Daniel Weisz

MS-based cross-linking analysis reveals the location of the PsbQ protein in cyanobacterial photosystem II

Significance Cyanobacteria, photosynthetic prokaryotes, were primarily responsible to create an oxygen-rich atmosphere on our planet. Photosystem II (PSII), a large membrane-bound pigment protein complex in cyanobacteria, uses light energy to oxidize water to dioxygen. X-ray crystal structures of cyanobacterial PSII have recently been determined. PsbQ, a protein that optimizes PSII-mediated oxygen evolution activity, is ubiquitously present in cyanobacteria. However, PsbQ is absent in the determined structures of PSII. By using protein cross-linking, mutagenesis, and advanced MS techniques, we showed that PsbQ in the model cyanobacterium Synechocystis 6803 binds to CP47 and PsbO, two known protein components of PSII. These results helped determine the location of PsbQ on the lumenal side, near the water oxidation site, of cyanobacterial PSII. PsbQ is a luminal extrinsic protein component that regulates the water splitting activity of photosystem II (PSII) in plants, algae, and cyanobacteria. However, PsbQ is not observed in the currently available crystal structures of PSII from thermophilic cyanobacteria. The structural location of PsbQ within the PSII complex has therefore remained unknown. Here, we report chemical cross-linking followed by immunodetection and liquid chromatography/tandem MS analysis of a dimeric PSII complex isolated from the model cyanobacterium, Synechocystis sp. PCC 6803, to determine the binding site of PsbQ within PSII. Our results demonstrate that PsbQ is closely associated with the PsbO and CP47 proteins, as revealed by cross-links detected between 120K of PsbQ and 180K and 59K of PsbO, and between 102K of PsbQ and 440D of CP47. We further show that genetic deletion of the psbO gene results in the complete absence of PsbQ in PSII complexes as well as the loss of the dimeric form of PSII. Overall, our data provide a molecular-level description of the enigmatic binding site of PsbQ in PSII in a cyanobacterium. These results also help us understand the sequential incorporation of the PsbQ protein during the PSII assembly process, as well as its stabilizing effect on the oxygen evolution activity of PSII.

Mass Spectrometry-based Footprinting Reveals Structural Dynamics of Loop E of the Chlorophyll-binding Protein CP43 during Photosystem II Assembly in the Cyanobacterium Synechocystis 6803

Background: The mechanism for association and dissociation of Psb27 and CP43 is poorly understood. Results: Loop E of CP43 undergoes significant conformational change upon D1 processing. Conclusion: D1 processing initiates the dissociation of Psb27 from CP43. Significance: The structural dynamics of the lumenal domain of CP43 plays a critical role in the assembly of functional Photosystem II centers. The PSII repair cycle is required for sustainable photosynthesis in oxygenic photosynthetic organisms. In cyanobacteria and higher plants, proteolysis of the precursor D1 protein (pD1) to expose a C-terminal carboxylate group is an essential step leading to coordination of the Mn4CaO5 cluster, the site of water oxidation. Psb27 appears to associate with both pD1- and D1-containing PSII assembly intermediates by closely interacting with CP43. Here, we report that reduced binding affinity between CP43 and Psb27 is triggered by the removal of the C-terminal extension of the pD1 protein. A mass spectrometry-based footprinting strategy was adopted to probe solvent-exposed aspartic and glutamic acid residues on the CP43 protein. By comparing the extent of footprinting between HT3ΔctpAΔ27PSII and HT3ΔctpAPSII, two genetically modified PSII assembly complexes, we found that Psb27 binds to CP43 on the side of Loop E distal to the pseudo-symmetrical D1-D2 axis. By comparing a second pair of PSII assembly complexes, we discovered that Loop E of CP43 undergoes a significant conformational rearrangement due to the removal of the pD1 C-terminal extension, altering the Psb27-CP43 binding interface. The significance of this conformational rearrangement is discussed in the context of recruitment of the PSII lumenal extrinsic proteins and Mn4CaO5 cluster assembly. In addition to CP43s previously known function as one of the core PSII antenna proteins, this work demonstrates that Loop E of CP43 plays an important role in the functional assembly of the Water Oxidizing Center (WOC) during PSII biogenesis.

Massive Tumor Embolism Occurring During Pneumonectomy

Abstract A case of gross tumor embolism occurring during pneumonectomy for giant cell carcinoma is presented. Seven similar cases of massive tumor embolism during pulmonary resection for malignant disease have been previously described. Ours is the first recorded patient to have survived such an episode.

Results of surgery for congenital supravalvular aortic stenosis.

Of eight children aged 3 to 15 years with surgical correction of severe supravalvular aortic stenosis, 6 were evaluated 7 to 44 months later by repeat cardiac catheterization and aortography. Prosthetic patch angioplasty was performed in all cases. Preoperative systolic gradients ranged from 40 to 90 mm Hg (average 70); postoperative gradients ranged from 0 to 20 mm Hg (average 11). The postoperative anglographic appearance of the ascending aorta was near normal in all six patients, and none had new aortic valve insufficiency. These results of surgery for supravalvular aortic stenosis are judged to be excellent.

THE ACS LCID PROJECT. XI. ON THE EARLY TIME RESOLUTION OF SFHs OF LOCAL GROUP DWARF GALAXIES: COMPARING THE EFFECTS OF REIONIZATION IN MODELS WITH OBSERVATIONS*

The analysis of the early star formation history (SFH) of nearby galaxies, obtained from their resolved stellar populations, is relevant as a test for cosmological models. However, the early time resolution of observationally derived SFHs is limited by several factors. Thus, direct comparison of observationally derived SFHs with those derived from theoretical models of galaxy formation is potentially biased. Here we investigate and quantify this effect. For this purpose, we analyze the duration of the early star formation activity in a sample of four Local Group dwarf galaxies and test whether they are consistent with being true fossils of the pre-reionization era; i.e., if the quenching of their star formation occurred before cosmic reionization by UV photons was completed. Two classical dSph (Cetus and Tucana) and two dTrans (LGS-3 and Phoenix) isolated galaxies with total stellar masses between 1.3 x 106 and 7.2 x 106 M☉ have been studied. Accounting for time resolution effects, the SFHs peak as much as 1.25 Gyr earlier than the optimal solutions. Thus, this effect is important for a proper comparison of model and observed SFHs. It is also shown that none of the analyzed galaxies can be considered a true fossil of the pre-reionization era, although it is possible that the outer regions of Cetus and Tucana are consistent with quenching by reionization.

The proteolysis adaptor, NblA, binds to the N-terminus of β-phycocyanin: Implications for the mechanism of phycobilisome degradation

Phycobilisome (PBS) complexes are massive light-harvesting apparati in cyanobacteria that capture and funnel light energy to the photosystem. PBS complexes are dynamically degraded during nutrient deprivation, which causes severe chlorosis, and resynthesized during nutrient repletion. PBS degradation occurs rapidly after nutrient step down, and is specifically triggered by non-bleaching protein A (NblA), a small proteolysis adaptor that facilitates interactions between a Clp chaperone and phycobiliproteins. Little is known about the mode of action of NblA during PBS degradation. In this study, we used chemical cross-linking coupled with LC-MS/MS to investigate the interactions between NblA and phycobiliproteins. An isotopically coded BS3 cross-linker captured a protein interaction between NblA and β-phycocyanin (PC). LC-MS/MS analysis identified the amino acid residues participating in the binding reaction, and demonstrated that K52 in NblA is cross-linked to T2 in β-PC. These results were modeled onto the existing crystal structures of NblA and PC by protein docking simulations. Our data indicate that the C-terminus of NblA fits in an open groove of β-PC, a region located inside the central hollow cavity of a PC rod. NblA may mediate PBS degradation by disrupting the structural integrity of the PC rod from within the rod. In addition, M1-K44 and M1-K52 cross-links between the N-terminus of NblA and the C-terminus of NblA are consistent with the NblA crystal structure, confirming that the purified NblA is structurally and biologically relevant. These findings provide direct evidence that NblA physically interacts with β-PC.

Multiple copies of the PsbQ protein in a cyanobacterial photosystem II assembly intermediate complex

Photosystem II (PSII) undergoes frequent damage owing to the demanding electron transfer chemistry it performs. To sustain photosynthetic activity, damaged PSII undergoes a complex repair cycle consisting of many transient intermediate complexes. By purifying PSII from the cyanobacterium Synechocystis sp. PCC 6803 using a histidine-tag on the PsbQ protein, a lumenal extrinsic subunit, a novel PSII assembly intermediate was isolated in addition to the mature PSII complex. This new complex, which we refer to as PSII-Q4, contained four copies of the PsbQ protein per PSII monomer, instead of the expected one copy. In addition, PSII-Q4 lacked two other lumenal extrinsic proteins, PsbU and PsbV, which are present in the mature PSII complex. We suggest that PSII-Q4 is a late PSII assembly intermediate that is formed just before the binding of PsbU and PsbV, and we incorporate these results into an updated model of PSII assembly.

Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b559 in Photosystem II

Significance Photosystem II (PSII) is a multisubunit membrane protein complex that catalyzes light-driven oxidation of water, supplying energy to nearly all life on Earth. Because of the challenging chemistry it performs, PSII undergoes frequent damage, prompting an intricate cycle of disassembly, repair, and reassembly. Psb28, a protein that binds transiently to the RC47 intermediate complex, protects PSII from light-induced damage. The location of Psb28 in PSII has not been determined. In this study, we used chemical cross-linking, mass spectrometry, and protein docking to demonstrate that Psb28 binds to cytochrome b559. Our results allow us to propose a mechanism by which Psb28 exerts its protective effect on the vulnerable RC47 complex. Photosystem II (PSII), a large pigment protein complex, undergoes rapid turnover under natural conditions. During assembly of PSII, oxidative damage to vulnerable assembly intermediate complexes must be prevented. Psb28, the only cytoplasmic extrinsic protein in PSII, protects the RC47 assembly intermediate of PSII and assists its efficient conversion into functional PSII. Its role is particularly important under stress conditions when PSII damage occurs frequently. Psb28 is not found, however, in any PSII crystal structure, and its structural location has remained unknown. In this study, we used chemical cross-linking combined with mass spectrometry to capture the transient interaction of Psb28 with PSII. We detected three cross-links between Psb28 and the α- and β-subunits of cytochrome b559, an essential component of the PSII reaction-center complex. These distance restraints enable us to position Psb28 on the cytosolic surface of PSII directly above cytochrome b559, in close proximity to the QB site. Protein–protein docking results also support Psb28 binding in this region. Determination of the Psb28 binding site and other biochemical evidence allow us to propose a mechanism by which Psb28 exerts its protective effect on the RC47 intermediate. This study also shows that isotope-encoded cross-linking with the “mass tags” selection criteria allows confident identification of more cross-linked peptides in PSII than has been previously reported. This approach thus holds promise to identify other transient protein–protein interactions in membrane protein complexes.

Reactive oxygen species leave a damage trail that reveals water channels in Photosystem II

This study illuminates the locations of water channels within Photosystem II, a large membrane protein complex. Photosystem II (PSII), a unique membrane-bound oxidoreductase, catalyzes light-driven oxidation of water to molecular oxygen. Although high-resolution structures of PSII are known, the exact path of the substrate water molecules to the catalytic Mn4CaO5 center within the PSII complex remains poorly understood. PSII produces reactive oxygen species (ROS), responsible for the frequent damage and turnover of this megacomplex that occur under physiological conditions. Such ROS are known to specifically modify PSII proteins. Using high-resolution tandem mass spectrometry, we identified oxidative modifications on 36 amino acid residues on the lumenal side of PSII, in the core PSII proteins D1, D2, and CP43 of the cyanobacterium Synechocystis sp. PCC 6803. Remarkably, these oxidized residues clustered into three nearly continuous formations, tracking the pathways of ROS diffusion from the manganese center all the way out to the surface of PSII. We suggest that these profiles of oxidized residues reveal the locations of water channels within PSII. Our results provide the most comprehensive experimental evidence to date of physiologically relevant oxidized residues in PSII and illuminate three possible channels for water between the catalytic Mn cluster in the PSII complex and the bulk medium around it.

The Use of Advanced Mass Spectrometry to Dissect the Life-Cycle of Photosystem II

Photosystem II (PSII) is a photosynthetic membrane-protein complex that undergoes an intricate, tightly regulated cycle of assembly, damage, and repair. The available crystal structures of cyanobacterial PSII are an essential foundation for understanding PSII function, but nonetheless provide a snapshot only of the active complex. To study aspects of the entire PSII life-cycle, mass spectrometry (MS) has emerged as a powerful tool that can be used in conjunction with biochemical techniques. In this article, we present the MS-based approaches that are used to study PSII composition, dynamics, and structure, and review the information about the PSII life-cycle that has been gained by these methods. This information includes the composition of PSII subcomplexes, discovery of accessory PSII proteins, identification of post-translational modifications and quantification of their changes under various conditions, determination of the binding site of proteins not observed in PSII crystal structures, conformational changes that underlie PSII functions, and identification of water and oxygen channels within PSII. We conclude with an outlook for the opportunity of future MS contributions to PSII research.