Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol.

Methanol, being electron rich and derivable from methane or CO2, is a potentially renewable one-carbon (C1) feedstock for microorganisms. Although the ribulose monophosphate (RuMP) cycle used by methylotrophs to assimilate methanol differs from the typical sugar metabolism by only three enzymes, turning a non-methylotrophic organism to a synthetic methylotroph that grows to a high cell density has been challenging. Here we reprogrammed E. coli using metabolic robustness criteria followed by laboratory evolution to establish a strain that can efficiently utilize methanol as the sole carbon source. This synthetic methylotroph alleviated a so far uncharacterized hurdle, DNA-protein crosslinking (DPC), by insertion sequence (IS)-mediated copy number variations (CNVs) and balanced the metabolic flux by mutations. Being capable of growing at a rate comparable with natural methylotrophs in a wide range of methanol concentrations, this synthetic methylotrophic strain illustrates genome editing and evolution for microbial tropism changes and expands the scope of biological C1 conversion.

Methylation of elongation factor 1A by yeast Efm4 or human eEF1A-KMT2 involves a beta-hairpin recognition motif and crosstalks with phosphorylation.

Translation elongation factor 1A (eEF1A) is an essential and highly conserved protein required for protein synthesis in eukaryotes. In both Saccharomyces cerevisiae and human, five different methyltransferases methylate specific residues on eEF1A, making eEF1A the eukaryotic protein targeted by the highest number of dedicated methyltransferases after histone H3. eEF1A methyltransferases are highly selective enzymes, only targeting eEF1A and each targeting just one or two specific residues in eEF1A. However, the mechanism of this selectivity remains poorly understood. To reveal how S. cerevisiae elongation factor methyltransferase 4 (Efm4) specifically methylates eEF1A at K316, we have used AlphaFold-Multimer modeling in combination with crosslinking mass spectrometry (XL-MS) and enzyme mutagenesis. We find that a unique beta-hairpin motif, which extends out from the core methyltransferase fold, is important for the methylation of eEF1A K316 in vitro. An alanine mutation of a single residue on this beta-hairpin, F212, significantly reduces Efm4 activity in vitro and in yeast cells. We show that the equivalent residue in human eEF1A-KMT2 (METTL10), F220, is also important for its activity towards eEF1A in vitro. We further show that the eEF1A guanine nucleotide exchange factor, eEF1Bα, inhibits Efm4 methylation of eEF1A in vitro, likely due to competitive binding. Lastly, we find that phosphorylation of eEF1A at S314 negatively crosstalks with Efm4-mediated methylation of K316. Our findings demonstrate how protein methyltransferases can be highly selective towards a single residue on a single protein in the cell.

A multiscale approach reveals the molecular architecture of the autoinhibited kinesin KIF5A.

Kinesin-1 is a microtubule motor that transports cellular cargo along microtubules. KIF5A is one of three kinesin-1 isoforms in humans, all of which are autoinhibited by an interaction between the motor and an IAK motif in the proximal region of the C-terminal tail. The C-terminal tail of KIF5A is ∼80 residues longer than the other two kinesin-1 isoforms (KIF5B and KIF5C) and it is unclear if it contributes to autoinhibition. Mutations in KIF5A cause neuronal diseases and could affect autoinhibition, as reported for a mutation that skips exon 27, altering its C-terminal sequence. Here, we combined negative-stain electron microscopy, crosslinking mass spectrometry (XL-MS) and AlphaFold2 structure prediction to determine the molecular architecture of the full-length autoinhibited KIF5A homodimer, in the absence of light chains. We show that KIF5A forms a compact, bent conformation, through a bend between coiled-coils 2 and 3, around P687. XL-MS of WT KIF5A revealed extensive interactions between residues in the motor, between coiled-coil 1 and the motor, between coiled-coils 1 and 2, with coiled-coils 3 and 4, and the proximal region of the C-terminal tail and the motor in the autoinhibited state, but not between the distal C-terminal region and the rest of the molecule. While negative-stain electron microscopy of exon-27 KIF5A splice mutant showed the presence of autoinhibited molecules, XL-MS analysis suggested that its autoinhibited state is more labile. Our model offers a conceptual framework for understanding how mutations within the motor and stalk domain may affect motor activity.

Egg envelope formation of medaka Oryzias latipes requires ZP proteins originating from both the liver and ovary.

Teleost oocytes are surrounded by a structure called chorion or egg envelopes, which is composed of zona pellucida (ZP) proteins. As a result of the gene duplication in teleost, the expression site of the zp genes, coding the major component protein of egg envelopes, changed from the ovary to the maternal liver. In Euteleostei, there are three liver-expressed zp genes, named choriogenin (chg) h, chg hm, and chg l, and the composition of the egg envelope is mostly made up of these Chgs. In addition, ovary-expressed zp genes are also conserved in the medaka genomes, and their proteins have also been found to be minor components of the egg envelopes. However, the specific role of liver-expressed versus ovary-expressed zp genes was unclear. In the present study, we showed that ovary-synthesized ZP proteins first form the base layer of the egg envelope and then Chgs polymerize inwardly to thicken the egg envelope. To analyze the effects of dysfunction of the chg gene, we generated some chg knockout medaka. All knockout females failed to produce normally fertilized eggs by the natural spawning. The egg envelopes lacking Chgs were significantly thinner, but layers formed by ZP proteins synthesized in the ovary were found in the thin egg envelope of knockout as well as wildtype eggs. These results suggest that the ovary-expressed zp gene is well conserved in all teleosts, including those species in which liver-derived ZP proteins are the major component, because it is essential for the initiation of egg envelope formation.

Structural basis of bidirectional allostery across the heme in a cytochrome P450 enzyme.

Cytochromes P450 (CYPs) are heme-containing enzymes that are present in all kingdoms of life and share a structurally homologous, globular protein fold. CYPs utilize structures distal to the heme to recognize and coordinate substrates, while the necessary interactions with redox partner proteins are mediated at the opposite, proximal surface. In the current study, we investigated the functional allostery across the heme for the bacterial enzyme CYP121A1, which utilizes a non-polar distal-to-distal dimer interface for specific binding of its dicyclotyrosine substrate. Fluorine-detected Nuclear Magnetic Resonance (19F-NMR) spectroscopy was combined with site-specific labeling of a distal surface residue (S171C of the FG-loop), one residue of the B-helix (N84C), and two proximal surface residues (T103C and T333C) with a thiol-reactive fluorine label. Adrenodoxin was used as a substitute redox protein and was found to promote a closed arrangement of the FG-loop, similar to the addition of substrate alone. Disruption of the protein-protein interface by mutagenesis of two CYP121 basic surface residues removed the allosteric effect. Moreover, 19F-NMR spectra of the proximal surface indicate that ligand-induced allostery modulates the environment at the C-helix but not the meander region of the enzyme. In light of the high degree of structural homology in this family of enzymes, we interpret the findings from this work to represent a conserved allosteric network in CYPs.

Extracellular intersubunit interactions modulate epithelial Na+ channel gating.

Epithelial Na+ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the ß-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking ßE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging ßE499C to αN510C or αQ441C altered the Na+ self-inhibition response via distinct mechanisms. Similar to bridging ßE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation.

Autoreactive B cells against malondialdehyde-induced protein cross-links are present in the joint, lung, and bone marrow of rheumatoid arthritis patients.

Autoantibodies to malondialdehyde (MDA) proteins constitute a subset of anti-modified protein autoantibodies in rheumatoid arthritis (RA), which is distinct from citrulline reactivity. Serum anti-MDA IgG levels are commonly elevated in RA and correlate with disease activity, CRP, IL6, and TNF-α. MDA is an oxidation-associated reactive aldehyde that together with acetaldehyde mediates formation of various immunogenic amino acid adducts including linear MDA-lysine, fluorescent malondialdehyde acetaldehyde (MAA)-lysine, and intramolecular cross-linking. We used single-cell cloning, generation of recombinant antibodies (n = 356 from 25 donors), and antigen-screening to investigate the presence of class-switched MDA/MAA+ B cells in RA synovium, bone marrow, and bronchoalveolar lavage. Anti-MDA/MAA+ B cells were found in bone marrow plasma cells of late disease and in the lung of both early disease and risk-individuals and in different B cell subsets (memory, double negative B cells). These were compared with previously identified anti-MDA/MAA from synovial memory and plasma cells. Seven out of eight clones carried somatic hypermutations and all bound MDA/MAA-lysine independently of protein backbone. However, clones with somatic hypermutations targeted MAA cross-linked structures rather than MDA- or MAA-hapten, while the germline-encoded synovial clone instead bound linear MDA-lysine in proteins and peptides. Binding patterns were maintained in germline converted clones. Affinity purification of polyclonal anti-MDA/MAA from patient serum revealed higher proportion of anti-MAA versus anti-MDA compared to healthy controls. In conclusion, IgG anti-MDA/MAA show distinct targeting of different molecular structures. Anti-MAA IgG has been shown to promote bone loss and osteoclastogenesis in vivo and may contribute to RA pathogenesis.

Pathogenetic Contributions and Therapeutic Implications of Transglutaminase 2 in Neurodegenerative Diseases.

Neurodegenerative diseases encompass a heterogeneous group of disorders that afflict millions of people worldwide. Characteristic protein aggregates are histopathological hallmark features of these disorders, including Amyloid ß (Aß)-containing plaques and tau-containing neurofibrillary tangles in Alzheimer's disease, α-Synuclein (α-Syn)-containing Lewy bodies and Lewy neurites in Parkinson's disease and dementia with Lewy bodies, and mutant huntingtin (mHTT) in nuclear inclusions in Huntington's disease. These various aggregates are found in specific brain regions that are impacted by neurodegeneration and associated with clinical manifestations. Transglutaminase (TG2) (also known as tissue transglutaminase) is the most ubiquitously expressed member of the transglutaminase family with protein crosslinking activity. To date, Aß, tau, α-Syn, and mHTT have been determined to be substrates of TG2, leading to their aggregation and implicating the involvement of TG2 in several pathophysiological events in neurodegenerative disorders. In this review, we summarize the biochemistry and physiologic functions of TG2 and describe recent advances in the pathogenetic role of TG2 in these diseases. We also review TG2 inhibitors tested in clinical trials and discuss recent TG2-targeting approaches, which offer new perspectives for the design of future highly potent and selective drugs with improved brain delivery as a disease-modifying treatment for neurodegenerative disorders.

Transglutaminases in fibrosis-overview and recent advances.

Transglutaminases (TGs) are a family of protein cross-linking enzymes that are capable of stiffening and insolubilizing proteins and creating protein networks, and thereby altering biological functions of proteins. Their role in fibrosis progression has been widely investigated with a focus on kidney, lung, liver, and heart where activity is triggered by various stimuli including hypoxia, inflammation, and hyperglycemia. TG2 has been considered one of the key enzymes in the pathogenesis of fibrosis mainly through transforming growth factor beta (TGF-beta) signaling and matrix cross-linking mechanisms. Although TG2 has been most widely studied in this context, the involvement of other TGs, TG1 and Factor XIII-A (FXIII-A), is beginning to emerge. This mini-review highlights the major steps taken in the TG and fibrosis research and summarizes the most recent advances and contributions of TG2, TG1, and FXIII-A to the progression of fibrosis in various animal models. Also, their mechanisms of action as well as therapeutic prospects are discussed.

Implications of conformational flexibility, lipid binding, and regulatory domains in cell-traversal protein CelTOS for apicomplexan migration.

Malaria and other apicomplexan-caused diseases affect millions of humans, agricultural animals, and pets. Cell traversal is a common feature used by multiple apicomplexan parasites to migrate through host cells and can be exploited to develop therapeutics against these deadly parasites. Here, we provide insights into the mechanism of the Cell-traversal protein for ookinetes and sporozoites (CelTOS), a conserved cell-traversal protein in apicomplexan parasites and malaria vaccine candidate. CelTOS has previously been shown to form pores in cell membranes to enable traversal of parasites through cells. We establish roles for the distinct protein regions of Plasmodium vivax CelTOS and examine the mechanism of pore formation. We further demonstrate that CelTOS dimer dissociation is required for pore formation, as disulfide bridging between monomers inhibits pore formation, and this inhibition is rescued by disulfide-bridge reduction. We also show that a helix-destabilizing amino acid, Pro127, allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C terminus of CelTOS is a negative regulator that limits pore formation. Finally, we highlight that lipid binding is a prerequisite for pore assembly as mutation of a phospholipids-binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. These findings identify critical regions in CelTOS and will aid in understanding the egress mechanism of malaria and other apicomplexan parasites as well as have implications for studying the function of other essential pore-forming proteins.

Inner membrane YfgM-PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation.

PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD-YfgM complex formation as well as the molecular mechanisms of PpiD-YfgM and PpiD/YfgM-Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD-YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon.

Characterization of interactions within the Igα/Igß transmembrane domains of the human B-cell receptor provides insights into receptor assembly.

The B-cell receptor (BCR), a complex comprised of a membrane-associated immunoglobulin and the Igα/ß heterodimer, is one of the most important immune receptors in humans and controls B-cell development, activity, selection, and death. BCR signaling plays key roles in autoimmune diseases and lymphoproliferative disorders, yet, despite the clinical significance of this protein complex, key regions (i.e., the transmembrane domains) have yet to be structurally characterized. The mechanism for BCR signaling also remains unclear and has been variously described by the mutually exclusive cross-linking and dissociation activation models. Common to these models is the significance of local plasma membrane composition, which implies that interactions between BCR transmembrane domains (TMDs) play a role in receptor functionality. Here we used an in vivo assay of TMD oligomerization called GALLEX alongside spectroscopic and computational methods to characterize the structures and interactions of human Igα and Igß TMDs in detergent micelles and natural membranes. We observed weak self-association of the Igß TMD and strong self-association of the Igα TMD, which scanning mutagenesis revealed was entirely stabilized by an E-X10-P motif. We also demonstrated strong heterotypic interactions between the Igα and Igß TMDs both in vitro and in vivo, which scanning mutagenesis and computational models suggest is multiconfigurational but can accommodate distinct interaction sites for self-interactions and heterotypic interactions of the Igα TMD. Taken together, these results demonstrate that the TMDs of the human BCR are sites of strong protein-protein interactions that may direct BCR assembly, endoplasmic reticulum retention, and immune signaling.

The cysteine residue in beta-lactoglobulin reacts with oxidized tyrosine residues in beta-casein to give casein-lactoglobulin dimers.

Proteins are modified during milk processing and storage, with sidechain oxidation and crosslinking being major consequences. Despite the prevalence and importance of proteins in milk, and particularly caseins (∼80% of total content), the nature of the cross-links formed by oxidation, and their mechanisms of formation, are poorly characterized. In this study, we investigated the formation and stability of cross-links generated by the nucleophilic addition of Cys residues to quinones generated on oxidation of Tyr residues. The mechanisms and stability of these adducts was explored using ubiquitin as a model protein, and ß-casein. Ubiquitin and ß-casein were oxidized using a rose Bengal/visible light/O2 system, or by the enzyme tyrosinase. The oxidized proteins were incubated with glutathione or ß-lactoglobulin (non-oxidized, but unfolded by treatment at 70 °C), before analysis by SDS-PAGE, immunoblotting and LC-MS. Our data indicate that Cys-quinone adducts are readily-formed, and are stable for >48 h. Thus, oxidized ß-casein reacts efficiently with the thermally unfolded ß-lactoglobulin, likely via Michael addition of the exposed Cys to a Tyr-derived quinone. These data provide a novel, and possibly general, mechanism of protein cross-link formation, and provides information of the stability of these species that have potential as markers of protein quality.

Piezo1 channel causes lens sclerosis via transglutaminase 2 activation.

Presbyopia is caused by age-related lenticular hardening, resulting in near vision loss, and it occurs in almost every individual aged ≥50 years. The lens experiences mechanical pressure during for focal adjustment to change its thickness. As lenticular stiffening results in incomplete thickness changes, near vision is reduced, which is known as presbyopia. Piezo1 is a mechanosensitive channel that constantly senses pressure changes during the regulation of visual acuity, and changes in Piezo1 channel activity may contribute to presbyopia. However, no studies have reported on Piezo1 activation or the onset of presbyopia. To elucidate the relevance of Piezo1 activation and cross-linking in the development of presbyopia, we analysed the function of Piezo1 in the lens. The addition of Yoda1, a Piezo1 activator, induced an increase in transglutaminase 2 (TGM2) mRNA expression and activity through the extra-cellular signal-regulated kinase (ERK) 1/2 and c-Jun-NH2-terminal kinase1/2 pathways. In ex vivo lenses, Yoda1 treatment induced γ-crystallin cross-linking via TMG2 activation. Furthermore, Yoda1 eye-drops in mice led to lenticular hardening via TGM2 induction and activation in vivo, suggesting that Yoda1-treated animals could serve as a model for presbyopia. Our findings indicate that this presbyopia-animal model could be useful for screening drugs for lens-stiffening inhibition.

Proximity-enhanced protein crosslinking through an alkene-tetrazine reaction.

The inverse electron demand Diels-Alder (iEDDA) reaction between a tetrazine and a strained alkene has been widely explored as useful bioorthogonal chemistry for selective labeling of biomolecules. In this work, we exploit the slow reaction between a non-conjugated terminal alkene and a tetrazine, and apply this reaction to achieving a proximity-enhanced protein crosslinking. In one protein subunit, a terminal alkene-containing amino acid was site-specifically incorporated in response to an amber nonsense codon. In another protein subunit, a tetrazine moiety was introduced through the attachment to a cysteine residue. Fast protein crosslinking was achieved due to a large increase in effective molarity of the two reactants that were brought to close proximity by the two interacting protein subunits. Such a proximity-enhanced protein crosslinking is useful for the study of protein-protein interactions.

Structural Investigations of Human A2M Identify a Hollow Native Conformation That Underlies Its Distinctive Protease-Trapping Mechanism.

Human α2-macroglobulin (A2M) is the most characterized protease inhibitor in the alpha-macroglobulin (αM) superfamily, but the structure of its native conformation has not been determined. Here, we combined negative stain electron microscopy (EM), small-angle X-ray scattering (SAXS), and cross-linking-mass spectrometry (XL-MS) to investigate native A2M and its collapsed conformations that are obtained through aminolysis of its thiol ester by methylamine or cleavage of its bait region by trypsin. The combined interpretation of these data resulted in a model of the native A2M tetramer and its conformational changes. Native A2M consists of two crescent-shaped disulfide-bridged subunit dimers, which face toward each other and surround a central hollow space. In native A2M, interactions across the disulfide-bridged dimers are minimal, with a single major interface between the linker (LNK) regions of oppositely positioned subunits. Bait region cleavage induces both intrasubunit domain repositioning and an altered configuration of the disulfide-bridged dimer. These changes collapse the tetramer into a more compact conformation, which encloses an interior protease-trapping cavity. A recombinant A2M with a modified bait region was used to map the bait region's position in native A2M by XL-MS. A second recombinant A2M introduced an intersubunit disulfide into the LNK region, demonstrating the predicted interactions between these regions in native A2M. Altogether, our native A2M model provides a structural foundation for understanding A2M's protease-trapping mechanism, its conformation-dependent receptor interactions, and the dissociation of native A2M into dimers due to inflammatory oxidative stress.

Photosealing of dural defects using a biocompatible patch.

PURPOSE: Photosealing of many biological tissues can be achieved using a biocompatible material in combination with a dye that is activated by visible light to chemically bond over the tissue defect via protein cross-linking reactions. The aim of this study was to test the efficacy of photosealing using a commercially available biomembrane (AmnioExcel Plus) to securely close dural defects in comparison to another sutureless method (fibrin glue) in terms of repair strength. METHODS: Two-millimeter diameter holes were created in dura harvested from New Zealand white rabbits and repaired ex vivo using one of two methods: (1) in n = 10 samples, photosealing was used to bond a 6-mm-diameter AmnioExcel Plus patch over the dural defect, and (2) in n = 10 samples, fibrin glue was used to attach the same patch over the dural defect. Repaired dura samples were then subjected to burst pressure testing. Histological analysis was also performed of photosealed dura. RESULTS: The mean burst pressures of rabbit dura repaired with photosealing and fibrin glue were 302 ± 149 mmHg and 26 ± 24 mmHg, respectively. The increased repair strength using photosealing was statistically significant and considerably higher than the normal intracranial pressure of ~ 20 mmHg. Histology demonstrated a tight union at the interface between the dura surface and patch with no disruption of the dura structure. CONCLUSION: The results of this study suggest that photosealing performs better than fibrin glue for the fixation of a patch for ex vivo repair of small dural defects. Photosealing is worthy of testing in pre-clinical models for the repair of dural defects.

Construction of metal-organic framework-based multienzyme system for L-tert-leucine production.

Chiral compounds are important drug intermediates that play a critical role in human life. Herein, we report a facile method to prepare multi-enzyme nano-devices with high catalytic activity and stability. The self-assemble molecular binders SpyCatcher and SpyTag were fused with leucine dehydrogenase and glucose dehydrogenase to produce sc-LeuDH (SpyCatcher-fused leucine dehydrogenase) and GDH-st (SpyTag-fused glucose dehydrogenase), respectively. After assembling, the cross-linked enzymes LeuDH-GDH were formed. The crosslinking enzyme has good pH stability and temperature stability. The coenzyme cycle constant of LeuDH-GDH was always higher than that of free double enzymes. The yield of L-tert-leucine synthesis by LeuDH-GDH was 0.47 times higher than that by free LeuDH and GDH. To further improve the enzyme performance, the cross-linked LeuDH-GDH was immobilized on zeolite imidazolate framework-8 (ZIF-8) via bionic mineralization, forming LeuDH-GDH @ZIF-8. The created co-immobilized enzymes showed even better pH stability and temperature stability than the cross-linked enzymes, and LeuDH-GDH@ZIF-8 retains 70% relative conversion rate in the first four reuses. In addition, the yield of LeuDH-GDH@ZIF-8 was 0.62 times higher than that of LeuDH-GDH, and 1.38 times higher than that of free double enzyme system. This work provides a novel method for developing multi-enzyme nano-device, and the ease of operation of this method is appealing for the construction of other multi-enzymes @MOF systems for the applications in the kinds of complex environment.

Unnatural Amino Acid Crosslinking for Increased Spatiotemporal Resolution of Chromatin Dynamics.

The utilization of an expanded genetic code and in vivo unnatural amino acid crosslinking has grown significantly in the past decade, proving to be a reliable system for the examination of protein-protein interactions. Perhaps the most utilized amino acid crosslinker, p-benzoyl-(l)-phenylalanine (pBPA), has delivered a vast compendium of structural and mechanistic data, placing it firmly in the upper echelons of protein analytical techniques. pBPA contains a benzophenone group that is activated with low energy radiation (~365 nm), initiating a diradical state that can lead to hydrogen abstraction and radical recombination in the form of a covalent bond to a neighboring protein. Importantly, the expanded genetic code system provides for site-specific encoding of the crosslinker, yielding spatial control for protein surface mapping capabilities. Paired with UV-activation, this process offers a practical means for spatiotemporal understanding of protein-protein dynamics in the living cell. The chromatin field has benefitted particularly well from this technique, providing detailed mapping and mechanistic insight for numerous chromatin-related pathways. We provide here a brief history of unnatural amino acid crosslinking in chromatin studies and outlooks into future applications of the system for increased spatiotemporal resolution in chromatin related research.

Manipulation of protein structure and bonding pattern to improve the gelling and textural quality of surimi gels from silver carp: incorporation of mosambi (Citrus limetta) peel extract.

BACKGROUND: This investigation focused on the use of mosambi peel extract (MPE) fortification (at 0% to 1.50%, w/w) in silver carp surimi to improve the gelling, textural, and other physicochemical properties of the surimi. RESULTS: The peels were extracted in ethanol (40-100% concentrations, v/v) and water. It was found that 100% ethanol had significantly (P < 0.05) higher yield and total phenolic, flavonoid, and tannin content. The fortification of MPE at optimum level (0.75%) improved the breaking force (55.1%) and gel strength (89.9%) significantly (P < 0.05) in comparison with 0% MPE gel samples. Moreover, 0.75% MPE-fortified gels had higher hydrogen and hydrophobic bonds, higher water-holding capacity, and lower sulfhydryl groups and free amino groups. The myosin heavy chain (MHC) bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) disappeared completely in the MPE-fortified gels. Fortification with MPE affected the secondary structures of protein as shifting of peaks was observed in Fourier-transform infrared (FTIR) spectra. Scanning electron microscopy (SEM) images showed relatively organized finer and denser gel networks in MPE-treated gels. CONCLUSION: The surimi gels fortified with 0.75% MPE demonstrated improved gelling properties, with an overall higher acceptability than the unfortified gels (0% MPE). The fortified gels also became enriched with bioactive polyphenols, which are generally not present in surimi. This study provides an efficient way to utilize mosambi peel to develop functional surimi and surimi-based products with improved gel ability. © 2023 Society of Chemical Industry.