Discovery and development of penicillin-binding protein-type thioesterases as biocatalysts.

One of the main targets of the structural characterization of elastomers so far has been the correlation of the polymerization conditions with the properties of the resulting polymers. The first step is the analysis of polymer structure, such as the chemical composition of copolymers, isomeric structure of diene polymers, degree of branching, extent of modification, functionality of end groups, amounts of abnormal groups, tacticity, and so on. Progress in nuclear magnetic resonance spectroscopy (NMR) makes possible the second step, which is the structural characterization of polymer chains, such as the sequence distribution of comonomer units in copolymer, isomeric units in diene polymers, configurational sequences in vinyl polymers, head and tail arrangement of monomer units. Recent development of FT-NMR spectroscopy, high-field spectroscopy from 300 MHz to 600 MHz at 1H-NMR, solid-state 13C-NMR, and two-dimensional NMR has facilitated a more detailed analysis of these structural features. The complexity of the structure of elastomers, which is derived from highly controlled copolymerization processes, leads to the widespread application of modern FT-NMR spectroscopy. It may reasonably be said that a fair number of structural problems in elastomers has been solved by NMR analysis. The high sensitivity of Fourier-transform infrared spectroscopy (FT-IR) has enabled one to determine trace structural changes in elastomers. Coupled on-line techniques such as gas-chromatography-mass-spectrometry combined with pyrolysis, liquid chromatography-NMR, and gel permeation chromatography-FT-IR will be powerful tools for the characterization of elastomers. Progress in analytical instruments has stimulated the development of high-performance elastomers, the synthesis of new speciality elastomers, and the elucidation of mechanisms for property enhancements. The use of modern instruments and a combination of ordinary methods of structural analysis have satisfied needs to some extent. However, a newer method of structural characterization is always desired in order to achieve higher orders of information. for example, the characterization of inhomogeneity along the polymer chain and between the polymer chains has become an important problem, especially in polymer blend systems. As to the former problem, spectroscopic methods provide only limited information. Although the NMR and FT-IR spectroscopies are very powerful tools for the analysis of short sequence distributions, it is difficult to characterize the distribution of long sequences and hybrid systems containing random and blocked sequences along the polymer chain. Gel permeation chromatography (GPC) is one of the most popular techniques for the analysis of molecular-weight distribution. However, it provides complicated information including molecular-weight distribution and chemical-composition distribution in the case of copolymers. Recent progress of high-performance liquid chromatography (HPLC) has provided a new powerful tool for the structural characterization of copolymers. It is appropriate to review the recent advances in structural characterization of elastomers, especially the characterization of microstructure of polymer chains, from the viewpoints of methodology and applicability of new methods. As to the NMR analysis of elastomers, reviews are available. Here, considerable attention is focused on the procedures for the assignment of signals, because the applicability of a NMR method is based on the reliability of the signal assignments. The other topics are selected to provide direct information for polymer synthesis or polymer properties.

Volume‐regulated anion channels (VRACs) encoded by the LRRC8 gene family play essential roles in diverse and fundamentally important physiological processes in vertebrate cells. The recent determination of high‐resolution cryo‐electron microscopy (cryo‐EM) structures of homomeric and heteromeric LRRC8 channel complexes has created unprecedented opportunities for understanding the molecular basis of VRAC structure, function and pharmacology. Native LRRC8 channels are obligatory heteromers composed of at least one LRRC8A subunit together with one of the other paralogues (LRRC8B‐E) with an unknown stoichiometry. This heteromeric nature of endogenously expressed VRACs and the difficulties associated with controlling the composition and stoichiometry of heterologously expressed LRRC8 channels present considerable experimental challenges. The development of LRRC8 chimeras, which exhibit normal functional and regulatory properties and that can be expressed as homomeric channels, circumvents many of these challenges. In this review, we discuss the recent advances in the structural characterization of LRRC8 channels, with a primary focus on the cryo‐EM structures of one such chimera, created by swapping 25 residues from LRRC8A subunits to LRRC8C subunits and termed as 8C‐8A(IL125).

Nucleation and crystallization of globular proteins — what we know and what is missing

Structural characterization of rubber from Hevea brasiliensis (NR) has been carried out to elucidate the biosynthesis mechanism of rubber molecule as well as to find the relationship between physical properties characteristic of NR and its structure. Recent advances of structural studies have provided a series of new information on the structure of long-chain branching based on the result of selective decomposition of branch-points by chemical and enzymatic treatments as well as physical treatment such as polar solvent treatments and washing of NR latex by centrifugation in the presence of a surfactant. The measurement of the resulting rubber with NMR, FTIR, SEC and dilute solution viscosity provided confirming evidence that the initiating terminal (ω-terminal) with an unidentified functional group and phosphate terminal (α-terminal) form branch-points by hydrogen bond, ionic bond or micelle formation of phospholipids linked to both terminal groups. Based on these results, the origin of green properties characteristic of NR has been explained and a new mechanism of storage hardening has been proposed. The purification method by saponification of NR latex developed for the structural characterization has been applied to produce purified NR latex free from Type I allergic reaction. Instantaneous coagulation of saponified latex by the use of a flocculant and formic acid has provided solid saponified NR having good green and cured rubber properties.

This paper presents some recent advances in structural characterization of periodic inversion domains in ferroelectric nonlinear optical crystals using novel techniques such as diffraction-space mapping, diffuse scattering imaging and phase-contrast imaging. The great advantages of using both highly coherent hard x-ray photons delivered by the third-generation synchrotron source and the well-defined x-ray beam provided by state-of-the-art laboratory diffractometer are addressed in terms of investigation of periodically domain-inverted LiNbO3 crystals. The microstructural characteristics of the periodic inversion domains in LiNbO3 are discussed, with an indication of their differences from naturally-occurring inversion domains.

Confluence of structural and chemical biology: plant polyketide synthases as biocatalysts for a bio-based future

Small-angle X-ray scattering (SAXS) is a method for examining the solution structure, oligomeric state, conformational changes, and flexibility of biomacromolecules at a scale ranging from a few Angstroms to hundreds of nanometers. Wide time scales ranging from real time (milliseconds) to minutes can be also covered by SAXS. With many advantages, SAXS has been extensively used, it is widely used in the structural characterization of biomacromolecules in food science and technology. However, the application of SAXS in charactering the structure of food biomacromolecules has not been reviewed so far. In the current review, the principle, theoretical calculations and modeling programs are summarized, technical advances in the experimental setups and corresponding applications of in situ capabilities: combination of chromatography, time-resolved, temperature, pressure, flow-through are elaborated. Recent applications of SAXS for monitoring structural properties of biomacromolecules in food including protein, carbohydrate and lipid are also highlighted, and limitations and prospects for developing SAXS based on facility upgraded and artificial intelligence to study the structural properties of biomacromolecules are finally discussed. Future research should focus on extending machine time, simplifying SAXS data treatment, optimizing modeling methods in order to achieve an integrated structural biology based on SAXS as a practical tool for investigating the structure-function relationship of biomacromolecules in food industry.

Recent advances in structural characterization of the HIV envelope glycoprotein (Env) have provided a high-resolution glimpse of the architecture of this target for neutralizing antibodies and the machinery responsible for mediating receptor binding and membrane fusion. These structures primarily capture the detailed organization of the receptor-naive, prefusion conformation of Env, but under native solution conditions Env is highly dynamic, sampling multiple conformational states as well as exhibiting local protein flexibility. Special emphasis is placed on the use of biophysical methods, including single-molecule fluorescence microscopy and hydrogen/deuterium-exchange mass spectrometry. Using novel biophysical approaches, striking isolate-specific differences in Env's dynamic profile have been revealed that appear to underlie phenotypic differences of the viral isolates such as neutralization sensitivity and CD4 receptor reactivity. Structural studies are complemented by novel biophysical investigations that enable visualization of the dynamics of HIV-1 Env under native conditions. These approaches will also enable us to gain new insights into the mechanisms of action of antibodies and drugs.

Macrolactamization of Glycosylated Peptide Thioesters by the Thioesterase Domain of Tyrocidine Synthetase

9] Solid-phase synthesis of cyclic homodetic peptides

V - THE SYNTHESIS OF CYCLIC PEPTIDES

Cyclic peptides exhibit advantages in binding protein targets with high affinity and competency in inhibiting protein-protein interactions. Cyclic peptide phage display with more than a billion variants is an invaluable tool in drug discovery. However, achieving efficient peptide cyclization on phages remains a challenge because of the limited availability of reaction sites, which also restrict scaffold diversity. Here, we report an isothiocyanate-derived cross-linker featuring dual reactive groups: a bromide that covalently attaches to cysteine thiols and a thiocyanogen that selectively forms a thiourea bridge with either the N-terminal amino group or ε-amines of lysine, depending on pH. This strategy enables pH-modulated cyclization. At pH 6.5, head-to-side chain cyclization occurs, and at pH 9.5, side chain-to-side chain ligation is performed. Both processes simultaneously generate thiourea scaffolds. To demonstrate the versatility and biocompatibility of this approach, we constructed cyclic peptide libraries using both cyclization methods and successfully selected binders for several targets, including cyclophilin D, murine double minute 2, and Keap1, with dissociation constants ranging from micromolar to nanomolar. Given the broad pharmacological potential of the thiourea moiety, this phage display library opens previously unidentified chemical space with high scaffold diversity and the integration of a proven pharmacophore for the development of cyclic peptide therapeutics.

In this article, I reflect on research on two ATPases. The first is F(1)F(0)-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization.

Cyclic peptides have been attracting a lot of attention in recent decades, especially in the area of drug discovery, as more and more naturally occurring cyclic peptides with diverse biological activities have been discovered. Chemical synthesis of cyclic peptides is essential when studying their structure-activity relationships. Conventional peptide cyclization methods via direct coupling have inherent limitations, like the susceptibility to epimerization at the C-terminus, poor solubility of fully protected peptide precursors, and low yield caused by oligomerization. In this regard, chemoselective ligation-mediated cyclization methods have emerged as effective strategies for cyclic peptide synthesis. The toolbox for cyclic peptide synthesis has been expanded substantially in the past two decades, allowing more efficient synthesis of cyclic peptides with various scaffolds and modifications. This Review will explore different chemoselective ligation technologies used for cyclic peptide synthesis that generate both native and unnatural peptide linkages. The practical issues and limitations of different methods will be discussed. The advance in cyclic peptide synthesis will benefit the biological and medicinal study of cyclic peptides, an important class of macrocycles with potentials in numerous fields, notably in therapeutics.

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