Computational protocol: Redox and Light Control the Heme-Sensing Activity of AppA

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Protocol publication

[…] Diffraction data were collected at the Berkeley beamline Advanced Light Source (ALS) 4.2.2. using remote data collection. The best anomalous data set was collected using selenium peak (4 selenium atoms in the SCHIC molecule) at 2.2-Å resolution. Data collection proceeded for 210° with a step of 1°. The crystal mosaicity was ~0.8. The best diffraction native data set was collected at 2.05-Å resolution. Initial indexing by HKL2000 provided primitive monoclinic space group P21. The initial phase estimates using SeMet data set were computed using Phaser (). Autobuild software of Phenix () then provided the beginning model. Coot () and Phenix were used for model building and final refinement. Final refinement statistics are shown in in the supplemental material. [...] The presence of reduced Cys sulfhydryl residues in AppA was determined by incubating ~10 µM AppA (either oxidized or 10 mM DTT-reduced) with 10 or 20 mM iodoacetamide at 22° C for 10 min. Excess iodoacetamide was then removed by passing through a MidiTrap G-25 (GE Healthcare) desalting column or a Zeba spin desalting column with a molecular weight cutoff of 7,000 (7K MWCO) (Thermo Scientific), depending on the sample volume. The samples were then treated with trypsin (protein/trypsin ratio is 10:1) at 37°C overnight. The digestion was stopped by the addition of trifluoroacetic acid (TFA) to a final concentration of 0.1% and desalted using ZipTip pipettes with C18 resin (Millipore). The samples eluted from ZipTip pipettes by 60% acetonitrile and 0.1% TFA were used for MALDI experiments with a Bruker Autoflex III MALDI-TOF mass spectrometer. To determine which cysteine residues are forming a disulfide bridge, 5 µM AppA was treated with 5 mM N-ethylmaleimide (NEM) for 45 min at 22°C, then with 10 mM DTT for 10 min at 22°C, and finally with 20 mM iodoacetamide for 30 min at 22°C. The double-labeled tryptic digest was analyzed by capillary-scale liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) (). An Eksigent two-dimensional (2D) Ultra liquid chromatograph (AB Sciex, Foster City, CA) injected 5 µl of extract onto a trapping column (100 by 0.1 mm) packed with 5-µm Michrom Magic C18 particles and desalted for 8 min at a flow rate of 4 µl/min using LC buffer A (0.1% [vol/vol] formic acid [Sigma-Aldrich], 3% [vol/vol] acetonitrile [EM Science] in high-performance liquid chromatography [HPLC]-grade water [EM Science]). The trapping column was then placed in line with a Picofrit column (150 by 0.075 mm) with an integrated ESI tip (New Objective, Woburn, MA) packed with the same material as in the trap. A 35-min gradient from 10 to 85% LC buffer B (0.1% [vol/vol] formic acid, 3% [vol/vol] water in acetonitrile) separated the peptide mixture prior to tandem mass spectrometry in an LTQ-Orbitrap XL mass spectrometer (Thermo, Waltham, MA). The mass/charge ratios of the intact peptide ions were measured at a resolving power of 15,000 in the orbitrap; the fragment ions were generated, and their masses were measured in the LTQ ion trap. The resulting mass spectra were manually interpreted; the MS-Product module of ProteinProspector (http://prospector.ucsf.edu) () was used to predict the fragment ion masses. […]

Pipeline specifications

Software tools PHENIX, Coot, Protein Prospector
Applications MS-based untargeted proteomics, Protein structure analysis
Chemicals Heme, Vitamin B 12