Orientation and nearest neighbor analysis of psbI gene product in the photosystem II reaction center complex using bifunctional cross-linkers

AbstractTwo distinct cross-linked products containing psbI gene product were generated in the photosystem II reaction center complex from spinach by treatment with bifunctional reagents directed to amino groups. The first product, which was generated by a 3,3'-dithiobis(succinimidyl propionate) treatment, is deduced to be formed between the ϵ-amino group of Lys3 of the psbI gene product, and a side-chain amino group present on the stromal extension of the D2 protein. The CNBr cleavage analysis of the cross-linked product predicted that the amino group of the D2 protein engaged in the cross-linking is either one of the three lysine residues on the N-terminal fragment (from N-terminus to Met19) or the Lys268 on the 6th fragment (from Val248 to Met275). The second product, which was also generated on the stromal side by a 1,6-hexamethylene diisocyanate treatment preferentially in alkaline conditions, is predicted to be formed between the ϵ-amino group of Lys3 of the psbI gene product and the N-terminal α-amino group of the α-subunit of cytochrome b559

Purification of ferredoxins and their reaction with purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Four ferredoxin (Fd) fractions, namely, FdA–D were purified from the green sulfur bacterium Chlorobium tepidum. Their absorption spectra are typical of 2[4Fe–4S] cluster type Fds with peaks at about 385 and 280 nm and a shoulder at about 305 nm. The A385/A280 ratios of the purified Fds were 0.76–0.80. Analysis of the N-terminal amino acid sequences of these Fds (15–25 residues) revealed that those of FdA and FdB completely agree with those deduced from the genes, fdx3 and fdx2, respectively, found in this bacterium (Chung and Bryant, personal communication). The N-terminal amino acid sequences of FdC and FdD (15 residues) were identical, and agree with that deduced from the gene fdx1 (Chung and Bryant, personal communication). The A385 values of these Fds were unchanged when they were stored for a month at −80°C under aerobic conditions and decreased by 10–15% when they were stored for 6 days at 4°C under aerobic conditions, indicating that they are not extremely unstable. In the presence of Fd-NADP+ reductase from spinach, and a purified reaction center (RC) preparation from C. tepidum composed of five kinds of polypeptides, these Fds supported the photoreduction of NADP+ at room temperature with the following Km and Vmax (in μmol NADP+ μmol BChl a−1 h−1): FdA, 2.0 μM and 258; FdB, 0.49 μM and 304; FdC, 1.13 μM and 226; FdD, 0.5 μM and 242; spinach Fd, 0.54 μM and 183. The Vmax value of FdB was more than twice that previously reported for purified RC preparations from green sulfur bacteria

Crosslinking between the 33 kDa extrinsic protein and the 47 kDa chlorophyll-carrying protein of the PS II reaction center core complex

AbstractUsing highly purified oxygen-evolving photosystem II complexes from spinach, the 33 kDa extrinsic protein was found to crosslink with the 47 kDa chlorophyll-carrying protein with a cleavable bifunctional crosslinking reagent, dithiobis(succinimidylpropionate)

Binding and Functional Properties of Five Extrinsic Proteins in Oxygen-evolving Photosystem II from a Marine Centric Diatom, Chaetoceros gracilis*

Oxygen-evolving photosystem II (PSII) isolated from a marine centric diatom, Chaetoceros gracilis, contains a novel extrinsic protein (Psb31) in addition to four red algal type extrinsic proteins of PsbO, PsbQ′, PsbV, and PsbU. In this study, the five extrinsic proteins were purified from alkaline Tris extracts of the diatom PSII by anion and cation exchange chromatographic columns at different pH values. Reconstitution experiments in various combinations with the purified extrinsic proteins showed that PsbO, PsbQ′, and Psb31 rebound directly to PSII in the absence of other extrinsic proteins, indicating that these extrinsic proteins have their own binding sites in PSII intrinsic proteins. On the other hand, PsbV and PsbU scarcely rebound to PSII alone, and their effective bindings required the presence of all of the other extrinsic proteins. Interestingly, PSII reconstituted with Psb31 alone considerably restored the oxygen evolving activity in the absence of PsbO, indicating that Psb31 serves as a substitute in part for PsbO in supporting oxygen evolution. A significant difference found between PSIIs reconstituted with Psb31 and with PsbO is that the oxygen evolving activity of the former is scarcely stimulated by Cl− and Ca2+ ions but that of the latter is largely stimulated by these ions, although rebinding of PsbV and PsbU activated oxygen evolution in the absence of Cl− and Ca2+ ions in both the former and latter PSIIs. Based on these results, we proposed a model for the association of the five extrinsic proteins with intrinsic proteins in diatom PSII and compared it with those in PSIIs from the other organisms

Induced pluripotent stem cells derived from a patient with familial idiopathic basal ganglia calcification (IBGC) caused by a mutation in SLC20A2 gene

Idiopathic basal ganglia calcification (IBGC), also known as Fahr disease or primary familial brain calcifications (PFBC), is a rare neurodegenerative disorder characterized by calcium deposits in basal ganglia and other brain regions, causing neuropsychiatric and motor symptoms. We established human induced pluripotent stem cells (iPSCs) from an IBGC patient. The established IBGC-iPSCs carried SLC20A2 c.1848G > A mutation (p.W616* of translated protein PiT2), and also showed typical iPSC morphology, pluripotency markers, normal karyotype, and the ability of in vitro differentiation into three-germ layers. The iPSC line will be useful for further elucidating the pathomechanism and/or drug development for IBGC