Monobromobimane labeling of sulfhydryl groups in spinach lumenal proteins and recombinant PsbO from Synechocystis Monobromobimane (mBBr) labeling of sulfhydryl groups in spinach lumenal proteins and recombinant PsbO from Synechocystis (isolated from strains 7S or 2RG) was performed essentially as described previously [77]. A sample of 10 mg of protein was incubated with 5 ml of 20 mM mBBr prepared in acetonitrile, in 50 mM buffer Tris-HCl, pH 8, with a final volume of 55 ml. The labeling reaction was conducted at room temperature for 15 min in darkness. To stop the reaction, 10 ml of 100 mM b- mercaptoethanol was added to the samples. Proteins were precipitated with five volumes of 100% acetone at 220uC for 2 h and recovered by centrifugation at 16 000 g for 15 min. The pellet was washed with acetone, air dried, solubilized in sample buffer, and heated at 80uC for 10 min. After SDS-PAGE, the fluorescence of the labeled proteins was visualized by UV, and the gels were stained with CBB. To attempt the total reduction of the disulfide bonds, PsbO samples were pre-incubated in the presence of 2.5 mM dithiotreitol (DTT) for 15 min at room temperature prior to mBBr treatment. To oxidize all the SH groups, samples of 6 mM recombinant PsbO were incubated in the presence of 35 mM CuSO4 for 2 h at room temperature and dialyzed against two changes of 50 mM Tris-HCl, pH 8, prior to mBBr treatment. Quantification of gels and blots signals The integrated density values of the bands was measured using Image J software, free available at http://rsb.info.nih.gov/ij/ index.html. Supporting Information Figure S1 Recombinant PsbO is reduced even in the absence of reducing agents. Recombinant PsbO of Synecho- cystis 6803 was isolated from the strains 7S or 2RG and incubated with monobromobiname (mBBr) to label the sulfhydryl groups. To reduce or oxidize PsbO, samples were pre-treated with 2.5 mM DTT or 35 mM CuSO4, respectively, previous to mBBr labeling. Ten micrograms of protein were loaded per lane. After SDS- PAGE proteins were visualized by UV (upper panel) and stained with CBB (lower panel). (TIF) Figure S2 Alignment of PsbO sequences. Multiple se- quence alignment of PsbO1 (ARAB 1, accession number AED98230.1) and PsbO2 (ARAB 2, accession number References 1. Lipinska B, Sharma S, Georgopoulos C (1988) Sequence analysis and regulation of the htrA gene of Escherichia coli: a s32-independent mechanism of heat- inducible transcription. Nucleic Acids Res 16: 10053–10067. 2. Strauch KL, Beckwith J (1988) An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc Natl Acad Sci USA 85: 1576–1580. 3. Gottesman S (1996) Proteases and their targets in Escherichia coli. Annu Rev Genet 30: 465–506. 4. Clausen T, Southan C, Ehrmann M (2002) The HtrA family of proteases: implications for protein composition and cell fate. Mol Cell 10: 443–455. 5. Doyle DA, Lee A, Lewis J, Kim E, Sheng M, et al. 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Asterisk indicates fully conserved residues, colon and period indicate strong and weak conservation, re- spectively, as defined by ClustalW2. (TIF) Figure S3 Time course degradation of PsbO in the presence of recombinant Deg proteases from Synecho- cystis sp. PCC 6803. PsbO was isolated from spinach leaves and incubated in the absence (PsbO) or the presence (PsbOred) of the complete thioredoxin system for 10 h without addition of recombinant proteases (A) or after addition of rHhoA (B), rHhoB (C) or rHtrA (D). Proteases were used at the standard enzyme to substrate ratio of 1:50 or at a ratio five times higher (1:10) referred as 5X. After SDS-PAGE the proteins (9 mg) were stained with CBB, the integrated density of the PsbO band was quantified using Image J software and represented as the residual amount taking the 0 h band as 100%. (TIF) Figure S4 Redox-dependent degradation of PsbO in the presence of combinations of recombinant Deg proteases from Synechocystis sp. PCC 6803. 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