Determining Macromolecular Structures Using Cryo-Electron Microscopy.

Structural insights into macromolecular and protein complexes provide key clues about the molecular basis of the function. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful structural biology method for studying protein and macromolecular structures at high resolution in both native and near-native states. Despite the ability to get detailed structural insights into the processes underlying protein function using cryo-EM, there has been hesitancy amongst plant biologists to apply the method for biomolecular interaction studies. This is largely evident from the relatively fewer structural depositions of proteins and protein complexes from plant origin in electron microscopy databank. Even though the progress has been slow, cryo-EM has significantly contributed to our understanding of the molecular biology processes underlying photosynthesis, energy transfer in plants, besides viruses infecting plants. This chapter introduces sample preparation for both negative-staining electron microscopy (NSEM) and cryo-EM for plant proteins and macromolecular complexes and data analysis using single particle analysis for beginners.

Cryo-EM structure of the plant 26S proteasome.

Targeted proteolysis is a hallmark of life. It is especially important in long-lived cells that can be found in higher eukaryotes, like plants. This task is mainly fulfilled by the ubiquitin-proteasome system. Thus, proteolysis by the 26S proteasome is vital to development, immunity, and cell division. Although the yeast and animal proteasomes are well characterized, there is only limited information on the plant proteasome. We determined the first plant 26S proteasome structure from Spinacia oleracea by single-particle electron cryogenic microscopy at an overall resolution of 3.3 Å. We found an almost identical overall architecture of the spinach proteasome compared with the known structures from mammals and yeast. Nevertheless, we noticed a structural difference in the proteolytic active ß1 subunit. Furthermore, we uncovered an unseen compression state by characterizing the proteasome's conformational landscape. We suspect that this new conformation of the 20S core protease, in correlation with a partial opening of the unoccupied gate, may contribute to peptide release after proteolysis. Our data provide a structural basis for the plant proteasome, which is crucial for further studies.

Atg12-Interacting Motif Is Crucial for E2-E3 Interaction in Plant Atg8 System.

Autophagy is an intracellular degradation system regulating cellular homeostasis. The two ubiquitin-like modification systems named the Atg8 system and the Atg12 system are essential for autophagy. Atg8 and Atg12 are ubiquitin-like proteins covalently conjugated with a phosphatidylethanolamine (PE) and Atg5, respectively, via enzymatic reactions. The Atg8-PE conjugate binds to autophagic membranes and recruits various proteins through direct interaction, whereas the Atg12-Atg5 conjugate recognizes Atg3, the E2 enzyme for Atg8, and facilitates Atg8-PE conjugation by functioning as the E3 enzyme. Although structural and biochemical analyses have well established the Atg8-family interacting motif (AIM), studies on the interacting sequence for Atg12 are rare (only one example for human ATG12-ATG3), thereby making it challenging to define a binding motif. Here we determined the crystal structure of the plant ATG12b as a complex with the ATG12b-binding region of ATG3 and revealed that ATG12b recognizes the aspartic acid (Asp)-methionine (Met) motif in ATG3 via a hydrophobic pocket and a basic residue, which we confirmed critical for the complex formation by mutational analysis. This recognition mode is similar to that reported between human ATG12 and ATG3, suggesting that the Asp-Met sequence is a conserved Atg12-interacting motif (AIM12). These data suggest that AIM12 mediates E2-E3 interaction during Atg8 lipidation and provide structural basis for developing chemicals that regulate autophagy by targeting Atg12-family proteins.

Insights into the multiscale structure and pasting properties of ball-milled waxy maize and waxy rice starches.

The effects of ball-milling on the pasting properties of waxy maize starch (WMS) and waxy rice starch (WRS) were investigated from a multiscale structural view. The results confirmed that ball-milling significantly destroyed the structures of the two waxy starches (especially WMS). Specifically, ball-milling led to obvious grooves on the surface of starch granules, a decrease in crystallinity and the degree of short-range order, and a reduction in double-helix components. Meanwhile, small-angle X-ray scattering results indicated that the semicrystalline lamellae of starch were disrupted after ball-milling. Ball-milling decreased the pasting temperatures. Furthermore, ball-milled starches exhibited lower peak and breakdown viscosity and weakened tendency to retrogradation. These results implied that ball-milling induced structural changes in starch that significantly affected its pasting properties. Hence, ball-milled starch may serve as food ingredients with low pasting temperature and paste viscosity as well as high paste stability under heating/cooling and shearing.

The Predicted Functional Compartmentation of Rice Terpenoid Metabolism by Trans-Prenyltransferase Structural Analysis, Expression and Localization.

Most terpenoids are derived from the basic terpene skeletons of geranyl pyrophosphate (GPP, C10), farnesyl-PP (FPP, C15) and geranylgeranyl-PP (GGPP, C20). The trans-prenyltransferases (PTs) mediate the sequential head-to-tail condensation of an isopentenyl-PP (C5) with allylic substrates. The in silico structural comparative analyses of rice trans-PTs with 136 plant trans-PT genes allowed twelve rice PTs to be identified as GGPS_LSU (OsGGPS1), homomeric G(G)PS (OsGPS) and GGPS_SSU-II (OsGRP) in Group I; two solanesyl-PP synthase (OsSPS2 and 3) and two polyprenyl-PP synthases (OsSPS1 and 4) in Group II; and five FPSs (OsFPS1, 2, 3, 4 and 5) in Group III. Additionally, several residues in "three floors" for the chain length and several essential domains for enzymatic activities specifically varied in rice, potentiating evolutionarily rice-specific biochemical functions of twelve trans-PTs. Moreover, expression profiling and localization patterns revealed their functional compartmentation in rice. Taken together, we propose the predicted topology-based working model of rice PTs with corresponding terpene metabolites: GPP/GGPPs mainly in plastoglobuli, SPPs in stroma, PPPs in cytosol, mitochondria and chloroplast and FPPs in cytosol. Our findings could be suitably applied to metabolic engineering for producing functional terpene metabolites in rice systems.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility.

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

Physicochemical and Antioxidative Characteristics of Potato Protein Isolate Hydrolysate.

This study investigated the physicochemical characteristics of potato protein isolate hydrolysate (PPIH) and its antioxidant activity. Potato protein isolate (PPI) was hydrolyzed into PPIH by the proteases bromelain, Neutrase, and Flavourzyme. Compared with PPI, the resulting PPIH had a lower molecular weight (MW, from 103.5 to 422.7 Da) and smaller particle size (<50 nm), as well as a higher solubility rate (>70%) under acidic conditions (pH 3-6). PPIH presented good solubility (73%) across the tested pH range of 3-6. As the pH was increased, the zeta potential of PPIH decreased from -7.4 to -21.6. Using the 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical-scavenging assay, we determined that the half-maximal effective concentration (EC50) values of ascorbic acid, PPIH, and PPI were 0.01, 0.89, and >2.33 mg/mL, respectively. Furthermore, PPIH (50 µg/mL) protected C2C12 cells from H2O2 oxidation significantly better than PPI (10.5% higher viability rate; p < 0.01). These findings demonstrated the possible use of PPIH as an antioxidant in medical applications.

Arginine-259 of UGT2B7 Confers UDP-Sugar Selectivity.

Enzymes of the human UDP-glycosyltransferase (UGT) superfamily typically catalyze the covalent addition of the sugar moiety from a UDP-sugar cofactor to relatively low-molecular weight lipophilic compounds. Although UDP-glucuronic acid (UDP-GlcUA) is most commonly employed as the cofactor by UGT1 and UGT2 family enzymes, UGT2B7 and several other enzymes can use both UDP-GlcUA and UDP-glucose (UDP-Glc), leading to the formation of glucuronide and glucoside conjugates. An investigation of UGT2B7-catalyzed morphine glycosidation indicated that glucuronidation is the principal route of metabolism because the binding affinity of UDP-GlcUA is higher than that of UDP-Glc. Currently, it is unclear which residues in the UGT2B7 cofactor binding domain are responsible for the preferential binding of UDP-GlcUA. Here, molecular dynamics (MD) simulations were performed together with site-directed mutagenesis and enzyme kinetic studies to identify residues within the UGT2B7 binding site responsible for the selective cofactor binding. MD simulations demonstrated that Arg259, which is located within the N-terminal domain, specifically interacts with UDP-GlcUA, whereby the side chain of Arg259 H-bonds and forms a salt bridge with the carboxylate group of glucuronic acid. Consistent with the MD simulations, substitution of Arg259 with Leu resulted in the loss of morphine, 4-methylumbelliferone, and zidovudine glucuronidation activity, but morphine glucosidation was preserved. SIGNIFICANCE STATEMENT: Despite the importance of uridine diphosphate glycosyltransferase (UGT) enzymes in drug and chemical metabolism, cofactor binding interactions are incompletely understood, as is the molecular basis for preferential glucuronidation by UGT1 and UGT2 family enzymes. The study demonstrated that long timescale molecular dynamics (MD) simulations with a UGT2B7 homology model can be used to identify critical binding interactions of a UGT protein with UDP-sugar cofactors. Further, the data provide a basis for the application of MD simulations to the elucidation of UGT-aglycone interactions.

Lupinus albus γ-Conglutin, a Protein Structurally Related to GH12 Xyloglucan-Specific Endo-Glucanase Inhibitor Proteins (XEGIPs), Shows Inhibitory Activity against GH2 ß-Mannosidase.

γ-conglutin (γC) is a major protein of Lupinus albus seeds, but its function is still unknown. It shares high structural similarity with xyloglucan-specific endo-glucanase inhibitor proteins (XEGIPs) and, to a lesser extent, with Triticum aestivum endoxylanase inhibitors (TAXI-I), active against fungal glycoside hydrolases GH12 and GH11, respectively. However, γC lacks both these inhibitory activities. Since ß-galactomannans are major components of the cell walls of endosperm in several legume plants, we tested the inhibitory activity of γC against a GH2 ß-mannosidase (EC 3.2.1.25). γC was actually able to inhibit the enzyme, and this effect was enhanced by the presence of zinc ions. The stoichiometry of the γC/enzyme interaction was 1:1, and the calculated Ki was 1.55 µM. To obtain further insights into the interaction between γC and ß-mannosidase, an in silico structural bioinformatic approach was followed, including some docking analyses. By and large, this work describes experimental findings that highlight new scenarios for understanding the natural role of γC. Although structural predictions can leave space for speculative interpretations, the full complexity of the data reported in this work allows one to hypothesize mechanisms of action for the basis of inhibition. At least two mechanisms seem plausible, both involving lupin-γC-peculiar structures.

Physical-Chemical Crosslinked Electrospun Colocasia esculenta Tuber Protein-Chitosan-Poly(Ethylene Oxide) Nanofibers with Antibacterial Activity and Cytocompatibility.

BACKGROUND: Electrospun nanofibers based on Colocasia esculenta tuber (CET) protein are considered as a promising material for wound dressing applications. However, the use of these nanofibers in aqueous conditions has poor stability. The present study was performed to obtain insights into the crosslinked electrospun CET's protein-chitosan (CS)-poly(ethylene oxide) (PEO) nanofibers and to evaluate their potential for wound dressing applications. METHODS: The electrospun nanofibers were crosslinked with glutaraldehyde (GA) vapor and heat treatment (HT) to enhance their physicochemical stability. The crosslinked nanofibers were characterized by protein profiles, morphology structures, thermal behavior, mechanical properties, and degradation behavior. Furthermore, the antibacterial properties and cytocompatibility were analyzed by antibacterial assessment and cell proliferation. RESULTS: The protein profiles of the electrospun CET's protein-CS-PEO nanofibers before and after HT crosslinking contained one major bioactive protein with a molecular weight of 14.4 kDa. Scanning electron microscopy images of the crosslinked nanofibers indicated preservation of the structure after immersion in phosphate buffered saline. The crosslinked nanofibers resulted in higher ultimate tensile strength and lower ultimate strain compared to the non-crosslinked nanofibers. GA vapor crosslinking showed higher water stability compared to HT crosslinking. The in vitro antibacterial activity of the crosslinked nanofibers showed a stronger bacteriostatic effect on Staphylococcus aureus than on Escherichia coli. Human skin fibroblast cell proliferation on crosslinked GA vapor and HT nanofibers with 1% (w/v) CS and 2% (w/v) CET's protein demonstrated the highest among all the other crosslinked nanofibers after seven days of cell culture. Cell proliferation and cell morphology results revealed that introducing higher CET's protein concentration on crosslinked nanofibers could increase cell proliferation of the crosslinked nanofibers. CONCLUSION: These results are promising for the potential use of the crosslinked electrospun CET's protein-CS-PEO nanofibers as bioactive wound dressing materials.

WDR90 is a centriolar microtubule wall protein important for centriole architecture integrity.

Centrioles are characterized by a nine-fold arrangement of microtubule triplets held together by an inner protein scaffold. These structurally robust organelles experience strenuous cellular processes such as cell division or ciliary beating while performing their function. However, the molecular mechanisms underlying the stability of microtubule triplets, as well as centriole architectural integrity remain poorly understood. Here, using ultrastructure expansion microscopy for nanoscale protein mapping, we reveal that POC16 and its human homolog WDR90 are components of the microtubule wall along the central core region of the centriole. We further found that WDR90 is an evolutionary microtubule associated protein. Finally, we demonstrate that WDR90 depletion impairs the localization of inner scaffold components, leading to centriole structural abnormalities in human cells. Altogether, this work highlights that WDR90 is an evolutionary conserved molecular player participating in centriole architecture integrity.

Cells are made up of compartments called organelles that perform specific roles. A cylindrical organelle called the centriole is important for a number of cellular processes, ranging from cell division to movement and signaling. Each centriole contains nine blades made up of protein filaments called microtubules, which link together to form a cylinder. This well-known structure can be found in a variety of different species. Yet, it is unclear how centrioles are able to maintain this stable architecture whilst carrying out their various different cell roles. In early 2020, a group of researchers discovered a scaffold protein at the center of centrioles that helps keep the microtubule blades stable. Further investigation suggested that another protein called WDR90 may also help centrioles sustain their cylindrical shape. However, the exact role of this protein was poorly understood. To determine the role of WDR90, Steib et al. ­ including many of the researchers involved in the 2020 study ­ used a method called Ultrastructure Expansion Microscopy to precisely locate the WDR90 protein in centrioles. This revealed that WDR90 is located on the microtubule wall of centrioles in green algae and human cells grown in the lab. Further experiments showed that the protein binds directly to microtubules and that removing WDR90 from human cells causes centrioles to lose their scaffold proteins and develop structural defects. This investigation provides fundamental insights into the structure and stability of centrioles. It shows that single proteins are key components in supporting the structural integrity of organelles and shaping their overall architecture. Furthermore, these findings demonstrate how ultrastructure expansion microscopy can be used to determine the role of individual proteins within a complex structure.

An ultrasonic-extracted arabinoglucan from Tamarindus indica L. pulp: A study on molecular and structural characterizations.

In this study, an ultrasonic-extracted polysaccharide (nCPTP-55) was obtained with the highest yield (61.08%, w/w) from tamarind pulp, which consisted chiefly of total sugar (85.98%, w/w) with few protein (2.10%, w/w). Monosaccharide analysis showed nCPTP-55 was mainly composed of arabinose (39.19 mol%) and glucose (50.48 mol%) with negligible GlcA (2.05 mol%), indicating the neutral nature of nCPTP-55, which was further elucidated structurally via GC-MS and NMR, i.e., an arabinoglucan composed of →3)-ß-D-Glcp-(1→ backbone with only T-α-L-Araf-(1→ branched at O-4 (27.82%) and O-6 (39.99%), resulting in relatively high A/G ratio (0.68-0.70). Based on MM2 minimized energy, the 3D schematic structures of nCPTP-55 could be considered as structural basis for its conformational behavior, which was preliminarily estimated via HPSEC-MALLS as between compact sphere and loosely hyper-branched chain (ρ = 0.84). Therefore, the relationship between molecular structure and conformational behavior was basically established for nCPTP-55, which was in a bid to have a better knowledge of its structure-property and structure-bioactivity relationships potentially required for more applications in food, cosmetic and pharmaceutical fields.

Effects of ultrasound-assisted enzyme hydrolysis on the microstructure and physicochemical properties of okara fibers.

This work focuses on the effects of different ultrasound power densities on the microstructural changes and physicochemical properties of okara fibers, which are composed of carbohydrate-based polymers. Okara suspensions were treated with ultrasound at different power densities (0, 1, 2, 3, 4, and 5 W/mL) for 30 min, after which the ultrasound-treated okara were hydrolyzed by trypsin to obtain okara fibers. The ultrasound treatment of the okara fibers induced structural disorganization and changes, evidenced mainly in their morphological characteristics and their relative crystallinity degrees. Increasing the ultrasound power broke the okara fibers into flaky and stacked structures. When the ultrasound power density reached 4 W/mL, the parenchyma became compact and the hourglass structure fractured. The mean particle size of the okara fiber was reduced from 82.24 µm to 53.96 µm, and the homogeneity was enhanced significantly. The relative crystallinity of the okara fibers was reduced from 55.14% to 36.47%. The okara fiber surface charge decreased when the ultrasound power was increased. However, after ultrasound treatment at 4 W/mL (800 W), the okara fiber suspension exhibited the highest viscosity value and a higher swelling capacity, water-holding capacity, and oil-holding capacity. Therefore, the results indicated that the selection of processing conditions for okara fibers is critical and that okara fiber modification using a high ultrasound treatment might improve their use in potential applications.

Mashes to Mashes, Crust to Crust. Presenting a novel microstructural marker for malting in the archaeological record.

The detection of direct archaeological remains of alcoholic beverages and their production is still a challenge to archaeological science, as most of the markers known up to now are either not durable or diagnostic enough to be used as secure proof. The current study addresses this question by experimental work reproducing the malting processes and subsequent charring of the resulting products under laboratory conditions in order to simulate their preservation (by charring) in archaeological contexts and to explore the preservation of microstructural alterations of the cereal grains. The experimentally germinated and charred grains showed clearly degraded (thinned) aleurone cell walls. The histological alterations of the cereal grains were observed and quantified using reflected light and scanning electron microscopy and supported using morphometric and statistical analyses. In order to verify the experimental observations of histological alterations, amorphous charred objects (ACO) containing cereal remains originating from five archaeological sites dating to the 4th millennium BCE were considered: two sites were archaeologically recognisable brewing installations from Predynastic Egypt, while the three broadly contemporary central European lakeshore settlements lack specific contexts for their cereal-based food remains. The aleurone cell wall thinning known from food technological research and observed in our own experimental material was indeed also recorded in the archaeological finds. The Egyptian materials derive from beer production with certainty, supported by ample contextual and artefactual data. The Neolithic lakeshore settlement finds currently represent the oldest traces of malting in central Europe, while a bowl-shaped bread-like object from Hornstaad-Hörnle possibly even points towards early beer production in central Europe. One major further implication of our study is that the cell wall breakdown in the grain's aleurone layer can be used as a general marker for malting processes with relevance to a wide range of charred archaeological finds of cereal products.

Cryo-EM structure of the RNA-rich plant mitochondrial ribosome.

The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs1. These organelles have a bacterial origin and were acquired during an early endosymbiosis event2. Mitochondria possess specialized gene expression systems composed of various molecular machines, including the mitochondrial ribosomes (mitoribosomes). Mitoribosomes are in charge of translating the few essential mRNAs still encoded by mitochondrial genomes3. While chloroplast ribosomes strongly resemble those of bacteria4,5, mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species6-10. In contrast to animals and trypanosomatids, plant mitoribosomes have unusually expanded ribosomal RNAs and have conserved the short 5S rRNA, which is usually missing in mitoribosomes11. We have previously characterized the composition of the plant mitoribosome6, revealing a dozen plant-specific proteins in addition to the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in the field, plant mitoribosomes remained elusive to high-resolution structural investigations and the plant-specific ribosomal features of unknown structures. Here, we present a cryo-electron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide repeat proteins (PPRs) that deeply interact with the plant-specific rRNA expansion segments. These additional rRNA segments and proteins reshape the overall structure of the plant mitochondrial ribosome, and we discuss their involvement in the membrane association and mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNA-constructive phase of mitoribosome evolution across eukaryotes.

Cell Wall Composition and Ultrastructural Immunolocalization of Pectin and Arabinogalactan Protein during Olea europaea L. Fruit Abscission.

Cell wall modification is integral to many plant developmental processes where cells need to separate, such as abscission. However, changes in cell wall composition during natural fruit abscission are poorly understood. In olive (Olea europaea L.), some cultivars such as 'Picual' undergo massive natural fruit abscission after fruit ripening. This study investigates the differences in cell wall polysaccharide composition and the localization of pectins and arabinogalactan protein (AGP) in the abscission zone (AZ) during cell separation to understand fruit abscission control in 'Picual' olive. To this end, immunogold labeling employing a suite of monoclonal antibodies to cell wall components (JIM13, LM5, LM6, LM19 and LM20) was investigated in olive fruit AZ. Cell wall polysaccharide extraction revealed that the AZ cell separation is related to the de-esterification and degradation of pectic polysaccharides. Moreover, ultrastructural localization showed that both esterified and unesterified homogalacturonans (HGs) localize mainly in the AZ cell walls, including the middle lamella and tricellular junction zones. Our results indicate that unesterified HGs are likely to contribute to cell separation in the olive fruit AZ. Similarly, immunogold labeling demonstrated a decrease in both galactose-rich and arabinose-rich pectins in AZ cell walls during ripe fruit abscission. In addition, AGPs were localized in the cell wall, plasma membrane and cytoplasm of AZ cells with lower levels of AGPs during ripe fruit abscission. This detailed temporal profile of the cell wall polysaccharide composition, and the pectins and AGP immunolocalization in the olive fruit AZ, offers new insights into cell wall remodeling during ripe fruit abscission.

Unusual features of the c-ring of F1FO ATP synthases.

Membrane integral ATP synthases produce adenosine triphosphate, the universal "energy currency" of most organisms. However, important details of proton driven energy conversion are still unknown. We present the first high-resolution structure (2.3 Å) of the in meso crystallized c-ring of 14 subunits from spinach chloroplasts. The structure reveals molecular mechanisms of intersubunit contacts in the c14-ring, and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane. Similar densities were found in all known high-resolution structures of c-rings of F1FO ATP synthases from archaea and bacteria to eukaryotes. The densities might originate from isoprenoid quinones (such as coenzyme Q in mitochondria and plastoquinone in chloroplasts) that is consistent with differential UV-Vis spectroscopy of the c-ring samples, unusually large distance between polar/apolar interfaces inside the c-ring and universality among different species. Although additional experiments are required to verify this hypothesis, coenzyme Q and its analogues known as electron carriers of bioenergetic chains may be universal cofactors of ATP synthases, stabilizing c-ring and prevent ion leakage through it.

Molecular basis for branched steviol glucoside biosynthesis.

Steviol glucosides, such as stevioside and rebaudioside A, are natural products roughly 200-fold sweeter than sugar and are used as natural, noncaloric sweeteners. Biosynthesis of rebaudioside A, and other related stevia glucosides, involves formation of the steviol diterpenoid followed by a series of glycosylations catalyzed by uridine diphosphate (UDP)-dependent glucosyltransferases. UGT76G1 from Stevia rebaudiana catalyzes the formation of the branched-chain glucoside that defines the stevia molecule and is critical for its high-intensity sweetness. Here, we report the 3D structure of the UDP-glucosyltransferase UGT76G1, including a complex of the protein with UDP and rebaudioside A bound in the active site. The X-ray crystal structure and biochemical analysis of site-directed mutants identifies a catalytic histidine and how the acceptor site of UGT76G1 achieves regioselectivity for branched-glucoside synthesis. The active site accommodates a two-glucosyl side chain and provides a site for addition of a third sugar molecule to the C3' position of the first C13 sugar group of stevioside. This structure provides insight on the glycosylation of other naturally occurring sweeteners, such as the mogrosides from monk fruit, and a possible template for engineering of steviol biosynthesis.

Food-grade gliadin microstructures obtained by electrohydrodynamic processing.

This paper presents a comprehensive study on the electrohydrodynamic processing of gliadin to develop food-grade delivery systems with different morphologies. The effects of biopolymer concentration, applied voltage and solution flow-rate on particle morphology, molecular organisation, crystallinity and thermal properties were investigated. Gliadin concentration influenced the apparent viscosity and conductivity of the solutions, giving raise to particle morphologies at 10 wt% gliadin and beaded-free fibers above 25 wt% gliadin. In general, increasing the voltage resulted in smaller average sizes of the obtained structures, while no significant differences in morphology were observed among the tested flow rates. Interestingly, the amide I position in the FTIR reflected changes in protein conformation which could be correlated with the final morphology attained. Moreover, the acetic acid used for solution preparation disrupted the original amino acid chain packing of the gliadin fraction, being the electrospun/electrosprayed samples amorphous. These gliadin-based microparticles and microfibers obtained could serve as food-grade delivery vehicles.

A Nucleosome Bridging Mechanism for Activation of a Maintenance DNA Methyltransferase.

DNA methylation and H3K9me are hallmarks of heterochromatin in plants and mammals, and are successfully maintained across generations. The biochemical and structural basis for this maintenance is poorly understood. The maintenance DNA methyltransferase from Zea mays, ZMET2, recognizes dimethylation of H3K9 via a chromodomain (CD) and a bromo adjacent homology (BAH) domain, which flank the catalytic domain. Here, we show that dinucleosomes are the preferred ZMET2 substrate, with DNA methylation preferentially targeted to linker DNA. Electron microscopy shows one ZMET2 molecule bridging two nucleosomes within a dinucleosome. We find that the CD stabilizes binding, whereas the BAH domain enables allosteric activation by the H3K9me mark. ZMET2 further couples recognition of H3K9me to an increase in the specificity for hemimethylated versus unmethylated DNA. We propose a model in which synergistic coupling between recognition of nucleosome spacing, H3K9 methylation, and DNA modification allows ZMET2 to maintain DNA methylation in heterochromatin with high fidelity.