Computational protocol: A new role for FBP21 as regulator of Brr2 helicase activity

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Protocol publication

[…] All NMR spectra were recorded on a Bruker Avance 700 MHz spectrometer equipped with a 5 mm triple resonance cryoprobe. Spectra were processed using TopSpin3.1 (Bruker) and analyzed using CCPNMR Analysis 2.2.2. (). Triple resonance spectra (HNCA, HNCOCA, HNCO, HNCACO and 1H–15N-NOESY) for backbone assignments were recorded with 2H–15N-13C-labeled Brr2C-Sec63 at concentrations of 200–500 μM in 10 mM sodium phosphate pH 7.0, 100 mM NaCl supplemented with 10% D2O at 300 K. Non-uniform sampling (25%) was used to reduce measurement time. Assignment of the HSQC signals was accomplished using CCPNMR Analysis 2.2.2 () and chemical shift predictions derived from the crystal structure of Brr2C-Sec63 (part of PDB ID 4F91) using ShiftX2 (). All 1H–15N-TROSY-HSQC spectra of amino acid-selectively labeled Brr2C-Sec63 (Leu, Ala (-Trp), Ile, Val, Gly (–Ser, –Cys), Trp–Tyr, Tyr–Phe) were measured at 100–300 μM in 10 mM sodium phosphate pH 7.0, 100 mM NaCl supplemented with 10% D2O at 300 K with 32–128 scans depending on the concentration and 1024 × 96 datapoints. All 1H–15N-TROSY-HSQC spectra of uniformly labeled proteins were measured at 100 μM in 10 mM Tris, 100 mM NaCl pH 7.5 supplemented with 10% D2O at 300 K with 32 scans and 1024 × 96 datapoints. NMR spectra of 15N-labeled FBP21276–376 were measured at a concentration of 100 μM with a 2-fold excess of Brr2C-Sec63. NMR spectra of 15N-labeled Brr2C-Sec63 were measured at a concentration of 100 μM with a two-fold excess of ligand (FBP21276–376, FBP21200–376 K357ER359E and FBP21326–376 and FBP21276–376), a four-fold excess of ligand (FBP21276–376) or a seven-fold excess of ligand (peptide 57 GVMADGVAPVFKKRRTENGK and peptide 58 GVAPVFKKRRTENGKSRNLR). For the NMR titrations, spectra were acquired with 100 μM Brr2C-Sec63 and 12.5, 25, 50, 75, 100, 150, 200 and 400 μM FBP21276–376. Assignments of the unbound Brr2C-Sec63 were transferred to the nearest neighbors of the bound state, employing amino-acid-type selectively-labeled Brr2C-Sec63 in complex with FBP21276–376 to help identifying longer shift distances. Chemical shift differences were calculated by {Δδ1H/15N = SQRT((δ1H)2 + (0.15*δ15N)2} and were considered to be strongly shifting when they exceeded the average plus standard deviation and weakly shifting when they exceeded the average chemical shift distance. The chemical shift changes were plotted on the structure using the PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC. For soluble paramagnetic relaxation enhancement (PRE) experiments, Gadopentetic acid (Gd(DTPA)2−) was used at 0, 2, 5 and 10 mM with Brr2C-Sec63 alone or in complex with FBP21276–376. All 1H–15N-TROSY-HSQC spectra for PRE experiments were recorded at 100 μM Brr2C-Sec63 and 200 μM FBP21276–376 (if applicable) in 10 mM Tris, 100 mM NaCl pH 7.5 supplemented with 10% D2O at 300 K with 32 scans and 1024 × 96 datapoints. Peak intensities of all single assigned peaks were extracted using CCPNMR Analysis (). To obtain a value for the intensity loss, the peak intensities of the Gd(DTPA)2−-samples were divided by the peak intensities of the spectrum without Gd(DTPA)2−, always with and without ligand. Subsequently, the intensity loss of each peak with ligand was subtracted from each peak without ligand. Values above the overall average difference plus standard deviation (0.33) were considered a strong protection from the PRE, values above the average difference (0.16) a weak protection. Likewise, values below the negative average difference minus standard deviation (−0.33) were considered a strong deprotection, values below the negative average difference (−0.16) a weak protection. […]

Pipeline specifications

Software tools CcpNmr, SHIFTX, PyMOL
Application NMR-based proteomics analysis