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Leghemoglobins enable the endosymbiotic fixation of molecular nitrogen (N2) in legume nodules by channeling O2 for bacterial respiration while maintaining a micro-oxic environment to protect O2-sensitive nitrogenase. We found that the NIN-like protein (NLP) transcription factors NLP2 and NIN directly activate the expression of leghemoglobins through a promoter motif, resembling a “double” version of the nitrate-responsive elements (NREs) targeted by other NLPs, that has conserved orientation and position across legumes. CRISPR knockout of the NRE-like element resulted in strongly decreased expression of the associated leghemoglobin. Our findings indicate that the origins of the NLP-leghemoglobin module for O2 buffering in nodules can be traced to an ancient pairing of NLPs with nonsymbiotic hemoglobins that function in hypoxia.

Objective Ferroptosis plays an important role in traumatic brain injury (TBI). The p53 protein is a major mediator of ferroptosis. However, the role of p53-mediated ferroptosis in TBI has not been studied. Sirtuin 2 (SIRT2) exerts a protective effects role in TBI, although the underlying mechanism of this protection remains unclear. In the present study, we tested the hypothesis that that SIRT2 mitigates TBI by regulating p53-mediated ferroptosis. Methods and results To model TBI in mice, we used the controlled cortical impact (CCI) injury method. We found that ferroptosis was significantly activated by CCI, and peaked 3 days following CCI, as evidenced by upregulation of GPX4 and SLC7A11, increased content of decreases glutathione, lipid peroxidation, malondialdehyde and ferrous ion. Inhibition of ferroptosis significantly alleviated neurological indications and brain edema. In addition, knockout of p53 significantly blocked ferroptosis following CCI. Furthermore, we found that inhibition of SIRT2 upregulated the acetylation of p53, as well as p53 expression, and exacerbated ferroptosis following CCI. Interestingly, knockout of p53 rescued the SIRT2 inhibition-induced exacerbation of ferroptosis. Conclusions These findings indicate that p53-mediated ferroptosis contributes to the pathogenesis of TBI. Furthermore, we demonstrate that SIRT2 exerts a neuroprotective effect against TBI by suppressing p53-mediated ferroptosis.

AimTo investigate the prevalence and factors associated with optic disc grey crescent (GC) in African Americans with glaucoma.MethodsStereo optic disc image features from subjects with glaucoma in the Primary Open-Angle African Ancestry Glaucoma Genetics Study were evaluated independently by non-physician graders and discrepancies adjudicated by an ophthalmologist. Risk factors for GC were evaluated by logistic regression models with intereye correlation accounted for by generalised estimating equations. Adjusted ORs (aORs) were generated.ResultsGC was present in 227 (15%) of 1491 glaucoma cases, with 57 (3.82%) bilateral and 170 (11.4%) unilateral. In multivariable analysis, factors associated with GC were younger age (aOR 1.27, 95% CI 1.11 to 1.43 for every decade younger in age, p=0.001), diabetes (aOR 1.46, 95% CI 1.09 to 1.96, p=0.01), optic disc tilt (aOR 1.84, 95% CI 1.36 to 2.48, pConclusionsMore than 1 in 10 glaucoma cases with African ancestry have GC, occurring more frequently in younger subjects, higher degrees of African ancestry and diabetes. GC was associated with several ocular features, including optic disc tilt and beta peripapillary atrophy. These associations should be considered when evaluating black patients with primary open-angle glaucoma.

Legumes, unlike other plants, have the ability to establish symbiosis with nitrogen-fixing rhizobia. It has been theorized that a unique property of legume root cortical cells enabled the initial establishment of rhizobial symbiosis1–3. Here we show that a SHORTROOT–SCARECROW (SHR–SCR) stem cell program in cortical cells of the legume Medicago truncatula specifies their distinct fate. Regulatory elements drive the cortical expression of SCR, and stele-expressed SHR protein accumulates in cortical cells of M. truncatula but not Arabidopsis thaliana. The cortical SHR–SCR network is conserved across legume species, responds to rhizobial signals, and initiates legume-specific cortical cell division for de novo nodule organogenesis and accommodation of rhizobia. Ectopic activation of SHR and SCR in legumes is sufficient to induce root cortical cell division. Our work suggests that acquisition of the cortical SHR–SCR module enabled cell division coupled to rhizobial infection in legumes. We propose that this event was central to the evolution of rhizobial endosymbiosis. Repurposing of an SHR–SCR stem cell program in the legume root cortex enables rhizobial symbiosis.

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0, resulting from decreased activity of 3-ketoacyl-ACP synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains >40% high-melting-point molecular species (HMP-PG; molecules that contain only 16:0, 16:1-trans, and 18:0 fatty acids)-a trait associated with chilling-sensitive plants-compared with

Recently, several studies have reported that the pharmacological effects exerted by cannabidiol (CBD) are partially related to the regulation of autophagy. Increasing evidence indicates that autophagy provides protection against ischemia-induced brain injury. However, the protective effect of CBD against mitochondrial-dependent apoptosis in hemorrhagic shock (HS)-induced brain injury has not been studied. In the present study, we observed the protective effects of CBD against neural mitochondrial-dependent apoptosis in a rat model of HS. In addition, CBD increased Beclin-1 and LC3II expression and reduced P62 expression, which were indicative of autophagy. CBD treatment attenuated the neural apoptosis induced by HS, as reflected by restoring mitochondrial dysfunction, downregulation of BAX, neuro-apoptosis ratio and NF-κB signaling activation, and upregulation of BCL2 in the cerebral cortex. Such protective effects were reversed by 3-Methyladenine, a specific autophagy inhibitor, indicating that the protective effects of CBD treatment involved autophagy. LY294002, a PI3K inhibitor, significantly inhibited CBD-induced autophagy, demonstrating that PI3K/AKT signaling is involved in the CBD's regulation of autophagy. Furthermore, we found that CBD treatment upregulated PI3K/AKT signaling via cannabinoid receptor 1. Therefore, these findings suggested that CBD treatment protects against cerebral injury induced by HS-mediated mitochondrial-dependent apoptosis by activating the PI3K/AKT signaling pathway to reinforce autophagy.

Purification and collection of industrial products from oil-water mixtures are commonly implemented processes. However, the efficiencies of such processes can be severely influenced by the presence of emulsifiers that induce the formation of small oil droplets dispersed in the mixtures. Understanding of this emulsifying effect and its counteractions which occur at the oil/water interface is therefore necessary for the improvement of designs of these processes. In this paper, we investigated the interfacial mechanisms of protein-induced emulsification and the opposing surfactant-induced demulsification related to corn oil refinement. At corn oil/water interfaces, the pH-dependent emulsifying function of zein protein, which is the major storage protein of corn, was elucidated by the surface/interface-sensitive sum frequency generation (SFG) vibrational spectroscopy technique. The effective stabilization of corn oil droplets by zein protein was illustrated and correlated to its ordered amide I group at the oil/water interface. Substantial decrease of this ordering with the addition of three industrial surfactants to corn oil-zein solution mixtures was also observed using SFG, which explains the surfactant-induced destabilization and coalescence of small oil droplets. Surfactant-protein interaction was then demonstrated to be the driving force for the disordering of interfacial proteins, either by disrupting protein layers or partially excluding protein molecules from the interface. The ordered zein proteins at the interface were therefore revealed to be the critical factor for the formation of corn oil-water emulsion.

Thermophilic fungi are eukaryotic species that grow at high temperatures, but little is known about the underlying basis of thermophily at cell and molecular levels. Here the proteome and N-glycoproteome of Chaetomium thermophilum at varying culture temperatures (30, 50, and 55°C) were studied using hydrophilic interaction liquid chromatography enrichment and high-resolution liquid chromatography–tandem mass spectroscopy analysis. With respect to the proteome, the numbers of differentially expressed proteins were 1,274, 1,374, and 1,063 in T50/T30, T55/T30, and T55/T50, respectively. The upregulated proteins were involved in biological processes, such as protein folding and carbohydrate metabolism. Most downregulated proteins were involved in molecular functions, including structural constituents of the ribosome and other protein complexes. For the N-glycoproteome, the numbers of differentially expressed N-glycoproteins were 160, 176, and 128 in T50/T30, T55/T30, and T55/T50, respectively. The differential glycoproteins were mainly involved in various types of N-glycan biosynthesis, mRNA surveillance pathway, and protein processing in the endoplasmic reticulum. These results indicated that an efficient protein homeostasis pathway plays an essential role in the thermophily of C. thermophilum, and N-glycosylation is involved by affecting related proteins. This is the novel study to reveal thermophilic fungi’s physiological response to high-temperature adaptation using omics analysis, facilitating the exploration of the thermophily mechanism of thermophilic fungi.

The formation of nitrogen-fixing nodules on legume roots requires the coordination of infection by rhizobia at the root epidermis with the initiation of cell divisions in the root cortex. During infection, rhizobia attach to the tip of elongating root hairs which then curl to entrap the rhizobia. However, the mechanism of root hair deformation and curling in response to symbiotic signals is still elusive. Here, we found that small GTPases (MtRac1/MtROP9 and its homologs) are required for root hair development and rhizobial infection in Medicago truncatula. Our results show that the Nod factor receptor LYK3 phosphorylates the guanine nucleotide exchange factor MtRopGEF2 at S73 which is critical for the polar growth of root hairs. In turn, phosphorylated MtRopGEF2 can activate MtRac1. Activated MtRac1 was found to localize at the tips of root hairs and to strongly interact with LYK3 and NFP. Taken together, our results support the hypothesis that MtRac1, LYK3, and NFP form a polarly localized receptor complex that regulates root hair deformation during rhizobial infection. This article is protected by copyright. All rights reserved.

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0, resulting from decreased activity of 3-ketoacyl-ACP synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains >40% high-melting-point molecular species (HMP-PG; molecules that contain only 16:0, 16:1-trans, and 18:0 fatty acids)—a trait associated with chilling-sensitive plants—compared with

This article focuses on the problem of near-far effects in pseudo-satellite ground-based navigation enhancement systems. First, the article analyzes the near-far effects from the perspective of signal power and from the perspective of correlation values, and briefly introduces several methods to suppress the near-far effects and their applicability. Then introduced the pseudo-code blind estimation algorithm against impact noise. In this paper, fractional low-order statistics combined with M-estimation enhanced projection approximation subspace tracking pseudocode sequence blind estimation algorithm is used to blindly estimate the pseudo-code sequence and simulate the anti-noise effect. The estimation results show that the impact component is basically eliminated and the estimation effect of the pseudo code sequence is very good, which shows that the algorithm has anti-impact noise characteristics and is suitable for high-performance pseudo code information estimation under the condition of low signal-to-noise ratio.

The oxygen reduction reaction (ORR) is of significant importance in the development of fuel cells. Now, cobalt-nitrogen-doped chiral carbonaceous nanotubes (l/d-CCNTs-Co) are presented as efficient electrocatalysts for ORR. The chiral template, N-stearyl-l/d-glutamic acid, induces the self-assembly of well-arranged polypyrrole and the formation of ordered graphene carbon with helical structures at the molecular level after the pyrolysis process. Co was subsequently introduced through the post-synthesis method. The obtained l/d-CCNTs-Co exhibits superior ORR performance, including long-term stability and better methanol tolerance compared to achiral Co-doped carbon materials and commercial Pt/C. DFT calculations demonstrate that the charges on the twisted surface of l/d-CCNTs are widely separated; as a result the Co atoms are more exposed on the chiral CCNTs. This work gives us a new understanding of the effects of helical structures in electrocatalysis.

The establishment of the symbiotic interaction between rhizobia and legumes involves the Nod factor signaling pathway. Nod factor recognition occurs through two plant receptors, NFR1 and NFR5. However, the signal transduction mechanisms downstream of NFR1-NFR5-mediated Nod factor perception remain largely unknown. Here, we report that a small guanosine triphosphatase (GTPase), GmROP9, and a guanine nucleotide exchange factor, GmGEF2, are involved in the soybean-rhizobium symbiosis. We show that GmNFR1α phosphorylates GmGEF2a at its N-terminal S86, which stimulates guanosine diphosphate (GDP)-to-GTP exchange to activate GmROP9 and that the active form of GmROP9 can associate with both GmNFR1α and GmNFR5α. We further show that a scaffold protein, GmRACK1, interacts with active GmROP9 and contributes to root nodule symbiosis. Collectively, our results highlight the symbiotic role of GmROP9-GmRACK1 and support the hypothesis that rhizobial signals promote the formation of a protein complex comprising GmNFR1, GmNFR5, GmROP9, and GmRACK1 for symbiotic signal transduction in soybean.

Legumes, unlike other plants, have the ability to establish symbiosis with nitrogen-fixing rhizobia. It has been theorized that a unique property of legume root cortical cells enabled the initial establishment of rhizobial symbiosis

Bimetallic sulfides with earth-abundant transition-metal elements are proposed to enhance the electrocatalytic activities. Further replacement of S atom by less electronegative P atom improves the electrocatalytic performance of OER and HER. Herein, hollow bimetallic zinc cobalt phosphosulfides (Zn0.3 Co2.7 S3 P) are synthesized by a two-step process. The optimal catalyst of Zn0.3 Co2.7 S3 P with particle size of 50 nm displays an excellent electroactivity and long-term durability toward efficient overall water splitting process in alkaline medium. The excellent bifunctional electrocatalytic performance may be ascribed to the synergistic effect of hollow structure, anion substitution tuning and unique size control.

Chilling stress is a major factor limiting plant development and crop productivity. Because the plant response to chilling is so complex, we are far from understanding the genes important in the response to chilling. To identify new genes important in chilling tolerance, we conducted a novel mutant screen, combining a confirmed SALK T-DNA insertion collection with traditional forward genetics. We screened a pool of more than 3700 confirmed homozygous SALK T-DNA insertion lines for visible defects under prolonged growth at 5°C. Of the chilling-sensitive mutants we observed, mutations at one locus were characterized in detail. This gene, At1g45231, encodes an Arabidopsis (Arabidopsis thaliana) trimethylguanosine synthase (TGS1), previously uncharacterized in the plant kingdom. We confirmed that Arabidopsis TGS1 is a functional ortholog of other trimethylguanosine synthases based both on its in vitro methyltransferase activity and on its ability to rescue the cold-growth inhibition of a Saccharomyces cerevisiae tgs1Δ mutant in vivo. While tgs1 mutant plants grew normally at 22°C, their vegetative and reproductive growth was severely compromised under chilling conditions. When we transgenically expressed TGS1 in the mutant plants, the chilling-sensitive phenotype was relieved, demonstrating that TGS1 is required for chilling tolerance.

Polar membrane glycerolipids occur in a mixture of molecular species defined by a polar head group and characteristic acyl groups esterified to a glycerol backbone. A molecular species of phosphatidylglycerol specific to chloroplasts of plants carries a Delta(3-trans) hexadecenoic acid in the sn-2 position of its core glyceryl moiety. The fad4-1 mutant of Arabidopsis thaliana missing this particular phosphatidylglycerol molecular species lacks the necessary fatty acid desaturase, or a component thereof. The overwhelming majority of acyl groups associated with membrane lipids in plants contains double bonds with a cis configuration. However, FAD4 is unusual because it is involved in the formation of a trans double bond introduced close to the carboxyl group of palmitic acid, which is specifically esterified to the sn-2 glyceryl carbon of phosphatidylglycerol. As a first step towards the analysis of this unusual desaturase reaction, the FAD4 gene was identified by mapping of the FAD4 locus and coexpression analysis with known lipid genes. FAD4 encodes a predicted integral membrane protein that appears to be unrelated to classic membrane bound fatty acid desaturases based on overall sequence conservation. However, the FAD4 protein contains two histidine motifs resembling those of metalloproteins such as fatty acid desaturases. FAD4 is targeted to the plastid. Overexpression of the cDNA in transgenic Arabidopsis led to increased accumulation of the Delta(3-trans) hexadecanoyl group in phosphatidylglycerol relative to wild type. Taken together these results are consistent with the hypothesis that FAD4 is the founding member of a novel class of fatty acid desaturases.

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0 due to decreased activity of 3-ketoacyl-acyl carrier protein (ACP) synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains up to 45% high-melting-point molecular species (molecules that contain only 16:0, 16:1-trans, and 18:0), a trait associated with chilling-sensitive plants, compared with less than 10% in wild-type Arabidopsis. Although they do not exhibit typical chilling sensitivity, when exposed to low temperatures (2°C–6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. A screen for suppressors of this low-temperature phenotype has identified 11 lines, some of which contain additional alterations in leaf-lipid composition relative to fab1. Here, we report the identification of two suppressor mutations, one in act1, which encodes the chloroplast acyl-ACP:glycerol-3-phosphate acyltransferase, and one in lpat1, which encodes the chloroplast acyl-ACP:lysophosphatidic acid acyltransferase. These enzymes catalyze the first two steps of the prokaryotic pathway for glycerolipid synthesis, so we investigated whether other mutations in this pathway would rescue the fab1 phenotype. Both the gly1 mutation, which reduces glycerol-3-phosphate supply to the prokaryotic pathway, and fad6, which is deficient in the chloroplast 16:1/18:1 fatty acyl desaturase, were discovered to be suppressors. Analyses of leaf-lipid compositions revealed that mutations at all four of the suppressor loci result in reductions in the proportion of high-melting-point molecular species of phosphatidylglycerol relative to fab1. We conclude that these reductions are likely the basis for the suppressor phenotypes.

When manganese stabilizing protein (MSP) was treated with 0.5 mM N-succinimidyl propionate (NSP), the rebinding ability and oxygen-releasing capabilities of the modified MSP were not altered, in spite of changes of MSP surface Lys residues. Furthermore, far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed that 0.5 mM NSP-modified MSP retained most of its native secondary and tertiary structure. Mapping of the sites of NSP modification by Staphylococcus V(8) protease digestion of the modified protein, as well as analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry, indicated that seven Lys residues were modified. The results suggested that these residues are not absolutely essential to the structure and function of MSP. However, when the NSP concentration was increased to 4 mM, the modified MSP was unable to bind photosystem II and completely lost its reactivating capability. Both far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed a clear conformational change in MSP after 4 mM NSP treatment, suggesting that some Lys residues are involved in maintaining the structure and function of MSP. Analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that another six Lys residues, namely Lys20, Lys101, Lys196, Lys207, Lys130 (or Lys137) and Lys66 (or Lys76), were modified by 4 mM NSP. Therefore, these six Lys residues are crucial in maintaining the structure and function of soluble MSP.

Silicon carbide (SiC) is an inorganic material which has non-linear conductivity characteristics. When mixed nano-size SiC into silicone rubber (SIR), the composites can achieve non-linear conductivity characteristics also. In this paper, the non-linear conductivity properties of 5wt%, 15wt%, 30wt%, 45wt% nano-SiC/SIR composites are investigated. The relationship between conductivity and electric field is obtained. In order to know the effect of non-linear SiC/SIR composites on homogenizing electric field, the electric stress distribution of nonlinear insulation cables termination is simulated by finite element method. The results show that the SiC/SIR composites can homogenize the electric field distribution of cable termination effectively. With the increase of non-linear conductivity level, the electric field distribution of cable termination with non-linear SiC/silicone rubber composite is more homogenization.

Members of the ATP-binding cassette (ABC) transporter family are essential proteins in species as diverse as archaea and humans. Their domain architecture has remained relatively fixed across these species, with rare exceptions. Here, we show one exception to be the trigalactosyldiacylglycerol 1, 2, and 3 (TGD1, -2, and -3) putative lipid transporter located at the chloroplast inner envelope membrane. TGD2 was previously shown to be in a complex of >500 kDa. We demonstrate that this complex also contains TGD1 and -3 and is very stable because it cannot be broken down by gentle denaturants to form a "core" complex similar in size to standard ABC transporters. The complex was purified from Pisum sativum (pea) chloroplast envelopes by native gel electrophoresis and examined by mass spectrometry. Identified proteins besides TGD1, -2, or -3 included a potassium efflux antiporter and a TIM17/22/23 family protein, but these were shown to be in separate high molecular mass complexes. Quantification of the complex components explained the size of the complex because 8-12 copies of the substrate-binding protein (TGD2) were found per functional transporter.

Vascular plants use two pathways to synthesize galactolipids, the predominant lipid species in chloroplasts—a prokaryotic pathway that resides entirely in the chloroplast, and a eukaryotic pathway that involves assembly in the endoplasmic reticulum. Mutants deficient in the endoplasmic reticulum pathway, trigalactosyldiacylglycerol (tgd1-1 and tgd2-1) mutants, had been previously identified with reduced contents of monogalactosyldiacylglycerol and digalactosyldiacylglycerol, and altered lipid molecular species composition. Here, we report that the altered lipid composition affected photosynthesis in lipid trafficking mutants. It was found that proton motive force as measured by electrochromic shift was reduced by ~40 % in both tgd mutants. This effect was accompanied by an increase in thylakoid conductance attributable to ATPase activity and so the rate of ATP synthesis was nearly unchanged. Thylakoid conductance to ions also increased in tgd mutants. However, gross carbon assimilation in tgd mutants as measured by gas exchange was only marginally affected. Rubisco activity, electron transport rate, and photosystem I and II oxidation status were not altered. Despite the large differences in proton motive force, responses to heat and high light stress were similar between tgd mutants and the wild type.

In most plants the assembly of the photosynthetic thylakoid membrane requires lipid precursors synthesized at the endoplasmic reticulum (ER). Thus, the transport of lipids from the ER to the chloroplast is essential for biogenesis of the thylakoids. TGD2 is one of four proteins in Arabidopsis required for lipid import into the chloroplast, and was found to bind phosphatidic acid in vitro. However, the significance of phosphatidic acid binding for the function of TGD2 in vivo and TGD2 interaction with membranes remained unclear. Developing three functional assays probing how TGD2 affects lipid bilayers in vitro, we show that it perturbs membranes to the point of fusion, causes liposome leakage and redistributes lipids in the bilayer. By identifying and characterizing five new mutant alleles, we demonstrate that these functions are impaired in specific mutants with lipid phenotypes in vivo. At the structural level, we show that TGD2 is part of a protein complex larger than 500 kDa, the formation of which is disrupted in two mutant alleles, indicative of the biological relevance of this TGD2-containing complex. Based on the data presented, we propose that TGD2, as part of a larger complex, forms a lipid transport conduit between the inner and outer chloroplast envelope membranes, with its N terminus anchored in the inner membrane and its C terminus binding phosphatidic acid in the outer membrane.

The 1, 4, and 8 tyrosine (Tyr) residues on the PSII extrinsic 23 kDa protein were modified with 5, 10 or 40 mM N-acetylimidazole (NAI) respectively. The amount of rebound NAI-modified extrinsic 23 kDa protein was 98%, 80%, and 5% of that in the unmodified protein, respectively. These results indicate that the Tyr residues are absolutely essential to reconstitution ability. Further, the fluorescence and circular dichroism spectra among native and NAI-modified extrinsic 23 kDa proteins were similar, suggesting that the modification by NAI did not markedly influence the basic secondary structure of the native conformation. Thus, we have concluded that the tyrosine residues in the extrinsic 23 kDa protein are important for interaction with PSII membranes. In addition, we found that the structure of the extrinsic 23 kDa protein is stable in suspension (pH 4-9 or Tm 25-55 degrees C).

To determine the contribution of charged amino acids to binding with the photosystem II complex (PSII), the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N- succinimidyl propionate (NSP) or glycine methyl ester (GME) in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4−10 and 10−14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3−4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.

To study its contribution to the assembly of the green plant manganese stabilizing protein (MSP) into photosystem II (PSII), tyrosine residues were specifically acetylated using N-acetylimidazole (NAI). In soluble MSP, three groups of Tyr residues could be differentiated by NAI acetylation: approximately 5 (actually approximately 5.2) Tyr residues could be easily acetylated (superficial), 1-2 Tyr residues could be acetylated when the NAI concentration was sufficiently high (superficially buried), and 1-2 Tyr residues could only be acetylated in the presence of the denaturant, urea (deeply buried). Acetylation of the 5.2 Tyr residues did not affect the reconstitution or oxygen-evolving activities of the MSP, and far-UV circular dichroism (CD) analysis showed that the altered MSP retained most of its native secondary structure. These results suggested that the 5.2 Tyr residues are not absolutely essential to the function of MSP. However, further modification of the 1-2 superficially buried Tyr residues (for a total acetylation of approximately 6.4 Tyr residues) completely abrogated the MSP rebinding and oxygen evolution activities. Finally, at least one tyrosine residue was inaccessible to NAI until MSP was completely unfolded by 8 M urea. Deacetylation of MSP with 6.4 or 8 acetylated Tyr residues with hydroxylamine restored most of the rebinding and oxygen-evolving activities. A prominent red shift in fluorescence spectra of MSP (excited at 280 or 295 nm) was observed after modification of 6.4 Tyr residues, and a further shift could be found after all 8 Tyr residues were modified, indicating a great loss of native secondary structure. Far-UV CD revealed that MSP was mostly unfolded when 6.4 Tyr residues were modified and completely unfolded when all 8 Tyr residues were modified. Fluorescence and far-UV CD studies revealed that loss of MSP rebinding to PSII membranes following NAI modification correlated well with conformational changes in MSP. Together, these results indicate that different tyrosine residues have different contributions to the binding and assembly of MSP into PSII.