, 5µm
                      particle size, 4.6x250mm, Thermo Scientific) using an
                      Ultimate 3000 HPLC system (Thermo Scientific, Waltham, MA,
                      USA) in HPLC-buffer containing 50 mM KH2PO4/K2HPO4 pH 6.0,
                      10 mM tetrabutylammonium bromide and $10-15\%$ acetonitrile.
                      Subsequently, samples were analyzed using the HPLC
                      integrator (Chromeleon 7.2, Thermo Scientific, Waltham, MA,
                      USA). Initial rates of GDP production were plotted against
                      the GTP concentration using GraFit5 (v.5.0.13, Erithacus
                      Software). The number of experiments is indicated in the
                      graph and data point is the average (±s.e.m.) of indicated
                      repetitions. The Michaelis-Menten equation was fitted to
                      determine KM (±s.e.) and kcat (±s.e.). Excel sheets used
                      for the calculation of means are provided. No values are
                      reported if the HPLC separation failed (e.g. unstable
                      baseline). Charcoal GTP hydrolysis assay. The [γ-32P]GTP
                      charcoal assay was performed as previously described (Bollag
                      and McCormick, 1995). Briefly, 0.1 µM full-length LRRK2 or
                      0.5 µM 6xHIS-MBP-RocCOR was incubated with different GTP
                      concentrations, ranging from 75 µM to 8 mM, in the presence
                      of [γ-32P] GTP in GTPase assay buffer (30 mM Tris pH 8, 150
                      mM NaCl, 10 mM MgCl2, $5\%$ (v/v) Glycerol and 3 mM DTT).
                      Samples were taken at different time-points and immediately
                      quenched with $5\%$ activated charcoal in 20 mM phosphoric
                      acid. All non-hydrolyzed GTP and proteins were stripped by
                      the activated charcoal and sedimented by centrifugation. The
                      radioactivity of the isolated inorganic phosphates was then
                      measured by scintillation counting. The initial rates of
                      γ-phosphate release and the Michaelis-Menten kinetics were
                      calculated as described above. (2) Profile plots (Raw data)
                      obtained for the Mass photometry analysis for T1343A vs WT
                      LRRK2. MP was performed as described in (Guaitoli et al.,
                      2023). Briefly, the dimer ratio of LRRK2 was determined on a
                      Refeyn Two MP instrument (Refeyn). Prior to the experiment,
                      a standard curve relating particle contrasts to molecular
                      weight was established using a Native molecular weight
                      standard (Invitrogen, 1:200 dilution in HEPES-based elution
                      buffer: 50 mM HEPES [pH 8.0], 150 mM NaCl supplemented with
                      200 µM desthiobiotin). Prior to mass photometry, the
                      proteins, either WT or T1343A LRRK2, were incubated with ATP
                      to induce autophophorylation. The LRRK2 protein was diluted
                      to 2x of the final concentration (end concentrations: 75 nM
                      and 100 nM) in elution buffer. The optical setup was focused
                      in 10 μl elution buffer before adding 10 µl of the
                      adjusted protein sample. Depending on the obtained count
                      numbers, acquisition times were chosen between 20 s to 1
                      min. The dimer ratio in each measurement was normalize
                      according to the equation. The measurement was perfomed in
                      triplicates. (3) AlphaFold3 model of LRRK2-pT1343 either
                      bound to GDP/Mg or GTP/Mg. Using AlphaFold3 (Abramson et
                      al., 2024), we modeled and compared the GDP vs the GTP-state
                      of phospho-T1343 LRRK2. Interestingly, the AlphaFold3 model
                      suggests, that the phosphate group of the pT1343 residue is
                      orientated inwards thereby substituting the gamma phosphate
                      of the GTP in the GDP-bound state of LRRK2. This finding is
                      in well agreement with MD simulations published recently
                      (Stormer et al., 2023).},
      keywords     = {structural modeling (Other) / LRRK2 (Other)},
      cin          = {AG Gloeckner},
      cid          = {I:(DE-2719)1210007},
      pnm          = {352 - Disease Mechanisms (POF4-352)},
      pid          = {G:(DE-HGF)POF4-352},
      typ          = {PUB:(DE-HGF)32},
      doi          = {10.5281/zenodo.13837193},
      url          = {https://pub.dzne.de/record/272967},
}