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Eluted protein was loaded on a 10 to 32 linear sucrose gradient ready in Tris-HCl (30 mM, pH 7.5), NaCl (50 mM), EDTA (1 mM), UDM (0.05 ), and protease inhibitors and centrifuged at 160000g for 1216 h 4 to separate the dimer in the monomer. Sodium dodecyl sulfate olyacrylamide gel electrophoresis (SDS AGE) analysis in the purified protein was performed on a 12 polyacrylamide gel using standard procedures. Purification with the b6f complicated from spinach and Nostoc PCC 7120 has been described previously.24,57 Genetic modification and purification of the cyt b6f complex from Synechocystis sp. PCC 6803 have been performed following protocols described for Synechococcus sp. PCC 7002.58 Clear native gel electrophoresis was performed as outlined by published protocols59 on a four to 12 gradient gel. Gel densitometry was performed applying AlphaView computer software from ProteinSimple.60 Briefly, the intensity of bands corresponding to cyt f, cyt b6, ISP, and subIV was determined, followed by scaling for the corresponding molecular masses to acquire the subunitAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptBiochemistry. Author manuscript; available in PMC 2015 December 08.Agarwal et al.Pagestoichiometry relative to cyt f. Two-dimensional (2D) clear native-PAGE was performed as outlined by a protocol described elsewhere.61 Assay of Electron Transfer Activity Thylakoid membranes and cytochrome b6f have been isolated from F. diplosiphon, as described in this section. Cytochrome b6f oxidoreductase activity was measured at room temperature inside a 20 mM Tricine-NaOH buffer (pH 8.0) containing 0.05 UDM. The reaction mixture containing 25 M decyl-plastoquinone, two.five M horse heart cytochrome c, and 250 M potassium ferricyanide was initiated by addition of ten nM cytochrome b6f complicated or thylakoids containing five g of chlorophyll. The reaction was monitored through the modify in ferricyanide absorbance at 420 nm, relative Title Loaded From File towards the background price, determined by a millimolar extinction coefficient mM of 1.02 mM-1 cm-1. Mass Spectrometric Evaluation The polypeptide composition in the Fremyella cytochrome b6f preparation was analyzed by liquid chromatography and mass spectrometry (Supporting Facts, Figures S2 9) with fraction collection as described previously (LC-MS+).62 Selected polypeptide masses had been additional analyzed by high-resolution Fourier transform mass spectrometry working with collisionally activated or electron capture/transfer dissociation (CAD/ETD) as described previously.63 The high-resolution information were collected utilizing good ion nanoelectrospray ionization on ion cyclotron resonance/orbitrap Fourier transform instruments (Thermo Scientific) externally calibrated to yield data at mass accuracy tolerances of 15 ppm. Data were interpreted applying Prosight Pc two.0 (Thermo Scientific) to transform raw files in to peak lists and match the resulting monoisotopic masses to protein structures with many posttranslational modifications.64 Structure Research of Intermonomer Contacts Intermonomer interactions were identified in the recently published 2.five crystal structure of your b6f complicated (PDB entry 4OGQ),32 as well as the 1.9 crystal structure of the cyt bc1 complex (PDB entry 3CX5).31 The crystallographic dimers on the b6f and bc1 complex were analyzed for intersubunit interactions and interfacial region calculations working with COCOMAPS.65 Residue pairs separated by more than 4 were classified as noninteracting. Sequence Alignment Multiple-sequence alignment was performed i.