Tailoring Synthetic Polypeptide Design for Directed Fibril Superstructure Formation and Enhanced Hydrogel Properties.

The biological significance of self-assembled protein filament networks and their unique mechanical properties have sparked interest in the development of synthetic filament networks that mimic these attributes. Building on the recent advancement of autoaccelerated ring-opening polymerization of amino acid N-carboxyanhydrides (NCAs), this study strategically explores a series of random copolymers comprising multiple amino acids, aiming to elucidate the core principles governing gelation pathways of these purpose-designed copolypeptides. Utilizing glutamate (Glu) as the primary component of copolypeptides, two targeted pathways were pursued: first, achieving a fast fibrillation rate with lower interaction potential using serine (Ser) as a comonomer, facilitating the creation of homogeneous fibril networks; and second, creating more rigid networks of fibril clusters by incorporating alanine (Ala) and valine (Val) as comonomers. The selection of amino acids played a pivotal role in steering both the morphology of fibril superstructures and their assembly kinetics, subsequently determining their potential to form sample-spanning networks. Importantly, the viscoelastic properties of the resulting supramolecular hydrogels can be tailored according to the specific copolypeptide composition through modulations in filament densities and lengths. The findings enhance our understanding of directed self-assembly in high molecular weight synthetic copolypeptides, offering valuable insights for the development of synthetic fibrous networks and biomimetic supramolecular materials with custom-designed properties.

Glutamate oxidase sheets-Prussian blue grafted amperometric biosensor for the real time monitoring of glutamate release from primary cortical neurons.

Glutamate (GLU) is a primary excitatory neurotransmitter, and its dysregulation is associated with several neurodegenerative disorders. A major challenge in GLU estimation is the existence of other biomolecules in the brain that could directly get oxidized at the electrode. Hence, highly selective electroenzymatic biosensors that enable rapid estimation of GLU are needed. Initially, a copolymer, poly(2-dimethylaminoethyl methacrylate- styrene) was synthesized through reversible addition-fragmentation chain transfer polymerization to noncovalently functionalize reduced graphene oxide (rGO), named DS-rGO. Glutamate oxidase macromolecule immobilized DS-rGO formed enzyme nanosheets, which was drop-coated over Prussian blue electrodeposited disposable electrodes to fabricate the GLU biosensor. The interconnectivity between the enzyme nanosheets and the Prussian blue endows the biosensor with enhanced conductivity and electrochemical activity. The biosensor exhibited a linearity: 3.25-250 µM; sensitivity: 3.96 µA mM-1 cm-2, and a limit of detection: 0.96 µM for GLU in the Neurobasal Medium. The biosensor was applied to an in vitro primary rat cortical model to discriminate GLU levels in Neurobasal Medium, before and after KCl mediated depolarization, which provides new insights for elucidating neuronal functioning in the brain.

Effect of Micellar Shape on Viscoelastic Behavior and Gel Network Structure of Hydrogel Formed by Surfactant-mediated Gelation Using Glutamic-acid-based Organogelator.

Surfactant-mediated gelation (SMG) is a technique used to form hydrogels by solubilizing water-insoluble low-molecular-weight organogelators in surfactant micelles. In this study, we investigated the viscoelastic behavior of SMG hydrogels and the effect of micellar shape on their gel network structure using a glutamic acid-based organogelator. Stress-strain curves obtained from static viscoelasticity measurements showed that a wormlike micelle-mediated gel (W-SMG) exhibited a higher stress than a spherical micelle-mediated gel (S-SMG). From the viscosity-shear rate curve (flow curve), we inferred that the SMG gel exhibited a shear thickening behavior, particularly W-SMG. Microscopic observations revealed that W-SMG formed a denser and more uniform gel network than S-SMG when subjected to strong shearing. W-SMG showed remarkable adhesiveness and a significantly higher tensile normal stress than S-SMG. The storage modulus and loss modulus of W-SMG and the wormlike micellar solution obtained from frequency sweep measurements of the dynamic viscoelasticity were analyzed by Maxwell fitting. The wormlike micellar solution produced a good fit with the single Maxwell model, whereas W-SMG produced the best fit with the generalized Maxwell model comprising two Maxwell elements. From the relaxation time characteristics obtained from the Maxwell model, W-SMG was found to be a viscoelastic material coexisting with a structure having a short relaxation time derived from the gel network and a long relaxation time derived from the wormlike micelle. Under the oscillation strain measured by a rheometer, W-SMG showed a greater normal stress than the wormlike micellar solution, indicating a significant Weissenberg effect.

Modulation of the self-assembly kinetics and digestibility of type 3 resistant starch particles by co-crystallization with amino acid.

The effect of eight different l-amino acids (L-AA) on type-3 resistant starch particles (rSPs) derived from short chain glucan (SCG) was investigated. The L-AA were categorized based on their charge and polarity. The results reveal that positively charged L-AA, such as lysine and arginine, decreased the nucleation and growth rate of rSPs, while non-charged L-AA have negligible effects. Negatively charged L-AA, such as glutamic acid and aspartic acid, had a significant impact on the morphology and crystallinity of the rSPs, resulting in particle size of around 3 µm and crystallinity of around 35%. This implies that charged L-AA influence the arrangement of SCG double helices in the particles. Furthermore, the complexation of SCG with charged L-AA reduced the level of RS in rSPs, indicating that L-AA could be useful in modulating the physical properties and digestibility of rSPs.

Amino acid substitutions in human growth hormone affect secondary structure and receptor binding.

The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has basic relevance to cancer and growth disorders, and hGH is the scaffold for Pegvisomant, an anti-acromegaly therapeutic. For the latter reason, hGH has been extensively engineered by early workers to improve binding and other properties. We are particularly interested in E174 which belongs to the hGH zinc-binding triad; the substitution E174A is known to significantly increase binding, but to now no explanation has been offered. We generated this and several computationally-selected single-residue substitutions at the hGHR-binding site of hGH. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. The E174A substitution induces a change in the Circular Dichroism spectrum that suggests the appearance of coiled-coiling. Here we show that E174A increases affinity of hGH against hGHR because the off-rate is slowed down more than the on-rate. For E174Y (and certain mutations at other sites) the slowdown in on-rate was greater than that of the off-rate, leading to decreased affinity. The results point to a link between structure, zinc binding, and hGHR-binding affinity in hGH.

Review: Organophosphorus toxicants, in addition to inhibiting acetylcholinesterase activity, make covalent adducts on multiple proteins and promote protein crosslinking into high molecular weight aggregates.

The acute effects of exposure to organophosphorus toxicants are explained by inhibition of acetylcholinesterase activity. However, the mechanisms that explain long term illness associated with organophosphorus exposure are still under investigation. We find that organophosphorus nerve agents and organophosphorus pesticides make covalent adducts not only on the serine from acetylcholinesterase, but also on tyrosine, lysine, glutamate, serine and threonine from a variety of proteins. Almost any protein can be modified by a high dose of organophosphorus toxicant. A low dose of 10 µM chlorpyrifos oxon added to the serum-free culture medium of human neuroblastoma SH-SY5Y cells resulted in tyrosine adducts on 48 proteins immunopurified from the cell lysate. We identified the adducted proteins by mass spectrometry after immunopurifying modified proteins with a rabbit anti-diethoxyphospho-tyrosine monoclonal antibody which biased this study for tyrosine adducts. In cultured cells, the primary organophosphate targets are abundant proteins. Organophosphate-modified proteins may disrupt physiological processes. In separate experiments we identified organophosphate adducts on lysine. Organophosphylation activates the lysine for protein crosslinking. The activated lysine reacts with glutamic acid or aspartic acid protein side chains to form an isopeptide bond between proteins, resulting in high molecular weight crosslinked proteins. Crosslinked proteins form insoluble aggregates that may lead to neurogenerative disease.

Simultaneously Enhanced Formation of Pyrazines and Furans during Thermal Degradation of the Glycyl-l-glutamine Amadori Compound by Selected Exogenous Amino Acids and Appropriate Elevated Temperatures.

The Amadori compound of glucose and glycyl-l-glutamine (Gly-Gln-ARP) was prepared and characterized by UPLC-MS/MS and NMR. Gly-Gln-ARP could be thermally degraded into Gly-Gln and other secondary reaction products like glycyl-l-glutamic acid and its ARP via deamidation. The thermal processing temperature exerted a tremendous influence on the flavor formation of ARP. Furans were mainly formed at 100 °C, while an elevated temperature of 120 °C facilitated the massive accumulation of α-dicarbonyl compounds through the retro-aldolization of deoxyglucosone, and then increased the formation of pyrazines. The extra-added amino acids further promoted the formation of pyrazines at 120 °C, especially Glu, Lys, and His, further increasing the total concentration of pyrazines to 457 ± 6.26, 563 ± 65.5, and 411 ± 59.2 µg/L, respectively, exceeding the pure heated control at 140 °C (296 ± 6.67 µg/L). The total concentration of furans was enhanced to (20.7 × 103) ± 8.17 µg/L by extra-added Gln. Different increasing effects were observed on the type and flavor intensity of formed pyrazines and furans from different extra-added amino acids.

The role of conformational change and key glutamic acid residues in the ClC-ec1 antiporter.

The triple glutamine (Q) mutant (QQQ) structure of a Cl-/H+ antiporter from Escherichia coli (ClC-ec1) displaying a novel backbone arrangement has been used to challenge the long-held notion that Cl-/H+ antiporters do not operate through large conformational motions. The QQQ mutant substitutes the glutamine residue for an external glutamate E148, an internal glutamate E203, and a third glutamate E113 that hydrogen-bonds with E203. However, it is unknown if QQQ represents a physiologically relevant state, as well as how the protonation of the wild-type glutamates relates to the global dynamics. We herein apply continuous constant-pH molecular dynamics to investigate the H+-coupled dynamics of ClC-ec1. Although any large-scale conformational rearrangement upon acidification would be due to the accumulation of excess charge within the protein, protonation of the glutamates significantly impacts mainly the local structure and dynamics. Despite the fact that the extracellular pore enlarges at acidic pHs, an occluded ClC-ec1 within the active pH range of 3.5-7.5 requires a protonated E148 to facilitate extracellular Cl- release. E203 is also involved in the intracellular H+ transfer as an H+ acceptor. The water wire connection of E148 with the intracellular solution is regulated by the charge states of the E113/E203 dyad with coupled proton titration. However, the dynamics extracted from our simulations are not QQQ-like, indicating that the QQQ mutant does not represent the behavior of the wild-type ClC-ec1. These findings reinforce the necessity of having a protonatable residue at the E203 position in ClC-ec1 and suggest that a higher level of complexity exists for the intracellular H+ transfer in Cl-/H+ antiporters.

Secondary Structures in Synthetic Poly(Amino Acids): Homo- and Copolymers of Poly(Aib), Poly(Glu), and Poly(Asp).

The secondary structure of poly(amino acids) is an excellent tool for controlling and understanding the functionality and properties of proteins. In this perspective article the secondary structures of the homopolymers of oligo- and poly-glutamic acid (Glu), aspartic acid (Asp), and α-aminoisobutyric acid (Aib) are discussed. Information on external and internal factors, such as the nature of side groups, interactions with solvents and interactions between chains is reviewed. A special focus is directed on the folding in hybrid-polymers consisting of oligo(amino acids) and synthetic polymers. Being part of the SFB TRR 102 "Polymers under multiple constraints: restricted and controlled molecular order and mobility" this overview is embedded into the cross section of protein fibrillation and supramolecular polymers. As polymer- and amino acid folding is an important step for the utilization and design of future biomolecules these principles guide to a deeper understanding of amyloid fibrillation.

Guanidinium-amino acid hydrogen-bonding interactions in protein crystal structures: implications for guanidinium-induced protein denaturation.

In the present work, 86 available high resolution X-ray structures of proteins that contain one or more guanidinium ions (Gdm+) are analyzed for the distribution and nature of noncovalent interactions between Gdm+ and amino-acid residues. A total of 1044 hydrogen-bonding interactions were identified, of which 1039 are N-H⋯O, and five are N-H⋯N. Acidic amino acids are more likely to interact with Gdm+ (46% of interactions, 26% Asp and 20% Glu), followed by Pro (19% of interactions). DFT calculations on the identified Gdm+-amino acid hydrogen-bonded pairs reveal that although Gdm+ interacts primarily with the backbone amides of nonpolar amino acids, Gdm+ does interact with the sidechains of polar and acidic amino acids. We classified the optimized Gdm+-amino acid pairs into parallel [p], bifurcated [b], single hydrogen bonded [s] and triple hydrogen bonded [t] types. The [p] and [t] type pairs possess higher average interaction strength that is stronger than that of [b] and [s] type pairs. Negatively charged aspartate and glutamate residues interact with Gdm+ ion exceptionally tightly (-76 kcal mol-1) in [p] type complexes. This work provides statistical and energetics insights to better describe the observed destabilization or denaturation process of proteins by guanidinium salts.

Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient ß-barrel pore formation.

Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging ß-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane ß-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane ß-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric ß-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional ß-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.

Design optimisation and characterisation of an amperometric glutamate oxidase-based composite biosensor for neurotransmitter l-glutamic acid.

A polymer/enzyme composite biosensor for monitoring neurochemical glutamate was performance optimised in vitro for sensitivity, selectivity and stability. This first generation Pt/glutamate oxidase-based sensor displayed appropriate sensitivity (90.4 ± 2.0 nA cm-2 µM-1). It also has ideal stability/biocompatibility with no significant decrease in response observed for repeated calibrations, exposure to electron beam sterilisation, or following storage at 4 °C either dry (28 days) or in ex-vivo rodent brain tissue (14 days). Potential non-glutamate contributing signals, generated by extracellular levels of the principal endogenous electroactive interferents, were typically <5% of the basal (10 µM) glutamate response. Changes in molecular oxygen (the natural enzyme mediator) over the normal brain tissue range of 40-80 µM had minimal effect on the glutamate signal for concentrations of 10 and 100 µM (Mean KMO2 = 1.86 ± 0.74 µM, [O2]90% = ca. 15 µM). Additionally, a low µM calculated limit of detection (0.44 ± 0.05) and rapid response time (ca. 1.67 ± 0.06 s), combined with no effect of pH and temperature changes over physiologically relevant ranges (7.2-7.6 and 34-40 °C respectively), collectively suggest that this composite biosensor should reliably detect l-glutamate when used for neurochemical monitoring. Preliminary experiments involving implantation in the striatum of freely moving rats demonstrated stable recording over several weeks, and reliable detection of physiological changes in glutamate in response to behavioural/neuronal activation (locomotor activity and restraint stress).

Hydrogel Formation by Glutamic-acid-based Organogelator Using Surfactant-mediated Gelation.

Hydrogels formed by low-molecular-weight gelators have reversible sol-gel transition and responsiveness to various stimuli, and are used in cosmetics and drug applications. It is challenging to obtain hydrogels using novel gelators because subtle differences in their molecular architecture affect gelation. Organogelators (which form organogels) are insoluble in water, and their use as hydrogelators has not previously been considered. However, a surfactant-mediated gelation method was reported in which organogelators were solubilized in water by surfactants to form hydrogels using 12-hydroxyoctadecanoic acid. To investigate whether this method can be applied with other organogelators, the formation of hydrogel using a glutamic-acid-based organogelator was studied here. Hydrogels were formed by solubilizing 1:1 mixtures of glutamate-based organogelators, N-lauroyl-L-glutamic acid dibuthylamide, and N-2-ethylhexanoyl-L-glutamic acid dibutylamide in aqueous micellar solutions of anionic surfactant (sodium lauroyl glutamate) and cationic surfactant (cetyltrimethylammonium chloride). The minimum gelation concentration of the hydrogel was ~0.2-0.6 wt%. By changing the molar fraction of cetyltrimethylammonium chloride in the mixed surfactant, either spherical or wormlike micelles were formed. The hydrogel with wormlike micelles had a higher sol-gel transition temperature than that with spherical micelles and formed fine self-assembled fibrillar networks. Additionally, the hydrogel with the spherical micelles was elastic, whereas that with wormlike micelles was viscoelastic, suggesting that networks of the organogelators and wormlike micelles coexisted in the hydrogel from the wormlike micellar solution. Moreover, the hydrogel suppressed the reduction in the storage modulus at higher temperatures compared with the micellar aqueous solution, indicating that the elastic properties of the organogelator networks were maintained at high temperatures. The gel fibers of the hydrogel partially formed a loosely aggregated structure as the temperature increased, the fibers bundled via hydrophobic interactions, and new cross-linking points formed spontaneously. This phenomenon corresponded with an inflection point in the temperature-dependent storage modulus of the hydrogel.

In silico binding affinity prediction for metabotropic glutamate receptors using both endpoint free energy methods and a machine learning-based scoring function.

Metabotropic glutamate receptors (mGluRs) play an important role in regulating glutamate signal pathways, which are involved in neuropathy and periphery homeostasis. mGluR4, which belongs to Group III mGluRs, is most widely distributed in the periphery among all the mGluRs. It has been proved that the regulation of this receptor is involved in diabetes, colorectal carcinoma and many other diseases. However, the application of structure-based drug design to identify small molecules to regulate the mGluR4 receptor is limited due to the absence of a resolved mGluR4 protein structure. In this work, we first built a homology model of mGluR4 based on a crystal structure of mGluR8, and then conducted hierarchical virtual screening (HVS) to identify possible active ligands for mGluR4. The HVS protocol consists of three hierarchical filters including Glide docking, molecular dynamic (MD) simulation and binding free energy calculation. We successfully prioritized active ligands of mGluR4 from a set of screening compounds using HVS. The predicted active ligands based on binding affinities can almost cover all the experiment-determined active ligands, with only one ligand missed. The correlation between the measured and predicted binding affinities is significantly improved for the MM-PB/GBSA-WSAS methods compared to the Glide docking method. More importantly, we have identified hotspots for ligand binding, and we found that SER157 and GLY158 tend to contribute to the selectivity of mGluR4 ligands, while ALA154 and ALA155 could account for the ligand selectivity to mGluR8. We also recognized other 5 key residues that are critical for ligand potency. The difference of the binding profiles between mGluR4 and mGluR8 can guide us to develop more potent and selective modulators. Moreover, we evaluated the performance of IPSF, a novel type of scoring function trained by a machine learning algorithm on residue-ligand interaction profiles, in guiding drug lead optimization. The cross-validation root-mean-square errors (RMSEs) are much smaller than those by the endpoint methods, and the correlation coefficients are comparable to the best endpoint methods for both mGluRs. Thus, machine learning-based IPSF can be applied to guide lead optimization, albeit the total number of actives/inactives are not big, a typical scenario in drug discovery projects.

Role of Water in Proton-Coupled Electron Transfer between Tyrosine and Cysteine in Ribonucleotide Reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides and is critical for DNA synthesis and repair in all organisms. Its mechanism requires radical transfer along a ∼32 Špathway through a series of proton-coupled electron transfer (PCET) steps. Previous simulations suggested that a glutamate residue (E623) mediates the PCET reaction between two stacked tyrosine residues (Y730 and Y731) through a proton relay mechanism. This work focuses on the adjacent PCET reaction between Y730 and a cysteine residue (C439). Quantum mechanical/molecular mechanical free energy simulations illustrate that when Y730 and Y731 are stacked, E623 stabilizes the radical on C439 through hydrogen bonding with the Y730 hydroxyl group. When Y731 is flipped away from Y730, a water molecule stabilizes the radical on C439 through hydrogen bonding with Y730 and lowers the free energy barrier for radical transfer from Y730 to C439 through electrostatic interactions with the transferring hydrogen but does not directly accept the proton. These simulations indicate that the conformational motions and electrostatic interactions of the tyrosines, cysteine, glutamate, and water strongly impact the thermodynamics and kinetics of these two coupled PCET reactions. Such insights are important for protein engineering efforts aimed at altering radical transfer in RNR.

How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein.

E. coli single-stranded-DNA binding protein (EcSSB) displays nearest-neighbor (NN) and non-nearest-neighbor (NNN)) cooperativity in binding ssDNA during genome maintenance. NNN cooperativity requires the intrinsically-disordered linkers (IDL) of the C-terminal tails. Potassium glutamate (KGlu), the primary E. coli salt, promotes NNN-cooperativity, while KCl inhibits it. We find that KGlu promotes compaction of a single polymeric SSB-coated ssDNA beyond what occurs in KCl, indicating a link of compaction to NNN-cooperativity. EcSSB also undergoes liquid-liquid phase separation (LLPS), inhibited by ssDNA binding. We find that LLPS, like NNN-cooperativity, is promoted by increasing [KGlu] in the physiological range, while increasing [KCl] and/or deletion of the IDL eliminate LLPS, indicating similar interactions in both processes. From quantitative determinations of interactions of KGlu and KCl with protein model compounds, we deduce that the opposing effects of KGlu and KCl on SSB LLPS and cooperativity arise from their opposite interactions with amide groups. KGlu interacts unfavorably with the backbone (especially Gly) and side chain amide groups of the IDL, promoting amide-amide interactions in LLPS and NNN-cooperativity. By contrast, KCl interacts favorably with these amide groups and therefore inhibits LLPS and NNN-cooperativity. These results highlight the importance of salt interactions in regulating the propensity of proteins to undergo LLPS.

Physiological synaptic activity and recognition memory require astroglial glutamine.

Presynaptic glutamate replenishment is fundamental to brain function. In high activity regimes, such as epileptic episodes, this process is thought to rely on the glutamate-glutamine cycle between neurons and astrocytes. However the presence of an astroglial glutamine supply, as well as its functional relevance in vivo in the healthy brain remain controversial, partly due to a lack of tools that can directly examine glutamine transfer. Here, we generated a fluorescent probe that tracks glutamine in live cells, which provides direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions. This mobilization is mediated by connexin43, an astroglial protein with both gap-junction and hemichannel functions, and is essential for synaptic transmission and object recognition memory. Our findings uncover an indispensable recruitment of astroglial glutamine in physiological synaptic activity and memory via an unconventional pathway, thus providing an astrocyte basis for cognitive processes.

Identification of bacterial lipo-amino acids: origin of regenerated fatty acid carboxylate from dissociation of lipo-glutamate anion.

The identification of bacterial metabolites produced by the microbiota is a key point to understand its role in human health. Among them, lipo-amino acids (LpAA), which are able to cross the epithelial barrier and to act on the host, are poorly identified. Structural elucidation of few of them was performed by high-resolution tandem mass spectrometry based on electrospray combined with selective ion dissociations reach by collision-induced dissociation (CID). The negative ions were used for their advantages of yielding only few fragment ions sufficient to specify each part of LpAA with sensitivity. To find specific processes that help structural assignment, the negative ion dissociations have been scrutinized for an LpAA: the N-palmitoyl acyl group linked to glutamic acid (C16Glu). The singular behavior of [C16Glu-H]¯ towards CID showed tenth product ions, eight were described by expected fragment ions. In contrast, instead of the expected product ions due to CONH-CH bond cleavage, an abundant complementary dehydrated glutamic acid and fatty acid anion pair were observed. Specific to glutamic moiety, they were formed by a stepwise dissociation via molecular isomerization through ion-dipole formation prior to dissociation. This complex dissociated by partner splitting either directly or after inter-partner proton transfer. By this pathway, surprising regeneration of deprotonated fatty acid takes place. Such regeneration is comparable to that occurred from dissociation to peptides containing acid amino-acid. Modeling allow to confirm the proposed mechanisms explaining the unexpected behavior of this glutamate conjugate.

The structure and function of modular Escherichia coli O157:H7 bacteriophage FTBEc1 endolysin, LysT84: defining a new endolysin catalytic subfamily.

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level - key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the ß(1-4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.