Laced by D1:130Q in the T. vucanus structure. It should

Laced by D1:130Q in the T. vucanus structure. It should be noted that in T. vulcanus, D1:130Q is present in the constitutively expressed D1? isoform while the D1? and D1? isoforms, which are expressed only under certain environmental conditions, contain D1:130E [31,32]. These residues have been reported to be hydrogen- bonded to PheoD1 [33,34]. Additionally, in Thermosynechococcus, D1:135Y is replaced by D1:135F (Table 1). Consequently, in the Thermo?synechococcus structure: PheoD1 ?2.9A ?D1:130Q ?D1:133L ?D1:135F (Fig. 4). The mass spectrum identifying this group of modified residues is shown in Fig. S3. These oxidized residues may be adjacent to a putative ROS exit pathway leading fromResultsFig. 1 illustrates the quality of the mass spectrometry data used for the identification of oxidized amino acid residues in the D1 and D2 proteins. In this figure the MS/MS data collected for the D2 peptide 235A?52R are illustrated. In Fig. 1A, the data from the unmodified peptide are illustrated, while in Fig. 1B, data from the peptide bearing oxidized 247M are shown. In this example and others (Figs. S1 2), both modified and unmodified versions of the target peptide were identified; in another example only the modified peptide was detected (Fig. S3). Using a p value #0.00001 assured extremely high quality peptide identifications with nearly complete y- and b-ion series being observed. In this communication, we have focused on the domains of the D1 and D2 proteins located at or near redox active cofactors located on the stromal face of the thylakoid membrane. A total of 10 oxidatively modified amino acid residues on the D1 and D2 proteins were observed in the MedChemExpress CI-1011 vicinity of these cofactors. These are summarized in Table 1 along with the type of oxidative modifications and the residue location (surface or buried and not in contact with cavity or channel) within the T. vulcanus crystal structure. Please note that these residues were originally reported to be modified in Frankel et al. [20], however, their close association with the reducing-side cofactors was not discussed in that communication. In general, mass spectrometry coverage of intrinsic membrane proteins is quite challenging. The overall sequence coverage observed in this study for the proteins examined was 24 for D1 and 27 for D2, values which are quite comparable to that observed for these proteins by other Calcitonin (salmon) cost investigators (see, for instance Nakamura et al. [30]). However, the coverage of the residues located in the stromally exposed domains of these proteins was significantly higher, 67 for D1 and 48 for D2. These are the domains of principal interest in this study, as ROS produced on the reducing side of the photosystem must transit these regions to exit the photosystem. Within this context it should be noted that most oxidative modifications to amino acid residues lying in the transmembrane helixes of these proteins would be difficult to identify and many would escape detection due to their high hydophobicity and consequent expected poor resolution during reversed-phase chromatography.Oxidized Amino Acids on the Reducing Side of PS IIFigure 1. Example Mass Spectrometry Data from the Unmodified Peptide. 235AFNPTQAEETYSMVTAN252R and the Oxidatively Modified Peptide 235AFNPTQAEETYS247M+16 VTAN252R of the D2 Protein A. Top, spectrum of the collision-induced dissociation (CID) of the unmodified peptide 235AFNPTQAEETYSMVTAN252R. Various identified ions are labeled. Bottom, table of all pred.Laced by D1:130Q in the T. vucanus structure. It should be noted that in T. vulcanus, D1:130Q is present in the constitutively expressed D1? isoform while the D1? and D1? isoforms, which are expressed only under certain environmental conditions, contain D1:130E [31,32]. These residues have been reported to be hydrogen- bonded to PheoD1 [33,34]. Additionally, in Thermosynechococcus, D1:135Y is replaced by D1:135F (Table 1). Consequently, in the Thermo?synechococcus structure: PheoD1 ?2.9A ?D1:130Q ?D1:133L ?D1:135F (Fig. 4). The mass spectrum identifying this group of modified residues is shown in Fig. S3. These oxidized residues may be adjacent to a putative ROS exit pathway leading fromResultsFig. 1 illustrates the quality of the mass spectrometry data used for the identification of oxidized amino acid residues in the D1 and D2 proteins. In this figure the MS/MS data collected for the D2 peptide 235A?52R are illustrated. In Fig. 1A, the data from the unmodified peptide are illustrated, while in Fig. 1B, data from the peptide bearing oxidized 247M are shown. In this example and others (Figs. S1 2), both modified and unmodified versions of the target peptide were identified; in another example only the modified peptide was detected (Fig. S3). Using a p value #0.00001 assured extremely high quality peptide identifications with nearly complete y- and b-ion series being observed. In this communication, we have focused on the domains of the D1 and D2 proteins located at or near redox active cofactors located on the stromal face of the thylakoid membrane. A total of 10 oxidatively modified amino acid residues on the D1 and D2 proteins were observed in the vicinity of these cofactors. These are summarized in Table 1 along with the type of oxidative modifications and the residue location (surface or buried and not in contact with cavity or channel) within the T. vulcanus crystal structure. Please note that these residues were originally reported to be modified in Frankel et al. [20], however, their close association with the reducing-side cofactors was not discussed in that communication. In general, mass spectrometry coverage of intrinsic membrane proteins is quite challenging. The overall sequence coverage observed in this study for the proteins examined was 24 for D1 and 27 for D2, values which are quite comparable to that observed for these proteins by other investigators (see, for instance Nakamura et al. [30]). However, the coverage of the residues located in the stromally exposed domains of these proteins was significantly higher, 67 for D1 and 48 for D2. These are the domains of principal interest in this study, as ROS produced on the reducing side of the photosystem must transit these regions to exit the photosystem. Within this context it should be noted that most oxidative modifications to amino acid residues lying in the transmembrane helixes of these proteins would be difficult to identify and many would escape detection due to their high hydophobicity and consequent expected poor resolution during reversed-phase chromatography.Oxidized Amino Acids on the Reducing Side of PS IIFigure 1. Example Mass Spectrometry Data from the Unmodified Peptide. 235AFNPTQAEETYSMVTAN252R and the Oxidatively Modified Peptide 235AFNPTQAEETYS247M+16 VTAN252R of the D2 Protein A. Top, spectrum of the collision-induced dissociation (CID) of the unmodified peptide 235AFNPTQAEETYSMVTAN252R. Various identified ions are labeled. Bottom, table of all pred.