Computational protocol: Structural, Kinetic and Proteomic Characterization of Acetyl Phosphate-Dependent Bacterial Protein Acetylation

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Protocol publication

[…] Mass spectrometric data was searched using the database search engine ProteinPilot (AB SCIEX Beta 4.5, revision 1656) with the Paragon algorithm (4.5.0.0, 1654). Mass spectral data sets also were analyzed using a Mascot server version 2.3.02 after initially generating peak lists with the AB SCIEX mgf data converter version 1.3. All extensive details including search parameters, fixed and variable modifications, enzyme specificity, databases used, scoring, and false discovery rate analysis (FDR) are described in the . [...] MS1 chromatogram-based quantitation was performed in Skyline, an open source software project (http://proteome.gs.washington.edu/software/skyline) using a MS1 ion intensity chromatogram processing called MS1 Filtering . Briefly, prior to MS1 Filtering, comprehensive spectral libraries were first generated in Skyline from database searches of the raw data files using the BiblioSpec algorithm . Second, all raw files acquired in data dependent acquisition mode (DDA) were directly imported into Skyline 1.4, and MS1 precursor ions were extracted for all peptides present in the MS/MS spectral libraries (the MS1 resolution setting in Skyline for precursor ion extraction is set at 10,000). Quantitative analysis was based on extracted ion chromatograms (XICs) and the resulting precursor ion peak areas for M, M+1, and M+2; final quantitative comparisons were typically based on only the highest ranked (naturally most abundant) precursor ion, which was then compared between samples. [...] For functional analysis and protein ontology analysis ‘The Database for Annotation, Visualization and Integrated Discovery’ (DAVID v.6.7) was used . For pathway enrichment analysis, we used the Bonferroni correction as adjustment to p-values to determine statistical significance. To generate graphical ontology displays, Panther v8.1 classification system was used . [...] Crystals were harvested from PACT D11 (0.2 M calcium chloride, 0.1 M Tris pH 8, 20% (w/v) PEG 6000) for native TpiA, Classics F11 (0.2 M NaCl, 0.1 M Bis-Tris pH 6.5, 25% (w/v) PEG 3350 for acP-treated TpiA, and PACT G2 0.2 M sodium bromide, 0.1 M Bis Tris propane 7.5, 20% (w/v) PEG 3350 for AcP-treated GapA. Crystals not treated with acP were transferred to 5 µl of reservoir solution and mounted and frozen in liquid nitrogen for data collection. The crystals from Classics F11 and PACT G2 were treated with acP by soaking using the following procedure: crystals were transferred to 5 µl of the reservoir solution, AcP powder was added directly to the solution (approximately 10 mM final concentration) using a pipette tip, and crystals were quickly drawn through the solution, mounted and flash frozen with liquid nitrogen. The time between adding the acP and plunging the crystal into liquid nitrogen was less than 30 sec. Data were collected at 100 K at the Life Sciences Collaborative Access Team (LS-CAT) 21ID-D, 21ID-F and 21ID-G beamlines at Argonne National Laboratory (Argonne, IL). HKL3000 was used for data processing, integration, and scaling. The GapA and TpiA structures were solved by Molecular Replacement with Phaser in the CCP4 suite , using the GapA structure from E. coli (PDB ID: 1S7C) and the TpiA structure from E. coli (PDB ID: 1TRE) as the search models. The models were refined using Refmac and acetylated lysines were modified using the CCP4 monomer library. Manual corrections were performed on workstations using COOT and the final models were validated using the validation server of the PDB. PDB Codes: 4K6A (native tpiA), 4MVA (acP-treated TpiA), and 4MVJ (acP-treated GapA). Figures were made using Pymol (Delano, 2002) and structure parameters are shown in . [...] pKa values for LpdA and RNAP subunits were calculated using PROPKA 3.1 at http://propka.ki.ku.dk/ . PROPKA also calculates surface accessibility, defining a parameter referred to as buried ratio. As input for PROPKA, the following PDB structural files were used, PDB ID: 4JDR for LpdA, PDB ID: 4IGC for RNAP chain A (RpoA), chain C (RpoB), chain D (RpoC), and chain X (RpoD). […]

Pipeline specifications

Software tools CCP4, Coot, PyMOL, PROPKA
Application Protein structure analysis
Organisms Escherichia coli
Chemicals Acetyl Coenzyme A, Glyceraldehyde 3-Phosphate, Phosphates