Structural Studies Research Articles

Comparative chloroplast-specific SNP and nSCoT markers analysis and population structure study in kiwifruit plants.

Kiwifruit (Actinidiaceae family) is an economically important fruit tree in China and New Zealand. It is a typical dioecious plant that has undergone frequent natural hybridization, along with chromosomal ploidy diversity within the genus Actinidia, resulting in higher genetic differences and horticultural diversity between interspecific and intraspecific traits. This diversity provides a rich genetic base for breeding. China is not only the original center of speciation for the Actinidia genus but also its distribution center, housing the most domesticated species: A. chinensis var. chinensis, A. chinensis var. deliciosa, A. arguta, and A. polygama. However, there have been relatively few studies on the application of DNA markers and the genetic basis of kiwifruit plants. By combining information from chloroplast-specific SNPs and nuclear SCoT (nSCoT) markers, we can uncover complementary aspects of genetic variation, population structure, and evolutionary relationships. In this study, one chloroplast DNA (cpDNA) marker pair was selected out of nine cpDNA candidate pairs. Twenty nSCoT markers were selected and used to assess the population structure and chloroplast-specific DNA haplotype diversity in 55 kiwifruit plants (Actinidia), including 20 samples of A. chinensis var. chinensis, 22 samples of A. chinensis var. deliciosa, 11 samples of A. arguta, and two samples of A. polygama, based on morphological observations collected from China. The average genetic distance among the 55 samples was 0.26 with chloroplast-specific SNP markers and 0.57 with nSCoT markers. The Mantel test revealed a very small correlation (r = 0.21). The 55 samples were categorized into different sub-populations using Bayesian analysis, the Unweighted Pair Group Method with the Arithmetic Mean (UPGMA), and the Principal Component Analysis (PCA) method, respectively. Based on the analysis of 205 variable sites, a total of 15 chloroplast-specific DNA haplotypes were observed, contributing to a higher level of polymorphism with an Hd of 0.78. Most of the chloroplast-specific DNA haplotype diversity was distributed among populations, but significant diversity was also observed within populations. H1 was shared by 24 samples, including 12 of A. chinensis var. chinensis and 12 of A. chinensis var. deliciosa, indicating that H1 is an ancient and dominant haplotype among the 55 chloroplast-specific sequences. H2 may not have evolved further.The remaining haplotypes were rare and unique, with some appearing to be exclusive to a particular variety and often detected in single individuals. For example, the H15 haplotype was found exclusively in A. polygama. The population genetic variation explained by chloroplast-specific SNP markers has greater power than that explained by nSCoTs, with chloroplast-specific DNA haplotypes being the most efficient. Gene flow appears to be more evident between A. chinensis var. chinensis and A. chinensis var. deliciosa, as they share chloroplast-specific DNA haplotypes, In contrast, A.arguta and A. polygama possess their own characteristic haplotypes, derived from the haplotype of A. chinensis var. chinensis. Compared with A. chinensis, the A.arguta and A. polygama showed better grouping. It also seems crucial to screen out, for each type of molecular marker, especially haplotypes, the core markers of the Actinidia genus.

A study of macroinvertebrate community structure and diversity in response to land use type in the Yiluo River Basin.

The Yiluo River is the largest tributary below Xiaolangdi Reservoir in the middle reaches of the Yellow River, and is one of the important water conservation areas in the Yellow River Basin. Studying the ecological status of the Yiluo River under varied land use types in this basin is crucial for both ecological protection and the high-quality development of the Yellow River Basin. This study investigated the impacts of land use types on the macroinvertebrate community and functional structure in the Yiluo River Basin and introduced the concept of the land use health index (LUI). During the survey period, a total of 11,894 macroinvertebrates were collected, and 143 species were identified, belonging to 4 phyla, 7 orders, 22 families, and 75 families. The results showed that LUI had the most significant impact on macroinvertebrate community structure, with substrate type, dry plant weight, total phosphorus, turbidity, and attached algae biomass also playing significant roles in affecting macroinvertebrate communities. The species richness, the Shannon-Wiener index, and the Margalef richness index exhibited a nonlinear positive correlation with LUI of the sampling site, increasing as LUI enhancing and eventually reaching a plateau. Functional richness showed a linear and positive correlation with LUI, increasing with its enhancement, while functional evenness and functional divergence exhibited a nonlinear correlation with LUI. Functional evenness initially increased and then decreased with the enhancement of LUI, while functional divergence decreased with LUI enhancement. This study can provide a scientific reference for river ecological management under various land use scenarios.

A novel variant in the FLNB gene associated with spondylocarpotarsal synostosis syndrome.

Genetic disorders involved in skeleton system arise due to the disturbance in skeletal development, growth and homeostasis. Filamin B is an actin binding protein which is large dimeric protein which cross link actin cytoskeleton filaments into dynamic structure. A single nucleotide changes in the FLNB gene causes spondylocarpotarsal synostosis syndrome, a rare bone disorder due to which the fusion of carpels and tarsals synostosis occurred along with fused vertebrae. In the current study we investigated a family residing in north-western areas of Pakistan. The whole exome sequencing of proband was performed followed by Sanger sequencing of all family members of the subject to validate the variant segregation within the family. Bioinformatics tools were utilized to assess the pathogenicity of the variant. Whole Exome Sequencing revealed a novel variant (NM_001457: c.209C>T and p.Pro70Leu) in the FLNB gene which was homozygous missense mutation in the FLNB gene. The variant was further validated and visualized by Sanger sequencing and protein structure studies respectively as mentioned before. The findings have highlighted the importance of the molecular diagnosis in SCT (spondylocarpotarsal synostosis syndrome) for genetic risk counselling in consanguineous families.

Structural and chemical study of complex silver patterns additively manufactured by multi-photon reduction.

Multi-photon reduction (MPR) based on femtosecond laser makes rapid prototyping and molding in micro-nano scale feasible, but is limited in material selectivity due to lack of the understanding of the reaction mechanism in MPR process. In this paper, additively manufacturing of complex silver-based patterns through MPR is demonstrated. The effects of laser parameters, including laser pulse energies and scanning speeds, on the structural and chemical characteristics of the printed structures are systematically investigated. The results show that the geometric size of printed cubes deviates from the designed size further by increasing laser pulse energies or decreasing scanning speeds. The reaction mechanism of MPR is revealed by studying the elemental composition and chemical structures of printed cubes. The evolution of Raman spectra upon the laser processing parameters suggests that the MPR process mainly includes two processes: reduction and decomposition. In the MPR process, silver ions are reduced and grow into particles by accepting the electrons from ethonal molecules; meanwhile carboxyl groups in polyvinylpyrrolidone are decomposed and form amorphous carbon that is attached on the surface of silver particles. The conductivity of silver wires fabricated by MPR reaches 2×105 S/m and stays relatively constant as varying their cross section area, suggesting excellent electrical conduction. The understanding of the MPR process would accelerate the development of MPR technology and the implementation of MPR in micro-electromechanical systems could therefore be envisioned.

Highly specific aptamer trap for extremophilic RNA polymerases

During transcription initiation, the holoenzyme of bacterial RNA polymerase (RNAP) specifically recognizes promoters using a dedicated σ factor. During transcription elongation, the core enzyme of RNAP interacts with nucleic acids mainly nonspecifically, by stably locking the DNA template and RNA transcript inside the main cleft. Here, we present a synthetic DNA aptamer that is specifically recognized by both core and holoenzyme RNAPs from extremophilic bacteria of the Deinococcus-Thermus lineage. The aptamer binds RNAP with subnanomolar affinities, forming extremely stable complexes even at high ionic strength conditions, blocks RNAP interactions with the DNA template and inhibits RNAP activity during transcription elongation. We propose that the aptamer binds at a conserved site within the downstream DNA-binding cleft of RNAP and traps it in an inactive conformation. The aptamer can potentially be used for structural studies to reveal RNAP conformational states, affinity binding of RNAP and associated factors, and screening of transcriptional inhibitors.

Structural Studies of a Complex of a CAG/CTG Repeat Sequence-Specific Binding Molecule and A–A-Mismatch-Containing DNA

Structural Studies of a Complex of a CAG/CTG Repeat Sequence-Specific Binding Molecule and A–A-Mismatch-Containing DNA

Antibodies expand the scope of angiotensin receptor pharmacology.

G-protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small-molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue and cellular levels. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays and structural studies, we develop maternally selective heavy-chain-only antibody ('nanobody') antagonists against the angiotensin II type I receptor and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to angiotensin II type I receptor with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.

Orbital-overlap-driven hybridization in 3d-transition metal perovskite oxides LaMO3 (M = Ti-Ni) and La2CuO4

The wide tunability of strongly correlated transition metal (TM) oxides stems from their complex electronic properties and the coupled degrees of freedom. Among the perovskite oxides family, LaMO3 (M = Ti-Ni) allows an M-dependent systematic study of the electronic structure within the same-structure-family motif. While most of the studies have been focusing on the 3d TMs and oxygen sites, the role of the rare-earth site has been far less explored. In this work, we use resonant inelastic X-ray scattering (RIXS) at the lanthanum N4,5 edges and density functional theory (DFT) to investigate the hybridization mechanisms in LaMO3. We link the spatial-overlap-driven hybridization to energetic-overlap-driven hybridization by comparing the RIXS chemical shifts and the DFT band widths. The scope is extended to highly covalent Ruddlesden-Popper perovskite La2CuO4 by intercalating lanthanum atoms to rock-salt layers. Our work evidences an observable contribution of localized lanthanum 5p and 4f orbitals in the band structure.

The β-subunit of tryptophan synthase is a latent tyrosine synthase.

Aromatic amino acids and their derivatives are diverse primary and secondary metabolites with critical roles in protein synthesis, cell structure and integrity, defense and signaling. All de novo aromatic amino acid production relies on a set of ancient and highly conserved chemistries. Here we introduce a new enzymatic transformation for L-tyrosine synthesis by demonstrating that the β-subunit of tryptophan synthase-which natively couples indole and L-serine to form L-tryptophan-can act as a latent 'tyrosine synthase'. A single substitution of a near-universally conserved catalytic residue unlocks activity toward simple phenol analogs and yields exclusive para carbon-carbon bond formation to furnish L-tyrosines. Structural and mechanistic studies show how a new active-site water molecule orients phenols for a nonnative mechanism of alkylation, with additional directed evolution resulting in a net >30,000-fold rate enhancement. This new biocatalyst can be used to efficiently prepare valuable L-tyrosine analogs at gram scales and provides the missing chemistry for a conceptually different pathway to L-tyrosine.

Enabling nucleophilic reactivity in molecular calcium fluoride complexes.

Calcium fluoride is the ultimate source of all fluorochemicals. Current synthetic approaches rely on the use of HF, generated from naturally occurring fluorspar and sulfuric acid. Methods for constructing E-F bonds directly from CaF2 have long been frustrated by its high lattice energy, low solubility and impaired fluoride ion nucleophilicity. Little fundamental understanding of the reactivity of Ca-F moieties is available to guide methodology development; well-defined molecular species containing Ca-F bonds are extremely rare, and existing examples are strongly aggregated and evidence no nucleophilic fluoride delivery. Here, by contrast, we show that by targeting anionic systems of the type [Ln(X)2CaF]-, monomeric calcium fluoride complexes containing single Ca-F bonds can be synthesized, including via routes involving fluoride abstraction from existing C-F bonds. Comparative structural and spectroscopic studies of mono- and dinuclear systems allow us to define structure-activity relationships for E-F bond formation from molecular calcium fluorides.

Engineering of a Baeyer-Villiger monooxygenase to improve substrate scope, stereoselectivity and regioselectivity.

Baeyer-Villiger monooxygenases belong to a family of flavin-binding proteins that catalyze the Baeyer-Villiger (BV) oxidation of ketones to produce lactones or esters, which are important intermediates in pharmaceuticals or sustainable materials. Phenylacetone monooxygenase (PAMO) from Thermobifida fusca with moderate thermostability catalyzes the oxidation of aryl ketone substrates, but is limited by high specificity and narrow substrate scope. In the present study, we applied loop optimization by loop swapping followed by focused saturation mutagenesis in order to evolve PAMO mutants capable of catalyzing the regioselective BV oxidation of cyclohexanone and cyclobutanone derivatives with formation of either normal or abnormal esters or lactones. We further modulated PAMO to increase enantioselectivity. Crystal structure studies indicate that rotation occurs in the NADP-binding domain and that the high B-factor region is predominantly distributed in the catalytic pocket residues. Computational analyses further revealed dynamic character in the catalytic pocket and reshaped hydrogen bond interaction networks, which is more favorable for substrate binding. Our study provides useful insights for studying enzyme-substrate adaptations.

A Structural Study on Absorption of Lysozyme in Amorphous Starch Microspheres.

The potential of using proteins as drugs is held back by their low stability in the human body and challenge of delivering them to the site of function. Extensive research is focused on drug delivery systems that can protect, carry, and release proteins in a controlled manner. Of high potential are cross-linked degradable starch microspheres (DSMs), as production of these is low-cost and environmentally friendly, and the products are degradable by the human body. Here, we demonstrate that DSMs can absorb the model protein lysozyme from an aqueous solution. At low amounts of lysozyme, its concentration in starch microspheres strongly exceeds the bulk concentration in water. However, at higher protein contents, the difference between concentrations in the two phases becomes small. This indicates that, at lower lysozyme contents, the absorption is driven by protein-starch interactions, which are counteracted by protein-protein electrostatic repulsion at high concentrations. By applying small-angle X-ray scattering (SAXS) to the DSM-lysozyme system, we show that lysozyme molecules are largely unaltered by the absorption in DSM. In the same process, the starch network is slightly perturbed, as demonstrated by a decrease in the characteristic chain to chain distance. The SAXS data modeling suggests an uneven distribution of the protein within the DSM particles, which can be dependent on the internal DSM structure and on the physical interactions between the components. The results presented here show that lysozyme can be incorporated into degradable starch microspheres without any dependence on electrostatic or specific interactions, suggesting that similar absorption would be possible for pharmaceutical proteins.

Quantification of native lignin structural features with gel‐phase 2D‐HSQC0 reveals lignin structural changes during extraction

Our ability to study and valorize the lignin fraction of biomass is hampered by the fundamental and still unmet challenge of precisely quantifying native lignin’s structural features. Here, we developed a rapid elevated‐temperature 1H–13C Heteronuclear Single‐Quantum Coherence Zero (HSQC0) NMR method that enables this precise quantification of native lignin structural characteristics even with whole plant cell wall (WPCW) NMR spectroscopy, overcoming fast spin relaxation in the gel phase. We also formulated a Gaussian fitting algorithm to perform automatic and reliable spectral integration. By combining HSQC0 measurements with yield measurements following depolymerization, we can confirm the combinatorial nature of radical coupling reactions during biosynthesis leading to a random sequential organization of linkages within a largely linear lignin chain. Such analyses illustrate how this analytical method can greatly facilitate the study of native lignin structure, which can then be used for fundamental studies or to understand lignin depolymerization methods like reductive catalytic fractionation or aldehyde‐assisted fractionation.

Wavy grain in sycamore maple (Acer pseudoplatanus) — a structural analysis of xylem and phloem

Summary Wavy grain (fiddle back) in trees has attracted the interest of both science and industry due to its aesthetic and economic importance. As the underlying causes of this particular structural feature in trees remain uncertain, this study investigates the possibility of non-invasively detecting wavy grain patterns within tree bark tissue of sycamore maple (Acer pseudoplatanus). Wavy grain, characterized by alternating light and dark stripes on the wood surface, is highly valued in the production of musical instruments. This research explores the potential of employing transmission light microscopy and microcomputer tomography (μCT) to examine the internal structure of phloem tissue to inspect whether the wavy grain structure found in the xylem continues in the bark. The investigation of phloem tissue proved challenging due to its irregular cellular arrangement. Nonetheless, slight phloem fibre deviations were observed in individual trees. However, no definite connection was found between the periodic oscillations of the xylem fibre tissue and the structure of the phloem in a given area. Variability in wavy grain expression among the xylem of the trees posed an additional challenge, with amplitudes and wavelengths differing across specimens. The application of μCT offered advantages over traditional sample preparation, enabling the study of the anatomical structure across larger areas and multiple depths without altering the specimen. This study sheds light on the potential of employing advanced imaging techniques to detect wavy grain patterns, providing insights that could benefit industries reliant on high-quality wood, such as musical instrument crafting and forestry.

Quantification of native lignin structural features with gel-phase 2D-HSQC0 reveals lignin structural changes during extraction.

Our ability to study and valorize the lignin fraction of biomass is hampered by the fundamental and still unmet challenge of precisely quantifying native lignin's structural features. Here, we developed a rapid elevated-temperature 1H-13C Heteronuclear Single-Quantum Coherence Zero (HSQC0) NMR method that enables this precise quantification of native lignin structural characteristics even with whole plant cell wall (WPCW) NMR spectroscopy, overcoming fast spin relaxation in the gel phase. We also formulated a Gaussian fitting algorithm to perform automatic and reliable spectral integration. By combining HSQC0 measurements with yield measurements following depolymerization, we can confirm the combinatorial nature of radical coupling reactions during biosynthesis leading to a random sequential organization of linkages within a largely linear lignin chain. Such analyses illustrate how this analytical method can greatly facilitate the study of native lignin structure, which can then be used for fundamental studies or to understand lignin depolymerization methods like reductive catalytic fractionation or aldehyde-assisted fractionation.

Antisense RNA C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms a triplex-like structure and binds small synthetic ligand.

The abnormal expansion of GGGGCC/GGCCCC hexanucleotide repeats (HR) in C9orf72 is associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Structural polymorphisms of HR result in the multifactorial pathomechanism of ALS/FTD. Consequently, many ongoing studies are focused at developing therapies targeting pathogenic HR RNA. One of them involves small molecules blocking sequestration of important proteins, preventing formation of toxic nuclear foci. However, rational design of potential therapeutics is hindered by limited number of structural studies of RNA-ligand complexes. We determined the crystal structure of antisense HR RNA in complex with ANP77 ligand (1.1 Å resolution) and in the free form (0.92 and 1.5 Å resolution). HR RNA folds into a triplex structure composed of four RNA chains. ANP77 interacted with two neighboring single-stranded cytosines to form pseudo-canonical base pairs by adopting sandwich-like conformation and adjusting the position of its naphthyridine units to the helical twist of the RNA. In the unliganded structure, the cytosines formed a peculiar triplex i-motif, assembled by trans C•C+ pair and a third cytosine located at the Hoogsteen edge of the C•C+ pair. These results extend our knowledge of the structural polymorphisms of HR and can be used for rational design of small molecules targeting disease-related RNAs.

Expression, Purification and Characterization of Recombinant Disintegrin from Gloydius Brevicaudus Venom in Escherichia Coli.

Disintegrins, a family of snake venom protein, which are capable of modulating the activity of integrins that play a fundamental role in the regulation of many physiological and pathological processes. The main purpose of this study is to obtain the recombinant disintegrin (r-DI) and evaluate its biological activity. In this study, we explored a high-level expression prokaryotic system and purification strategy for r-DI. Then, r-DI was treated to assay effects on cell growth, migration, and invasion. The affinity for the interactions of r-DI with integrin was determined using Surface plasmon resonance (SPR) analyses. The r-DI can be expressed in Escherichia coli and purified by one-step chromatography. The r-DI can inhibit B16F10 cells proliferation, migration, and invasion. Also, we found that r-DI could interact with the integrin αIIbβ3 (GPIIb/IIIa). The r-DI can be expressed, purified, characterized through functional assays, and can also maintain strong biological activities. Thus, this study showed potential therapeutic effects of r-DI for further functional and structural studies.

Structural insights into drug transport by an aquaglyceroporin

Pentamidine and melarsoprol are primary drugs used to treat the lethal human sleeping sickness caused by the parasite Trypanosoma brucei. Cross-resistance to these two drugs has recently been linked to aquaglyceroporin 2 of the trypanosome (TbAQP2). TbAQP2 is the first member of the aquaporin family described as capable of drug transport; however, the underlying mechanism remains unclear. Here, we present cryo-electron microscopy structures of TbAQP2 bound to pentamidine or melarsoprol. Our structural studies, together with the molecular dynamic simulations, reveal the mechanisms shaping substrate specificity and drug permeation. Multiple amino acids in TbAQP2, near the extracellular entrance and inside the pore, create an expanded conducting tunnel, sterically and energetically allowing the permeation of pentamidine and melarsoprol. Our study elucidates the mechanism of drug transport by TbAQP2, providing valuable insights to inform the design of drugs against trypanosomiasis.

Pikeperch muscle tissues: a comparative study of structure, enzymes, genes, and proteins in wild and farmed fish.

Pikeperch (Sander lucioperca) is a freshwater species and an internationally highly demanded fish in aquaculture. Despite intensive research efforts on this species, fundamental knowledge of skeletal muscle biology and structural characteristics is missing. Therefore, we conducted a comprehensive analysis of skeletal muscle parameters in adult pikeperch from two different origins, wild-caught specimens from a lake and those reared in a recirculating aquaculture system. The analyses comprised the biochemical characteristics (nucleic acid, protein content), enzyme activities (creatine kinase, lactate dehydrogenase, NADP-dependent isocitrate dehydrogenase), muscle-specific gene and protein expression (related to myofibre formation, regeneration and permanent growth, muscle structure), and muscle fibre structure. The findings reveal distinct differences between the skeletal muscle of wild and farmed pikeperch. Specifically, nucleic acid content, enzyme activity, and protein expression varied significantly. The higher enzyme activity observed in wild pikeperch suggests greater metabolically activity in their muscles. Conversely, farmed pikeperch indicated a potential for pronounced muscle growth. As the data on pikeperch skeletal muscle characteristics is sparse, the purpose of our study is to gain fundamental insights into the characteristics of adult pikeperch muscle. The presented data serve as a foundation for further research on percids' muscle biology and have the potential to contribute to advancements and adaptations in aquaculture practices.

Anion-Binding Properties of Short Linear Homopeptides

A comprehensive thermodynamic and structural study of the complexation affinities of tetra (L1), penta (L2), and hexaphenylalanine (L3) linear peptides towards several inorganic anions in acetonitrile (MeCN) and N,N-dimethylformamide (DMF) was carried out. The influence of the chain length on the complexation thermodynamics and structural changes upon anion binding are particularly addressed here. The complexation processes were characterized by means of spectrofluorimetric, 1H NMR, microcalorimetric, and circular dichroism spectroscopy titrations. The results indicate that all three peptides formed complexes of 1:1 stoichiometry with chloride, bromide, hydrogen sulfate, dihydrogen phosphate (DHP), and nitrate anions in acetonitrile and DMF. In the case of hydrogen sulfate and DHP, anion complexes of higher stoichiometries were observed as well, namely those with 1:2 and 2:1 (peptide:anion) complexes. Anion-induced peptide backbone structural changes were studied by molecular dynamic simulations. The anions interacted with backbone amide protons and one of the N-terminal amine protons through hydrogen bonding. Due to the anion binding, the main chain of the studied peptides changed its conformation from elongated to quasi-cyclic in all 1:1 complexes. The accomplishment of such a conformation is especially important for cyclopeptide synthesis in the head-to-tail macrocyclization step, since it is most suitable for ring closure. In addition, the studied peptides can act as versatile ionophores, facilitating transmembrane anion transport.