Cross-linking Amino Acids Research Articles

Superior proton conductivity of amino acid-modified UiO-66-(COOH)2 embedded in chitosan: Mechanistic insights into the acid-base interactions

We focus on optimizing acid-base interactions and hydrogen bonding networks to achieve enhanced proton conductivity under low relative humidity (RH) and high temperature. By functionalizing UiO-66-(COOH)2 with glutamic acid (Glu) and lysine (Lys), we generate Glu-UiO-66-(COOH)2 and Lys-UiO-66-(COOH)2. These modified materials are subsequently incorporated into chitosan (CS) to produce the composites Glu-UiO-66-(COOH)2@CS and Lys-UiO-66-(COOH)2@CS. The successful incorporation of amino acids and cross-linking between -COOH and -NH2 groups in the composites, confirmed by FTIR and PXRD analyses, significantly enhances the structural integrity and durability by strengthening the network, and reducing polymer chain mobility. Proton conductivity assessments reveal that Lys-UiO-66-(COOH)2@CS-7 exhibits a remarkable conductivity of 0.022 S/cm at 100 % RH and 363 K, outperforming Glu-UiO-66-(COOH)2@CS-7. The lower activation energy (Ea) of 0.28 eV for Lys-UiO-66-(COOH)2@CS-7, compared to 0.336 eV for Glu-UiO-66-(COOH)2@CS-7, highlights the significant improvement in intramolecular acid-base interactions and hydrogen bonding. Furthermore, Lys-UiO-66-(COOH)2@CS-7 maintains notable proton conductivity at 43 % RH, with an Ea of 0.216 eV, demonstrating its efficacy in low-humidity conditions. These findings underscore the profound impact of amino acid modifications and cross-linking on proton conductivity by reinforcing acid-base interactions, hydrogen bonding networks, and proton transfer efficiency.

The Role of the Extracellular Matrix in the Pathogenesis and Treatment of Pulmonary Emphysema.

Pulmonary emphysema involves progressive destruction of alveolar walls, leading to enlarged air spaces and impaired gas exchange. While the precise mechanisms responsible for these changes remain unclear, there is growing evidence that the extracellular matrix plays a critical role in the process. An essential feature of pulmonary emphysema is damage to the elastic fiber network surrounding the airspaces, which stores the energy needed to expel air from the lungs. The degradation of these fibers disrupts the mechanical forces involved in respiration, resulting in distension and rupture of alveolar walls. While the initial repair process mainly consists of elastin degradation and resynthesis, continued alveolar wall injury may be associated with increased collagen deposition, resulting in a mixed pattern of emphysema and interstitial fibrosis. Due to the critical role of elastic fiber injury in pulmonary emphysema, preventing damage to this matrix component has emerged as a potential therapeutic strategy. One treatment approach involves the intratracheal administration of hyaluronan, a polysaccharide that prevents elastin breakdown by binding to lung elastic fibers. In clinical trials, inhalation of aerosolized HA decreased elastic fiber injury, as measured by the release of the elastin-specific cross-linking amino acids, desmosine, and isodesmosine. By protecting elastic fibers from enzymatic and oxidative damage, aerosolized HA could alter the natural history of pulmonary emphysema, thereby reducing the risk of respiratory failure.

Engineered Proteins and Materials Utilizing Residue-Specific Noncanonical Amino Acid Incorporation.

The incorporation of noncanonical amino acids into proteins and protein-based materials has significantly expanded the repertoire of available protein structures and chemistries. Through residue-specific incorporation, protein properties can be globally modified, resulting in the creation of novel proteins and materials with diverse and tailored characteristics. In this review, we highlight recent advancements in residue-specific incorporation techniques as well as the applications of the engineered proteins and materials. Specifically, we discuss their utility in bio-orthogonal noncanonical amino acid tagging (BONCAT), fluorescent noncanonical amino acid tagging (FUNCAT), threonine-derived noncanonical amino acid tagging (THRONCAT), cross-linking, fluorination, and enzyme engineering. This review underscores the importance of noncanonical amino acid incorporation as a tool for the development of tailored protein properties to meet diverse research and industrial needs.

Stereoselective Synthesis of meso- and l,l-Diaminopimelic Acids from Enone-Derived α-Amino Acids.

The stereoselective synthesis of meso-diaminopimelic acid (meso-DAP), the key cross-linking amino acid of the peptidoglycan cell wall layer in Gram-negative bacteria, and its biological precursor, l,l-DAP, is described. The key step involved stereoselective reduction of a common enone-derived amino acid by substrate- or reagent-based control. Overman rearrangement of the resulting allylic alcohols, concurrent alkene hydrogenation and trichloroacetamide reduction, and subsequent ruthenium-catalyzed arene oxidation completed the synthesis of each stereoisomer. The synthetic utility of this approach was demonstrated with the efficient preparation of an l,l-DAP-derived dipeptide.

Synthesis of neodesmosine-d4 and quantitative LC-MS/MS analysis of neodesmosine in biological samples

Elastin is an important protein responsible for elasticity of cells and tissues in animal bodies. Although its detailed structure has not yet been elucidated, its crosslinking structure is considered to play an important role in expressing elasticity. Neodesmosine, which was also isolated from eggshell membrane, is one of the crosslinking amino acids of elastin. In this study, the synthesis of an isotopically labeled form of neodesmosine, neodesmosine-d4 (1-d4), was completed in four steps in 20 % overall yield. Quantitative LC-MS/MS analysis of neodesmosine in elastin and eggshell membranes of chicken and quail using 1-d4 as an internal standard was then conducted, revealing that these eggshell membranes contained 0.195 and 0.287 μg of neodesmosine, respectively, while the amount was almost double (0.539 μg) in elastin of bovine ligamentum. This result will aid in the elucidation of the structures and the correlation between elastin and eggshell membranes.

Synthesis of desmosine-KLH conjugated antigen via isoChichibabin pyridinium synthesis

Desmosine is a crosslinking amino acid of the extracellular matrix protein elastin that is contained in tissues such as ligamentum, skin, alveoli, and blood vessels. Quantitative LC-MS/MS analysis of desmosine in human dermis, plasma, and skipjack tuna has been employed as a useful biomarker. The aim of this study is to produce desmosine antibodies to develop an ELISA system that is simpler than LC-MS/MS and can measure many samples in a shorter time. In our previous work, the synthesis of isodesmosine with keyhole limpet hemocyanin (KLH) conjugated antigen using Chichibabin pyridinium synthesis was achieved. In this work, synthesis of desmosine with KLH as the conjugated antigen, which is a new model, using isoChichibabin pyridinium synthesis, is reported.

Flavin mononucleotide in visible light photoinitiating systems for multiple-photocrosslinking and photoencapsulation strategies

Visible light-induced photocrosslinking techniques have attracted significant attention for their flexibility, controllability, safety, and energy conservation, especially in tissue engineering and biofabrication, compared to UV photocrosslinking. Despite these advantages, current photoinitiators are constrained by various challenges, including inadequate photoinitiation efficiency, low biocompatibility, poor water solubility, and limited compatibility with diverse crosslinking systems. Here, a water-soluble derivative of riboflavin, flavin mononucleotide (FMN−), was used to assess its potential as an initiator in multiple-photocrosslinking systems, including radical photopolymerization, dityrosine, and ditryptophan coupling crosslinking, under blue light irradiation. Blue light irradiation facilitated an efficient electron transfer reaction between FMN− and persulfate, owing to their suitable spectral compatibility and photoactivity. The resulting oxidizing free radicals and excited triplet state of FMN− served as initiating active species for the multiple-photocrosslinking reactions. The combination of FMN− and potassium persulfate (KPS) exhibited exceptional photoinitiation efficiency for various biomaterials, including silk fibroin, gelatin, poly(ethylene glycol) diacrylate, and carboxymethyl cellulose modified with amino acids. Furthermore, the cytocompatibility of the FMN−/KPS photoinitiator was demonstrated by the survival rates of 3T3-LI fibroblasts encapsulated in it, which exceeded 95 % when compared to a commercial initiator. Statement of significanceBy introducing persulfate, the photoinitiation efficiency of flavin mononucleotide was significantly improved. The application scenarios of flavin mononucleotide and persulfate combinations were also greatly extended, including radical photopolymerization, dityrosine, diphenylalanine, and ditryptophan coupling crosslinking. Among them, the coupling crosslinking of amino acids (di-phenylalanine, and di-tryptophan) modified carboxymethyl cellulose, to our knowledge, was first reported. The excellent cytocompatibility of cell encapsulation further proved that the combinations of flavin mononucleotide and persulfate have great potential in tissue engineering.

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.

Fabrication of Insoluble Elastin by Enzyme-Free Cross-Linking.

Elastin is an essential extracellular matrix protein that enables tissues and organs such as arteries, lungs, and skin, which undergo continuous deformation, to stretch and recoil. Here, we describe an approach to fabricate artificial elastin with close-to-native molecular and mechanical characteristics. We polymerized recombinantly produced tropoelastin through coacervation and allysine-mediated cross-linking induced by pyrroloquinoline quinone (PQQ). We developed a technique that allows the recovery and repeated use of PQQ for protein cross-linking by covalent attachment to magnetic Sepharose beads. The produced material closely resembles natural elastin in its molecular, biochemical and mechanical properties, enabled by the occurrence of the cross-linking amino acids desmosine, isodesmosine and merodesmosine. It possesses elevated resistance against tryptic proteolysis and its Young's modulus ranging between 1-2MPa is similar to that of natural elastin. The approach described herein enables the engineering of mechanically resilient, elastin-like materials for biomedical applications. This article is protected by copyright. All rights reserved.

Enzymatic Crosslinking of Amino Acids Improves the Repair Effect of Keratin on Hair Fibre.

Although keratin can effectively repair hair fibres and enhance their moisture content and flexibility, it has a relatively low affinity for hair. In this study, the effects of transglutaminase (TGase)-commonly used to catalyse crosslinking of proteins or amino acids-in crosslinking serine and hydrolysed wool keratin to repair damaged hair and protect healthy hair were studied. Treatment with a repair solution containing hydrolysed wool keratin, serine, and TGase improved the physical and chemical properties of damaged hair samples. The alkali solubility of damaged hair samples decreased by 50.53%, fracture stress increased from 1.031 to 1.806 N, and fracture strain increased from 9.51 to 19.88 mm. Fourier transform infrared spectroscopy and X-ray analysis showed that amide bonds increased in damaged hair samples treated with the repair solution and hair crystallinity increased. Differential scanning calorimetry showed that the repair solution improved the thermal stability of damaged hair. After five cycles of washing, the effects of the repair solution were still apparent in damaged hair samples. The enzymatic solution had stronger repair effects than general hair care products and reduced water loss rates in damaged hair samples; repaired hair samples were also softer and brighter. The repair solution was effective in protecting healthy hair samples against chemical damage. The materials used to prepare the repair solution are all bio-based, and treatment with this product is safer and longer lasting.

Synthesis of lysinonorleucine and mass spectrometric analysis of lysinonorleucine and merodesmosine in bovine ligament and eggshell membrane

Elastin is an important extracellular matrix protein that contributes to the elasticity of cells, tissues, and organs. Although crosslinking amino acids such as desmosine and isodesmosine have been identified in elastin, details regarding the structure remain unclear. In this study, an elastin crosslinker, lysinonorleucine, was chemically synthesized and detected in hydrolyzed bovine ligament and eggshell membrane samples utilizing tandem mass spectrometry. Merodesmosine, another crosslinker of elastin, was also measured in the same samples using the same analytical method. The resulting data should aid in the elucidating the crosslinking structure of elastin and eggshell membranes.

Structural details of a Class B GPCR-arrestin complex revealed by genetically encoded crosslinkers in living cells

Understanding the molecular basis of arrestin-mediated regulation of GPCRs is critical for deciphering signaling mechanisms and designing functional selectivity. However, structural studies of GPCR-arrestin complexes are hampered by their highly dynamic nature. Here, we dissect the interaction of arrestin-2 (arr2) with the secretin-like parathyroid hormone 1 receptor PTH1R using genetically encoded crosslinking amino acids in live cells. We identify 136 intermolecular proximity points that guide the construction of energy-optimized molecular models for the PTH1R-arr2 complex. Our data reveal flexible receptor elements missing in existing structures, including intracellular loop 3 and the proximal C-tail, and suggest a functional role of a hitherto overlooked positively charged region at the arrestin N-edge. Unbiased MD simulations highlight the stability and dynamic nature of the complex. Our integrative approach yields structural insights into protein-protein complexes in a biologically relevant live-cell environment and provides information inaccessible to classical structural methods, while also revealing the dynamics of the system.

Insight into the intrinsically disordered transactivation domain of NFkB as a regulator of DNA binding using UV-induced unnatural amino acid crosslinking.

Within our vast genome, specific transcription factors like NFkB manage to find target sequences amongst overwhelming nonspecific options and help elicit timely cellular responses. A transcription factor's binding affinity for specific vs. nonspecific DNA must therefore be in a delicate balance to facilitate this search. Key in vitro experiments that described these binding affinities for the dominant NFkB dimer RelA/p50 were carried out with a truncated version of RelA - excluding a large intrinsically disordered transactivation domain (TAD) that was believed to be important for transcription activation through cofactor interactions but not impactful for DNA binding. This fit with a traditional view of protein domains acting as modular units. More recently, however, we discovered a novel function of the TAD: binding experiments revealed that the TAD's presence increased RelA/p50's affinity for DNA, with the strongest increase in binding affinity observed for non-specific DNA sequences lacking kB consensus sites. Here we follow up these findings with an investigation of the TAD's mechanism of action. We present preliminary results using an unnatural amino acid crosslinker (p-benzoyl-l-phenylalanine or pBpF) in combination with mass spectrometry to probe TAD interactions that lead to the observed changes in DNA binding affinity and specificity. pBpF is incorporated into specific positions in the TAD using AMBER codon suppression, and crosslinking is initiated by UV light. Interactions are analyzed by mass spectrometry to identify key sites of contact between the TAD and the natively folded Rel-Homology Domain.

Gelation profile of laccase-crosslinked Bambara groundnut (Vigna subterranea) protein isolate

Enzymatic crosslinking has gained attention in improving plant protein heat-induced gels, which are composed of weak network structures. The aim of the study was to investigate the effect of laccase crosslinking on the rheological and microstructural properties of heat-induced Bambara groundnut protein gels. The rheological properties of laccase-modified Bambara groundnut protein isolate (BPI11BPI- Bambara groundnut protein isolate.) gel formed in situ were investigated. Changes in viscoelastic properties were monitored during heating and cooling ramps and gel structure fingerprints were analyzed by frequency sweep. Laccase addition induced an initial protein structure breakdown (G″>G′) at an enzyme dose-dependent (1–3 U/g) before gel formation and stabilization. Gel point temperatures were significantly decreased from 85°C to 29°C (∼3 folds) with increasing laccase activity (0 to 3 U/g protein, respectively). For laccase crosslinked gels, G′ was substantially greater than G” (>1 log) with no dependency on angular frequency, which suggests the formation of relatively well-structured gels. The highest gel strength (tan δ of 0.09, G* of 555.51 kPa & An of 468.04 kPa) was recorded at a laccase activity of 2 U/g protein and the gels formed at this activity appeared homogeneous with compact lath sheet-like structure. The crosslinking effects of laccase were corroborated by the decrease in thiol and phenolic contents as well as the crosslinking of amino acids in model reactions. Overall, the use of laccase improved gel properties and significantly altered the gelation profile of BPI. Laccase-modified Bambara groundnut protein gels have potential to be used in food texture improvement and development of new food products. For instance, they can be used in plant-based milk products such as yoghurt and cheese.

A combined spectroscopic and molecular modeling Study on structure-function-dynamics under chemical modification: Alpha-chymotrypsin with formalin preservative.

Enzyme function can be altered via modification of its amino acid residues, side chains and large-scale domain modifications. Herein, we have addressed the role of residue modification in catalytic activity and molecular recognition of an enzyme alpha-chymotrypsin (CHT) in presence of a covalent cross-linker formalin. Enzyme assay reveals reduced catalytic activity upon increased formalin concentration. Polarization gated anisotropy studies of a fluorophore 8-Anilino-1-naphthalenesulfonic acid (ANS) in CHT show a dip rise pattern in presence of formalin which is consistent with the generation of multiple ANS binding sites in the enzyme owing to modifications of its local amino acid residues. Molecular docking study on amino acid residue modifications in CHT also indicate towards the formation of multiple ANS binding site. The docking model also predicted no change in binding behavior for the substrate Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) at the active site upon formalin induced amino acid cross-linking.

Total synthesis of merodesmosine

Elastin is an important extracellular matrix protein responsible for the elasticity of tissues such as ligamentum, arteries, and lungs. Its three-dimensional crosslinking structure plays an important role in its elasticity. However, the precise structure has not yet been clarified since it is difficult to analyze due to its insoluble nature. Merodesmosine is one of the possible crosslinking amino acids in elastin. In this work, the first total synthesis of merodesmosine was achieved in five steps in 21% overall yield from protected allysine. This achievement will aid in the elucidation of the crosslinking structure of elastin.

Maillard reaction products and amino acid cross-links in liquid infant formula: Effects of UHT treatment and storage

The formation of Maillard reaction products, including Amadori compounds (determined as furosine), advanced glycation end products (AGEs), α-dicarbonyl and furfural compounds, as well as amino acid cross-links (lysinoalanine and lanthionine) was investigated in direct (DI) and indirect (IN) UHT-treated experimental liquid infant formula (IF) during storage at 40 °C. IN-IF had higher concentrations of all investigated compounds compared to DI-IF and low pasteurized IF. IN UHT treatment induced significantly higher concentrations of α-dicarbonyl compounds (glyoxal, methylglyoxal, 3-deoxyglucosone and 3-deoxygalactosone) compared to DI, which facilitated increased formation of AGEs (N-Ɛ-(carboxymethyl)lysine, methylglyoxal- and glyoxal-derived hydroimidazolones) in unstored IFs. During storage for 6 months, concentrations of furosine and AGEs increased while α-dicarbonyl compounds decreased. Principal component analysis indicated that differences between IN-IF and DI-IF disappeared after 2 months of storage. IN-IF had higher concentrations of lysinoalanine and lanthionine and lower concentrations of available lysine and arginine than DI-IF indicating higher loss of protein quality in IN-IF.

Synthesis of Amino Acid α-Thioethers and Late-Stage Incorporation into Peptides

The accelerating discovery of structurally distinct peptide natural products bearing α-thioether cross-links, such as the family of sactipeptide natural products, highlights the need for strategies to synthesize this underexplored functional motif. Herein, we describe the preparation of orthogonally protected, cross-linked amino acid α-thioether building blocks and probe their stability toward conventional solid-phase peptide synthesis. We overcome challenges with linkage lability by developing a late-stage, on-resin approach to α-thioethers, providing important proof-of-principle for sactipeptide synthesis.

Synthesis of desmosine-BSA/KLH conjugates via Sonogashira/Negishi cross-coupling reactions

Desmosine is an elastin crosslinking amino acid that is expected to be a useful biomarker of diseases related to elastin degradation including chronic obstructive pulmonary disease (COPD). In this study, conjugates of desmosine and carrier proteins, such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), with linkers were designed and prepared for the creation of an effective antibody to be used in the development of an enzyme-linked immunosorbent assay (ELISA) system. Synthesis of desmosine along with the linkers having appropriate lengths was achieved via stepwise and chemo/regioselective palladium-catalyzed Sonogashira and Negishi cross-coupling reactions starting from an alkyne with protecting groups over nine steps. Subsequently, Michael addition with a thiolated carrier protein (BSA/KLH) was carried out using Traut’s reagent to afford seven types of desmosine-BSA/KLH conjugates.

IsoChichibabin desmosine-13C3,15N1 synthesis and quantitative LC-MS/MS analysis of desmosine and isodesmosine in human skin

Desmosine and isodesmosine are crosslinking amino acids of elastin, which is an essential component of the dermal extracellular matrix protein. Quantitative analysis of crosslinker desmosines in human skin dermis has not been fully achieved due to the insoluble nature of elastin protein. In the present study, chemical synthesis of isotopically labeled desmosine, desmosine-13C3,15N1, was carried out via isoChichibabin pyridinium synthesis starting from corresponding isotopically labeled amino acids. Isotope-dilution LC-MS/MS analysis of desmosine and isodesmosine utilizing synthetic desmosine-13C3,15N1 enabled the quantitative analysis of desmosines in human skin for the first time. Thus, ca. 1.43 μg of desmosines was detected from analysis of 1 mg of dry human skin.