Ligand Presentation Inside Protein Crystal Nanopores: Tunable Interfacial Adhesion Noncovalently Modulates Cell Attachment.

Protein crystals with sufficiently large solvent pores can non-covalently adsorb polymers in the pores. In principle, if these polymers contain cell adhesion ligands, the polymer-laden crystals could present ligands to cells with tunable adhesion strength. Moreover, porous protein crystals can store an internal ligand reservoir, so that the surface can be replenished. In this study, we demonstrate that poly(ethylene glycol) terminated with a cyclic cell adhesion ligand peptide (PEG-RGD) can be loaded into porous protein crystals by diffusion. Through atomic force microscopy (AFM), force-distance correlations of the mechanical interactions between activated AFM tips and protein crystals were precisely measured. The activation of AFM tips allows the tips to interact with PEG-RGD that was pre-loaded in the protein crystal nanopores, mimicking how a cell might attach to and pull on the ligand through integrin receptors. The AFM experiments also simultaneously reveal the detailed morphology of the buffer-immersed nanoporous protein crystal surface. We also show that porous protein crystals (without and with loaded PEG-RGD) serve as suitable substrates for attachment and spreading of adipose-derived stem cells. This strategy can be used to design surfaces that non-covalently present multiple different ligands to cells with tunable adhesive strength for each ligand, and with an internal reservoir to replenish the precisely defined crystalline surface.

Site-directed integration of exogenous DNA into the soybean genome by LbCas12a fused to a plant viral HUH endonuclease.

High efficiency site-directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach to mitigate this problem is to increase the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carries their recognition motifs. When fused to a CRISPR-associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis by using chimeric proteins between LbCas12a as a CRISPR-associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean (Glycine max). Two fusion protein configurations were tested to integrate a 70-nt oligonucleotide donor into a commercially important target site using protoplasts and in planta transformation. Site-directed integration rates of the donor DNA, when tethered to the fusion protein, reached about 26% in plants and were up to four-fold higher than in untethered controls. Integrations via canonical homology-directed repair or non-homologous end joining were promoted by tethering in a similar fashion. This study is the first demonstration of HUH EN-associated tethering to improve site-directed DNA integration in plants.

Measuring interactions of DNA with nanoporous protein crystals by atomic force microscopy.

Crosslinked porous protein crystals are a new biomaterial that can be engineered to encapsulate, stabilize, and organize guest molecules, nanoparticles, and biological moieties. In this study, for the first time, the combined interactions of DNA strands with porous protein crystals are quantitatively measured by high-resolution atomic force microscopy (AFM) and chemical force microscopy. The surface structure of protein crystals with unusually large pores was observed in liquid via high-resolution AFM. Force-distance (F-D) curves were also obtained using AFM tips modified to present or capture DNA. The modification of AFM tips allowed the tips to covalently bind DNA that was pre-loaded in the protein crystal nanopores. The modified tips enabled the interactions of DNA molecules with protein crystals to be quantitatively studied while revealing the morphology of the buffer-immersed protein crystal surface in detail, thereby preserving the structure and properties of protein crystals that could be disrupted or destroyed by drying. The hexagonal space group was manifest at the crystal surface, as were the strong interactions between DNA and the porous protein crystals in question. In sum, this study furthered our understanding of how a new protein-based biomaterial can be used to bind guest DNA assemblies.

Sequence-based Prediction of Protein-Protein Interactions Using Gray Wolf Optimizer-Based Relevance Vector Machine.

Protein-protein interactions (PPIs) are essential to a number of biological processes. The PPIs generated by biological experiment are both time-consuming and expensive. Therefore, many computational methods have been proposed to identify PPIs. However, most of these methods are limited as they are difficult to compute and rely on a large number of homologous proteins. Accordingly, it is urgent to develop effective computational methods to detect PPIs using only protein sequence information. The kernel parameter of relevance vector machine (RVM) is set by experience, which may not obtain the optimal solution, affecting the prediction performance of RVM. In this work, we presented a novel computational approach called GWORVM-BIG, which used Bi-gram (BIG) to represent protein sequences on a position-specific scoring matrix (PSSM) and GWORVM classifier to perform classification for predicting PPIs. More specifically, the proposed GWORVM model can obtain the optimum solution of kernel parameters using gray wolf optimizer approach, which has the advantages of less control parameters, strong global optimization ability, and ease of implementation compared with other optimization algorithms. The experimental results on yeast and human data sets demonstrated the good accuracy and efficiency of the proposed GWORVM-BIG method. The results showed that the proposed GWORVM classifier can significantly improve the prediction performance compared with the RVM model using other optimizer algorithms including grid search (GS), genetic algorithm (GA), and particle swarm optimization (PSO). In addition, the proposed method is also compared with other existing algorithms, and the experimental results further indicated that the proposed GWORVM-BIG model yields excellent prediction performance. For facilitating extensive studies for future proteomics research, the GWORVMBIG server is freely available for academic use at http://219.219.62.123:8888/GWORVMBIG.

Evaluation of glyphosate resistance in Arabidopsis thaliana expressing an altered target site EPSPS.

BACKGROUND: Glyphosate-resistant goosegrass has recently evolved and is homozygous for the double mutant of EPSPS (T102 I, P106 S or TIPS). These same mutations combined with EPSPS overexpression, have been used to create transgenic glyphosate-resistant crops. Arabidopsis thaliana (Wt EPSPS Ki ∼ 0.5 µM) was engineered to express a variant AtEPSPS-T102 I, P106 A (TIPA Ki = 150 µM) to determine the resistance magnitude for a more potent variant EPSPS that might evolve in weeds. RESULTS: Transgenic A. thaliana plants, homozygous for one, two or four copies of AtEPSPS-TIPA, had resistance (IC50 values, R/S) as measured by seed production ranging from 4.3- to 16-fold. Plants treated in reproductive stage were male sterile with a range of R/S from 10.1- to 40.6-fold. A significant hormesis (∼ 63% gain in fresh weight) was observed for all genotypes when treated at the initiation of reproductive stage with 0.013 kg ha-1 . AtEPSPS-TIPA enzyme activity was proportional to copy number and correlated with resistance magnitude. CONCLUSIONS: A. thaliana, as a model weed expressing one copy of AtEPSPS-TIPA (300-fold more resistant), had only 4.3-fold resistance to glyphosate for seed production. Resistance behaved as a single dominant allele. Vegetative tissue resistance was 4.7-fold greater than reproductive tissue resistance and was linear with gene copy number. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Glyphosate resistance in Ambrosia trifida: Part 1. Novel rapid cell death response to glyphosate.

BACKGROUND: Glyphosate-resistant (GR) Ambrosia trifida is now present in the midwestern United States and in southwestern Ontario, Canada. Two distinct GR phenotypes are known, including a rapid response (GR RR) phenotype, which exhibits cell death within hours after treatment, and a non-rapid response (GR NRR) phenotype. The mechanisms of resistance in both GR RR and GR NRR remain unknown. Here, we present a description of the RR phenotype and an investigation of target-site mechanisms on multiple A. trifida accessions. RESULTS: Glyphosate resistance was confirmed in several accessions, and whole-plant levels of resistance ranged from 2.3- to 7.5-fold compared with glyphosate-susceptible (GS) accessions. The two GR phenotypes displayed similar levels of resistance, despite having dramatically different phenotypic responses to glyphosate. Glyphosate resistance was not associated with mutations in EPSPS sequence, increased EPSPS copy number, EPSPS quantity, or EPSPS activity. CONCLUSION: These encompassing results suggest that resistance to glyphosate in these GR RR A. trifida accessions is not conferred by a target-site resistance mechanism. © 2017 Society of Chemical Industry.

Glyphosate resistance in Ambrosia trifida: Part 2. Rapid response physiology and non-target-site resistance.

BACKGROUND: The glyphosate-resistant rapid response (GR RR) resistance mechanism in Ambrosia trifida is not due to target-site resistance (TSR) mechanisms. This study explores the physiology of the rapid response and the possibility of reduced translocation and vacuolar sequestration as non-target-site resistance (NTSR) mechanisms. RESULTS: GR RR leaf discs accumulated hydrogen peroxide within minutes of glyphosate exposure, but only in mature leaf tissue. The rapid response required energy either as light or exogenous sucrose. The combination of phenylalanine and tyrosine inhibited the rapid response in a dose-dependent manner. Reduced glyphosate translocation was observed in GR RR, but only when associated with tissue death caused by the rapid response. Nuclear magnetic resonance studies indicated that glyphosate enters the cytoplasm and reaches chloroplasts, and it is not moved into the vacuole of GR RR, GR non-rapid response or glyphosate-susceptible A. trifida. CONCLUSION: The GR RR mechanism of resistance is not associated with vacuole sequestration of glyphosate, and the observed reduced translocation is likely a consequence of rapid tissue death. Rapid cell death was inhibited by exogenous application of aromatic amino acids phenylalanine and tyrosine. The mechanism by which these amino acids inhibit rapid cell death in the GR RR phenotype remains unknown, and it could involve glyphosate phytotoxicity or other agents generating reactive oxygen species. Implications of these findings are discussed. The GR RR mechanism is distinct from the currently described glyphosate TSR or NTSR mechanisms in other species. © 2017 Society of Chemical Industry.

Computational methods using weighed-extreme learning machine to predict protein self-interactions with protein evolutionary information.

Self-interactions Proteins (SIPs) is important for their biological activity owing to the inherent interaction amongst their secondary structures or domains. However, due to the limitations of experimental Self-interactions detection, one major challenge in the study of prediction SIPs is how to exploit computational approaches for SIPs detection based on evolutionary information contained protein sequence. In the work, we presented a novel computational approach named WELM-LAG, which combined the Weighed-Extreme Learning Machine (WELM) classifier with Local Average Group (LAG) to predict SIPs based on protein sequence. The major improvement of our method lies in presenting an effective feature extraction method used to represent candidate Self-interactions proteins by exploring the evolutionary information embedded in PSI-BLAST-constructed position specific scoring matrix (PSSM); and then employing a reliable and robust WELM classifier to carry out classification. In addition, the Principal Component Analysis (PCA) approach is used to reduce the impact of noise. The WELM-LAG method gave very high average accuracies of 92.94 and 96.74% on yeast and human datasets, respectively. Meanwhile, we compared it with the state-of-the-art support vector machine (SVM) classifier and other existing methods on human and yeast datasets, respectively. Comparative results indicated that our approach is very promising and may provide a cost-effective alternative for predicting SIPs. In addition, we developed a freely available web server called WELM-LAG-SIPs to predict SIPs. The web server is available at http://219.219.62.123:8888/WELMLAG/ .

Robust and accurate prediction of protein self-interactions from amino acids sequence using evolutionary information.

Self-interacting proteins (SIPs) play an essential role in cellular functions and the evolution of protein interaction networks (PINs). Due to the limitations of experimental self-interaction proteins detection technology, it is a very important task to develop a robust and accurate computational approach for SIPs prediction. In this study, we propose a novel computational method for predicting SIPs from protein amino acids sequence. Firstly, a novel feature representation scheme based on Local Binary Pattern (LBP) is developed, in which the evolutionary information, in the form of multiple sequence alignments, is taken into account. Then, by employing the Relevance Vector Machine (RVM) classifier, the performance of our proposed method is evaluated on yeast and human datasets using a five-fold cross-validation test. The experimental results show that the proposed method can achieve high accuracies of 94.82% and 97.28% on yeast and human datasets, respectively. For further assessing the performance of our method, we compared it with the state-of-the-art Support Vector Machine (SVM) classifier, and other existing methods, on the same datasets. Comparison results demonstrate that the proposed method is very promising and could provide a cost-effective alternative for predicting SIPs. In addition, to facilitate extensive studies for future proteomics research, a web server is freely available for academic use at .

Transcriptional responses indicate maintenance of photosynthetic proteins as key to the exceptional chilling tolerance of C4 photosynthesis in Miscanthus × giganteus.

Miscanthus × giganteus is exceptional among C4 plants in its ability to acclimate to chilling (≤14 °C) and maintain a high photosynthetic capacity, in sharp contrast to maize, leading to very high productivity even in cool temperate climates. To identify the mechanisms that underlie this acclimation, RNA was isolated from M × giganteus leaves in chilling and nonchilling conditions and hybridized to microarrays developed for its close relative Zea mays. Among 21 000 array probes that yielded robust signals, 723 showed significant expression change under chilling. Approximately half of these were for annotated genes. Thirty genes associated with chloroplast membrane function were all upregulated. Increases in transcripts for the lhcb5 (chlorophyll a/b-binding protein CP26), ndhF (NADH dehydrogenase F, chloroplast), atpA (ATP synthase alpha subunit), psbA (D1), petA (cytochrome f), and lhcb4 (chlorophyll a/b-binding protein CP29), relative to housekeeping genes in M. × giganteus, were confirmed by quantitative reverse-transcription PCR. In contrast, psbo1, lhcb5, psbA, and lhcb4 were all significantly decreased in Z. mays after 14 days of chilling. Western blot analysis of the D1 protein and LHCII type II chlorophyll a/b-binding protein also showed significant increases in M. × giganteus during chilling and significant decreases in Z. mays. Compared to other C4 species, M. × giganteus grown in chilling conditions appears to counteract the loss of photosynthetic proteins and proteins protecting photosystem II typically observed in other species by increasing mRNA levels for their synthesis.

Gene amplification confers glyphosate resistance in Amaranthus palmeri.

The herbicide glyphosate became widely used in the United States and other parts of the world after the commercialization of glyphosate-resistant crops. These crops have constitutive overexpression of a glyphosate-insensitive form of the herbicide target site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Increased use of glyphosate over multiple years imposes selective genetic pressure on weed populations. We investigated recently discovered glyphosate-resistant Amaranthus palmeri populations from Georgia, in comparison with normally sensitive populations. EPSPS enzyme activity from resistant and susceptible plants was equally inhibited by glyphosate, which led us to use quantitative PCR to measure relative copy numbers of the EPSPS gene. Genomes of resistant plants contained from 5-fold to more than 160-fold more copies of the EPSPS gene than did genomes of susceptible plants. Quantitative RT-PCR on cDNA revealed that EPSPS expression was positively correlated with genomic EPSPS relative copy number. Immunoblot analyses showed that increased EPSPS protein level also correlated with EPSPS genomic copy number. EPSPS gene amplification was heritable, correlated with resistance in pseudo-F(2) populations, and is proposed to be the molecular basis of glyphosate resistance. FISH revealed that EPSPS genes were present on every chromosome and, therefore, gene amplification was likely not caused by unequal chromosome crossing over. This occurrence of gene amplification as an herbicide resistance mechanism in a naturally occurring weed population is particularly significant because it could threaten the sustainable use of glyphosate-resistant crop technology.

Cool C4 photosynthesis: pyruvate Pi dikinase expression and activity corresponds to the exceptional cold tolerance of carbon assimilation in Miscanthus x giganteus.

The bioenergy feedstock grass Miscanthus x giganteus is exceptional among C(4) species for its high productivity in cold climates. It can maintain photosynthetically active leaves at temperatures 6 degrees C below the minimum for maize (Zea mays), which allows it a longer growing season in cool climates. Understanding the basis for this difference between these two closely related plants may be critical in adapting maize to colder weather. When M. x giganteus and maize grown at 25 degrees C were transferred to 14 degrees C, light-saturated CO(2) assimilation and quantum yield of photosystem II declined by 30% and 40%, respectively, in the first 48 h in these two species. The decline continued in maize but arrested and then recovered partially in M. x giganteus. Within 24 h of the temperature transition, the pyruvate phosphate dikinase (PPDK) protein content per leaf area transiently declined in M. x giganteus but then steadily increased, such that after 7 d the enzyme content was significantly higher than in leaves growing in 25 degrees C. By contrast it declined throughout the chilling period in maize leaves. Rubisco levels remained constant in M. x giganteus but declined in maize. Consistent with increased PPDK protein content, the extractable PPDK activity per unit leaf area (V(max)(,ppdk)) in cold-grown M. x giganteus leaves was higher than in warm-grown leaves, while V(max,ppdk) was lower in cold-grown than in warm-grown maize. The rate of light activation of PPDK was also slower in cold-grown maize than M. x giganteus. The energy of activation (E(a)) of extracted PPDK was lower in cold-grown than warm-grown M. x giganteus but not in maize. The specific activities and E(a) of purified recombinant PPDK from M. x giganteus and maize cloned into Escherichia coli were similar. The increase in PPDK protein in the M. x giganteus leaves corresponded to an increase in PPDK mRNA level. These results indicate that of the two enzymes known to limit C(4) photosynthesis, increase of PPDK, not Rubisco content, corresponds to the recovery and maintenance of photosynthetic capacity. Functionally, increased enzyme concentration is shown to increase stability of M. x giganteus PPDK at low temperature. The results suggest that increases in either PPDK RNA transcription and/or the stability of this RNA are important for the increase in PPDK protein content and activity in M. x giganteus under chilling conditions relative to maize.

Can the cold tolerance of C4 photosynthesis in Miscanthus x giganteus relative to Zea mays be explained by differences in activities and thermal properties of Rubisco?

The previous investigations show that the amount and activity of Rubisco appears the major limitation to effective C(4) photosynthesis at low temperatures. The chilling-tolerant and bioenergy feedstock species Miscanthus x giganteus (M. x giganteus) is exceptionally productive among C(4) grasses in cold climates. It is able to develop photosynthetically active leaves at temperatures 6 degrees C below the minimum for maize, and achieves a productivity even at 52 degrees N that exceeds that of the most productive C(3) crops at this latitude. This study investigates whether this unusual low temperature tolerance can be attributed to differences in the amount or kinetic properties of Rubisco relative to maize. An efficient protocol was developed to purify large amounts of functional Rubisco from C(4) leaves. The maximum carboxylation activities (V(max)), activation states, catalytic rates per active site (K(cat)) and activation energies (E(a)) of purified Rubisco and Rubisco in crude leaf extracts were determined for M. x giganteus grown at 14 degrees C and 25 degrees C, and maize grown at 25 degrees C. The sequences of M. x giganteus Rubisco small subunit mRNA are highly conserved, and 91% identical to those of maize. Although there were a few differences between the species in the translated protein sequences, there were no significant differences in the catalytic properties (V(max), K(cat), and E(a)) for purified Rubisco, nor was there any effect of growth temperature in M. x giganteus on these kinetic properties. Extracted activities were close to the observed rates of CO(2) assimilation by the leaves in vivo. On a leaf area basis the extracted activities and activation state of Rubisco did not differ significantly, either between the two species or between growth temperatures. The activation state of Rubisco in leaf extracts showed no significant difference between warm and cold-grown M. x giganteus. In total, these results suggest that the ability of M. x giganteus to be productive and maintain photosynthetically competent leaves at low temperature does not result from low temperature acclimation or adaptation of the catalytic properties of Rubisco.

Regulation of Rubisco activase and its interaction with Rubisco.

The large, alpha-isoform of Rubisco activase confers redox regulation of the ATP/ADP response of the ATP hydrolysis and Rubisco activation activities of the multimeric activase holoenzyme complex. The alpha-isoform has a C-terminal extension that contains the redox-sensitive cysteine residues and is characterized by a high content of acidic residues. Cross-linking and site-directed mutagenesis studies of the C-terminal extension that have provided new insights into the mechanism of redox regulation are reviewed. Also reviewed are new details about the interaction between activase and Rubisco and the likely mechanism of 'activation' that resulted from mutagenesis in a 'Sensor 2' domain of activase that AAA(+) proteins often use for substrate recognition. Two activase residues in this domain were identified that are involved in Rubisco recognition. The results directly complement earlier studies that identified critical residues for activase recognition in the large subunit of Rubisco.

A novel nucleus-encoded chloroplast protein, PIFI, is involved in NAD(P)H dehydrogenase complex-mediated chlororespiratory electron transport in Arabidopsis.

A transient rise in chlorophyll fluorescence after turning off actinic light reflects nonphotochemical reduction of the plastoquinone (PQ) pool. This process is dependent on the activity of the chloroplast NAD(P)H dehydrogenase (NDH) complex, which mediates electron flow from stromal reductants to the PQ pool. In this study, we characterized an Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutant pifi (for postillumination chlorophyll fluorescence increase), which possesses an intact NDH complex, but lacks the NDH-dependent chlorophyll fluorescence increase after turning off actinic light. The nuclear gene PIFI (At3g15840) containing the T-DNA insertion encodes a chloroplast-targeted protein localized in the stroma and is annotated as a protein of unknown function. The pifi mutant exhibited a lower capacity for nonphotochemical quenching, but similar CO(2) assimilation rates, photosystem II (PSII) quantum efficiencies (PhiPSII), and reduction levels of the primary electron acceptor of PSII (1 - qL) as compared with the wild type. The pifi mutant grows normally under optimal conditions, but exhibits greater sensitivity to photoinhibition and long-term mild heat stress than wild-type plants, which is consistent with lower capacity of nonphotochemical quenching. We conclude that PIFI is a novel component essential for NDH-mediated nonphotochemical reduction of the PQ pool in chlororespiratory electron transport.

Increased sensitivity of oxidized large isoform of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase to ADP inhibition is due to an interaction between its carboxyl extension and nucleotide-binding pocket.

In Arabidopsis, oxidation of the large (46-kDa) isoform activase to form a disulfide bond in the C-terminal extension (C-extension) significantly increases its ADP sensitivity for both ATP hydrolysis and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation, thereby decreasing both activities at physiological ratios of ADP/ATP. In this study, we demonstrate that the C-extension of the oxidized large activase isoform can be cross-linked with regions containing residues that contribute to the nucleotide-binding pocket, with a higher efficiency in the presence of ADP or the absence of nucleotides than with ATP. Coupled with measurements demonstrating a redox-dependent protease sensitivity of the C-extension and a lower ATP or adenosine 5'-O-(thiotriphosphate) (ATPgammaS) affinity of the oxidized large isoform than either the reduced form or the smaller isoform, the results suggest that the C-extension plays an inhibitory role in ATP hydrolysis, regulated by redox changes. In contrast, the ADP affinities of the small isoform and the reduced or oxidized large isoform were similar, which indicates that the C-extension selectively interferes with the proper binding of ATP, possibly by interfering with the coordination of the gamma-phosphate. Furthermore, replacement of conserved, negatively charged residues (Asp390, Glu394, and Asp401) in the C-extension with alanine significantly reduced the sensitivities of the mutants to ADP inhibition, which suggests the involvement of electrostatic interactions between them and positively charged residues in or near the nucleotide-binding pocket. These studies provide new insights into the mechanism of redox regulation of activase by the C-extension in the large isoform.

Two conserved tryptophan residues are responsible for intrinsic fluorescence enhancement in Rubisco activase upon ATP binding.

Two species-invariant tryptophan residues at positions 109 and 250 of tobacco Rubisco activase were identified by site-directed mutagenesis as being responsible for the increase in intrinsic fluorescence upon addition of ATP, which has been previously attributed to increased self-association. Substitution of W109, which is immediately prior to a 'P-loop' sequence in the ATP catalytic motif, with aromatic residues (Tyr or Phe), Cys or Lys eliminated both ATP hydrolysis and the intrinsic fluorescence enhancement. Although the W109 mutants bound ATP, ATP did not provide a partial protection against proteolysis by trypsin that was observed with the recombinant wild-type enzyme. In contrast, substitution of W250 with Tyr or Phe abolished about half (44%) of the increase in intrinsic fluorescence with ATP, but had little effect on ATP hydrolysis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation or proteolytic protection with ATP. The substitution of the other tryptophan residues, W16 and W305, with phenylalanine did not significantly alter the change in intrinsic fluorescence upon addition of ATP. Therefore, W109 and W250 are the residues reporting the conformational change that increases the intrinsic fluorescence.

Identification of critical arginine residues in the functioning of Rubisco activase.

Rubisco activase is a member of the AAA(+) family in which arginines located in the Box VII and Sensor 2 domains are a recurrent feature and typically contribute to ATP-binding/hydrolysis or an inter-subunit interface. Replacement of R241 or R244 in Box VII or R294 or R296 in Sensor 2 with alanine in tobacco activase did not greatly alter the binding of ATP or ADP. However, ATP hydrolysis was minimal (R241A and R244A) or greatly diminished (R296A) and none of these mutants were able to activate Rubisco. R241, R244 and R296 were also required for nucleotide-dependent conformational changes detected by intrinsic fluorescence and limited proteolysis. ATP-induced oligomerization, monitored by gel filtration, was not observed with the wild type and mutant tobacco activases in contrast to spinach activase and a R239A mutant (corresponding to R244A in tobacco). Thus, there is not a strict correlation of oligomerization with ATP hydrolysis and intrinsic fluorescence.

Difference between Biological Activities and Secondary Structures of Expression Product of atpE Gene from Broad Bean and Maize Chloroplast.

The chloroplast atpE genes from broad bean and maize were overexpressed in E.coli, respectively. After proper refolding and purification, two types of epsilon-subunit proteins with biological activity were obtained. When reconstituted with CF(1)(-epsilon)from different chloroplast ATPase, the effects of the reconstructed epsilon-subunit protein of maize CF(1) to the Ca(2+)-ATPase activity of epsilon-deficient CF(1) and to the proton leakage through CF(0) were markedly stronger than that of broad bean CF(1). It was also observed that the enhancing effect of maize epsilon-subunit protein to the chloroplast photophosphorylation activity was greater than that of broad bean epsilon-subunit protein. The results indicate that (1)the inhibition of epsilon-subunit is closely related to the its affinity with other parts of CF(1) (2)both functions of epsilon-subunit as inhibitor of ATPase and as proton pathway are closely linked. The circular dichroism was performed to show the differences in their secondary structure of these epsilon-subunit proteins based on the deduced primary sequences. Unconstrained analysis of the CD spectrum for the maize epsilon-subunit protein gave 22.6% alpha-helix, 30.6% beta-sheet, 9.3% beta-turn, and 37.7% unordered structure and 31.4%alpha-helix, 22.3% beta-sheet, 13.8% beta-turn, and 32.4% unordered structure for the epsilon-subunit from broad bean.