Molecular basis of Arginine and Lysine DNA sequence-dependent thermo-stability modulation.

We have used a variety of theoretical and experimental techniques to study the role of four basic amino acids-Arginine, Lysine, Ornithine and L-2,4-Diaminobutyric acid-on the structure, flexibility and sequence-dependent stability of DNA. We found that the presence of organic ions stabilizes the duplexes and significantly reduces the difference in stability between AT- and GC-rich duplexes with respect to the control conditions. This suggests that these amino acids, ingredients of the primordial soup during abiogenesis, could have helped to equalize the stability of AT- and GC-rich DNA oligomers, facilitating a general non-catalysed self-replication of DNA. Experiments and simulations demonstrate that organic ions have an effect that goes beyond the general electrostatic screening, involving specific interactions along the grooves of the double helix. We conclude that organic ions, largely ignored in the DNA world, should be reconsidered as crucial structural elements far from mimics of small inorganic cations.

Porphyrin-Schiff Base Conjugates Bearing Basic Amino Groups as Antimicrobial Phototherapeutic Agents.

New porphyrin-Schiff base conjugates bearing one (6) and two (7) basic amino groups were synthesized by condensation between tetrapyrrolic macrocycle-containing amine functions and 4-(3-(N,N-dimethylamino)propoxy)benzaldehyde. This approach allowed us to easily obtain porphyrins substituted by positive charge precursor groups in aqueous media. These compounds showed the typical Soret and four Q absorption bands with red fluorescence emission (ΦF ~ 0.12) in N,N-dimethylformamide. Porphyrins 6 and 7 photosensitized the generation of O2(1Δg) (ΦΔ ~ 0.44) and the photo-oxidation of L-tryptophan. The decomposition of this amino acid was mainly mediated by a type II photoprocess. Moreover, the addition of KI strongly quenched the photodynamic action through a reaction with O2(1Δg) to produce iodine. The photodynamic inactivation capacity induced by porphyrins 6 and 7 was evaluated in Staphylococcus aureus, Escherichia coli, and Candida albicans. Furthermore, the photoinactivation of these microorganisms was improved using potentiation with iodide anions. These porphyrins containing basic aliphatic amino groups can be protonated in biological systems, which provides an amphiphilic character to the tetrapyrrolic macrocycle. This effect allows one to increase the interaction with the cell wall, thus improving photocytotoxic activity against microorganisms.

Effects of Basic Amino Acids and Their Derivatives on SARS-CoV-2 and Influenza-A Virus Infection.

Amino acids have been implicated with virus infection and replication. Here, we demonstrate the effects of two basic amino acids, arginine and lysine, and their ester derivatives on infection of two enveloped viruses, SARS-CoV-2, and influenza A virus. We found that lysine and its ester derivative can efficiently block infection of both viruses in vitro. Furthermore, the arginine ester derivative caused a significant boost in virus infection. Studies on their mechanism of action revealed that the compounds potentially disturb virus uncoating rather than virus attachment and endosomal acidification. Our findings suggest that lysine supplementation and the reduction of arginine-rich food intake can be considered as prophylactic and therapeutic regimens against these viruses while also providing a paradigm for the development of broad-spectrum antivirals.

Role of surface-exposed charged basic amino acids (Lys, Arg) and guanidination in insulin on the interaction and stability of insulin-insulin receptor complex.

Naturally occurring proteins are emerging as novel therapeutics in the protein-based biopharmaceutical industry for the treatment of diabetes and obesity. However, proteins are not suitable for oral delivery due to short half-life, reduced physical and chemical stability and low permeability across the membrane. Chemical modification has been identified as a formulation strategy to enhance the stability and bioavailability of protein drugs. The present study aims to study the effect of charge-specific modification of basic amino acids (Lys, Arg) and guanidination on the interaction of insulin with its receptor using molecular modelling. Our investigation revealed that the guanidination of insulin (Lys-NHC = NHNH2) enhanced and exerted stronger binding of the protein to its receptor through electrostatic interaction than native insulin (Lys-NH3+). Point mutations of Lys and Arg (R22, K29; R22K, K29; R22, K29R; R22K, K29R) were attempted and the effects on the interaction and stability between insulin/modified insulins and insulin receptor were also analyzed in this study. The findings from the study are expected to provide a better understanding of the possible mechanism of action of the modified protein at a molecular level before advancing to real experiments.

Comparative chemical examination of inclusion complexes formed with ß-cyclodextrin derivatives and basic amino acids.

In this study, we have investigated the host-guest inclusion complexes between ß-cyclodextrin (ßCD), 2-hydroxypropyl-ß-cyclodextrin (2-HPßCD), and mono-6-tosyl-ß-cyclodextrin (TS-ßCD) excipients and two amino acids, such as L-arginine (L-Arg) and L-lysine (L-Lys). The formation of inclusion complexes was detected, and a comparative study was conducted at different pH, density, and viscosity. A physical mixture, comprising equal amount of amino acids was used to prepare the complex in a solid-state form. The experimental parameters, such as apparent molar volume, limiting apparent molar volume, partial molar volume were analyzed by measuring density at infinite dilution. The other quantities, such as dynamic viscosity, kinematic viscosity, relative viscosity, intrinsic viscosity, spatial viscosity, activation energy were determined for amino acid/ßCD complexes at various mass fractions of ßCDs and different temperatures. Finally, we found moderate (R2 > 0.5) and strong (R2 > 0.7) linear relationships (p-value < 0.0001) between the dynamic viscosity and the temperature: the temperature evaluation promotes the decrease in dynamic viscosity for amnio acid-ßCD (its derivatives) complexes. The results of this study emphasize important properties of analyzed complexes that can be utilized in the development of controlled drug delivery vectors.

Analyzing the driving forces of insulin stability in the basic amino acid solutions: A perspective from hydration dynamics.

Amino acids having basic side chains, as additives, are known to increase the stability of native-folded state of proteins, but their relative efficiency and the molecular mechanism are still controversial and obscure as well. In the present work, extensive atomistic molecular dynamics simulations were performed to investigate the hydration properties of aqueous solutions of concentrated arginine, histidine, and lysine and their comparative efficiency on regulating the conformational stability of the insulin monomer. We identified that in the aqueous solutions of the free amino acids, the nonuniform relaxation of amino acid-water hydrogen bonds was due to the entrapment of water molecules within the amino acid clusters formed in solutions. Insulin, when tested with these solutions, was found to show rigid conformations, relative to that in pure water. We observed that while the salt bridges formed by the lysine as an additive contributed more toward the direct interactions with insulin, the cation-π was more prominent for the insulin-arginine interactions. Importantly, it was observed that the preferentially more excluded arginine, compared to histidine and lysine from the insulin surface, enriches the hydration layer of the protein. Our study reveals that the loss of configurational entropy of insulin in arginine solution, as compared to that in pure water, is more as compared to the entropy loss in the other two amino acid solutions, which, moreover, was found to be due to the presence of motionally bound less entropic hydration water of insulin in arginine solution than in histidine or lysine solution.

Basic Amino Acid Conjugates of 1,2-Diselenan-4-amine with Protein Disulfide Isomerase-like Functions as a Manipulator of Protein Quality Control.

Protein disulfide isomerase (PDI) can assist immature proteins to correctly fold by controlling cysteinyl disulfide (SS)-relating reactions (i. e., SS-formation, SS-cleavage, and SS-isomerization). PDI controls protein quality by suppressing protein aggregation, as well as functions as an oxidative folding catalyst. Following the amino acid sequence of the active center in PDI, basic amino acid conjugates of 1,2-diselenan-4-amine (1), which show oxidoreductase- and isomerase-like activities for SS-relating reactions, were designed as a novel PDI model compound. By conjugating the amino acids, the diselenide reduction potential of compound 1 was significantly increased, causing improvement of the catalytic activities for all SS-relating reactions. Furthermore, these compounds, especially histidine-conjugated one, remarkably suppressed protein aggregation even at low concertation (0.3 mM∼). Thus, it was demonstrated that the conjugation of basic amino acids into 1 simultaneously achieves the enhancement of the redox reactivity and the capability to suppress protein aggregation.

Interaction of the neutral amino acid transporter ASCT2 with basic amino acids.

Glutamine transport across cell membranes is performed by a variety of transporters, including the alanine serine cysteine transporter 2 (ASCT2). The substrate-binding site of ASCT2 was proposed to be specific for small amino acids with neutral side chains, excluding basic substrates such as lysine. A series of competitive inhibitors of ASCT2 with low µM affinity were developed previously, on the basis of the 2,4-diaminobutyric acid (DAB) scaffold with a potential positive charge in the side chain. Therefore, we tested whether basic amino acids with side chains shorter than lysine can interact with the ASCT2 binding site. Molecular docking of L-1,3-diaminopropionic acid (L-DAP) and L-DAB suggested that these compounds bind to ASCT2. Consistent with this prediction, L-DAP and L-DAB, but not ornithine, lysine or D-DAP, elicited currents when applied to ASCT2-expressing cells. The currents were carried by anions and showed the hallmark properties of ASCT2 currents induced by transported substrates. The L-DAP response could be eliminated by a competitive ASCT2 inhibitor, suggesting that binding occurs at the substrate binding site. The KM for L-DAP was weakly voltage dependent. Furthermore, the pH dependence of the L-DAP response showed that the compound can bind in several protonation states. Together, these results suggest that the ASCT2 binding site is able to recognize L-amino acids with short, basic side chains, such as the L-DAP derivative ß-N-methylamino-l-Alanine (BMAA), a well-studied neurotoxin. Our results expand the substrate specificity of ASCT2 to include amino acid substrates with positively charged side chains.

Enantioselective Synthesis of a Tricyclic, sp3 -Rich Diazatetradecanedione: an Amino Acid-Based Natural Product-Like Scaffold.

6-, 7-, and 8-membered rings are assembled from a linear precursor by successive cyclisation reactions to construct a tricyclic diazatricyclo[6.5.1.04, 9 ]-tetradecanedione scaffold. Advanced building blocks based on d-aspartic acid and l-pyroglutamic acid were combined by a sp3 -sp2 Negishi coupling. A carbamate-guided syn-diastereoselective epoxidation followed by an intramolecular epoxide opening allowed the construction of the piperidine ring. An efficient one-pot hydroxyl-group protection twofold deprotection reaction prepared the ground for the cyclisation to the bicycle. A final deprotection of the orthogonal protecting groups and lactamisation led to the novel, sp3 -rich tricycle. The final compound is a substrate mimic of peptidyl-prolyl cis-trans isomerases featuring a locked trans-amide bond. Cheminformatic analysis of 179 virtual derivatives indicates favourable physicochemical properties and drug-like characteristics. As proof of concept we, show a low micromolar activity in a fluorescence polarisation assay towards the FK506-binding protein 12.

Channel from bacterial virus T7 DNA packaging motor for the differentiation of peptides composed of a mixture of acidic and basic amino acids.

Protein mutations can result in dysfunctional cell signaling pathways; therefore it is of significance to develop a robust platform for the detection of protein mutations. Here, we report that the channel of bacterial virus T7 DNA packaging motor is able to discriminate peptides containing a mixture of acidic (negatively charged) and basic (positively charged) amino acids. Peptides were differentiated based on their current signatures created by their unique charge compositions. In combination with protease digestion, peptides with the locational differences of single amino acid were also identified. The results suggest that the T7 motor channel has the potential for peptide differentiation, mutation verification, and analysis of protein sequence.

Rhamnolipids functionalized with basic amino acids: Synthesis, aggregation behavior, antibacterial activity and biodegradation studies.

Rhamnolipids have been intensively studied due to their remarkable properties; however, the biosynthesis of RLs cannot compete commercially with the production of synthetic surfactants. Here, novel cationic rhamnolipids (RLs) derivatives containing arginine and lysine were prepared for the first time using a straightforward synthetic procedure. The RLs used to prepare these new cationic derivatives were produced by Pseudomonas aeruginosa using waste frying oil as carbon source. It was found that the amino acid-based RLs form aggregates at very low concentrations, even below the CMC. Biodegradation studies indicate that these cationic RLs can be classified as readily biodegradable. Interestingly, the RL arginine conjugates exhibited notable DNA binding affinity and good antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, which increases the potential applications of these compounds. Consequently, the use of low-cost substrates and the added value of the final product constitute a more cost-effective rhamnolipid production.

Physical quantity of residue electrostatic energy in flavin mononucleotide binding protein dimer.

The electrostatic (ES) energy of each residue was for the first time quantitatively evaluated in a flavin mononucleotide binding protein (FBP). A residue electrostatic energy (RES) was obtained as the sum of the ES energies between atoms in each residue and all other atoms in the FBP dimer using atomic coordinates obtained by a molecular dynamics (MD) simulation. ES is one of the most important energies among the interaction energies in a protein. It is determined from the RES, the residues which mainly contribute to stabilize the structure of each subunit, and the binding energy between two subunits can be estimated. The RES of all residues in subunit A (Sub A) and subunit B (Sub B) were attractive forces, even though the residues contain net negative or positive charges. This reveals that the ES energies of any of the residues can contribute to stabilize the protein structure. The total binding ES energy over all residues among the subunits was distributed between -0.2 to -1.2 eV (mean = -0.67 eV) from the MD simulation time.

Water-Soluble Nanoparticle Receptors Supramolecularly Coded for Acidic Peptides.

Sequence-specific recognition of peptides is of enormous importance to many chemical and biological applications, but has been difficult to achieve due to the minute differences in the side chains of amino acids. Acidic peptides are known to play important roles in cell growth and gene expression. In this work, we report molecularly imprinted micelles coded with molecular recognition information for the acidic and hydrophobic side chains of acidic peptides. The imprinted receptors could distinguish acidic amino acids from other polar and nonpolar amino acids, with dissociation constants of tens of nanomolar for biologically active peptides containing up to 18 amino acids.

Evidence for the role of basic amino acids in the coat protein arm region of Cucumber necrosis virus in particle assembly and selective encapsidation of viral RNA.

Cucumber necrosis virus (CNV) is a T = 3 icosahedral virus with a (+)ssRNA genome. The N-terminal CNV coat protein arm contains a conserved, highly basic sequence ("KGRKPR"), which we postulate is involved in RNA encapsidation during virion assembly. Seven mutants were constructed by altering the CNV "KGRKPR" sequence; the four basic residues were mutated to alanine individually, in pairs, or in total. Virion accumulation and vRNA encapsidation were significantly reduced in mutants containing two or four substitutions and virion morphology was also affected, where both T = 1 and intermediate-sized particles were produced. Mutants with two or four substitutions encapsidated significantly greater levels of truncated RNA than that of WT, suggesting that basic residues in the "KGRKPR" sequence are important for encapsidation of full-length CNV RNA. Interestingly, "KGRKPR" mutants also encapsidated relatively higher levels of host RNA, suggesting that the "KGRKPR" sequence also contributes to selective encapsidation of CNV RNA.

Supramolecular Amino Acid Based Hydrogels: Probing the Contribution of Additive Molecules using NMR Spectroscopy.

Supramolecular hydrogels are composed of self-assembled solid networks that restrict the flow of water. l-Phenylalanine is the smallest molecule reported to date to form gel networks in water, and it is of particular interest due to its crystalline gel state. Single and multi-component hydrogels of l-phenylalanine are used herein as model materials to develop an NMR-based analytical approach to gain insight into the mechanisms of supramolecular gelation. Structure and composition of the gel fibres were probed using PXRD, solid-state NMR experiments and microscopic techniques. Solution-state NMR studies probed the properties of free gelator molecules in an equilibrium with bound molecules. The dynamics of exchange at the gel/solution interfaces was investigated further using high-resolution magic angle spinning (HR-MAS) and saturation transfer difference (STD) NMR experiments. This approach allowed the identification of which additive molecules contributed in modifying the material properties.

Activity of human kallikrein-related peptidase 6 (KLK6) on substrates containing sequences of basic amino acids. Is it a processing protease?

Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R↓R-ACC and Z-R↓R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates.

Characterization of the Wild-Type and Truncated Forms of a Neutral GH10 Xylanase from Coprinus cinereus: Roles of C-Terminal Basic Amino Acid-Rich Extension in Its SDS Resistance, Thermostability, and Activity.

A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase (CcXyn-Δ5C) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but CcXyn-Δ5C contained less α-helices (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature (45°C) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and CcXyn-Δ5C, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, CcXyn-Δ5C exhibited a much lower Km value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of CcXyn-Δ5C was 2.4-times higher than that of CcXyn. These properties make CcXyn-Δ5C a good model for the structure-function study of (α/ß)8-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.

Specially-Made Lipid-Based Assemblies for Improving Transmembrane Gene Delivery: Comparison of Basic Amino Acid Residue Rich Periphery.

Cationic lipid based assemblies provide a promising platform for effective gene condensation into nanosized particles, and the peripheral properties of the assemblies are vital for complexation and interaction with physical barriers. Here, we report three cationic twin head lipids, and each of them contains a dioleoyl-glutamate hydrophobic tail and a twin polar head of lysine, arginine, or histidine. Such lipids were proven to self-assemble in aqueous solution with well-defined nanostructures and residual amino-, guanidine-, or imidazole-rich periphery, showing strong buffering capacity and good liquidity. The assemblies with arginine (RL) or lysine (KL) periphery exhibited positive charges (∼+35 mV) and complete condensation of pDNA into nanosized complexes (∼120 nm). In contrast, assemblies composed of histidine-rich lipids (HL) showed relatively low cationic electric potential (∼+10 mV) and poor DNA binding ability. As expected, the designed RL assemblies with guanidine-rich periphery enhanced the in vitro gene transfection up to 190-fold as compared with the golden standard PEI25k and Lipofectamine 2000, especially in the presence of serum. Meanwhile, interaction with cell and endo/lysosome membrane also revealed the superiority of RL complexes, that the guanidine-rich surface efficiently promoted transmembrane process in cellular internalization and endosomal disruption. More importantly, RL complexes also succeeded beyond others in vivo with significantly (∼7-fold) enhanced expression in HepG2 tumor xenografts in mice, as well as stronger green fluorescence protein imaging in isolated tumors and tumor frozen sections.

Characterization of basic amino acids-conjugated PAMAM dendrimers as gene carriers for human adipose-derived mesenchymal stem cells.

Since mesenchymal stem cells (MSCs) can self-renew and differentiate into multiple cell types, the delivery of genes to this type of cell can be an important tool in the emerging field of tissue regeneration and engineering. However, development of more efficient and safe nonviral vectors for gene delivery to stem cells in particular still remains a great challenge. In this study, we describe a group of nonviral gene delivery vectors, conjugated PAMAM derivatives (PAMAM-H-R, PAMAM-H-K, and PAMAM-H-O), displaying affinity toward human adipose-derived mesenchymal stem cells (AD-MSCs). Transfection efficiency using pDNA encoding for luciferase (Luc) and enhanced green fluorescent protein (EGFP), and cytotoxicity assays were performed in human AD-MSCs. The results show that transfection efficiencies of conjugated PAMAM derivatives are improved significantly compared to native PAMAM dendrimer, and that among PAMAM derivatives, cytotoxicity of PAMAM-H-K and PAMAM-H-O were very low. Also, treatment of human AD-MSCs to polyplex formation in conjugated PAMAM derivatives, their cellular uptake and localization were analyzed by flow cytometry and confocal microscopy.

Kinetics of CO2 Absorption into Aqueous Basic Amino Acid Salt: Potassium Salt of Lysine Solution.

Aqueous amino acid salts are considered as an attractive alternative to alkanolamine solvents (e.g., MEA) for carbon dioxide (CO2) absorption. The kinetics of CO2 into unloaded aqueous solutions of potassium lysinate (LysK) was studied using a wetted wall column at concentrations ranging from 0.25 to 2.0 M and temperatures from 298 to 333 K. Physicochemical properties of aqueous LysK solutions such as density, viscosity, and physical solubility of CO2 were measured to evaluate the reaction rate constants. The reaction pathway is described using zwitterion mechanism taking into account the effect of ionic strength on the reaction rate. Under the fast pseudo-first-order regime, the reaction rate parameters were obtained and correlated in a power-law reaction rate expression. LysK shows higher chemical reactivity toward CO2 than the industrial standard MEA and most of amino acid salts. Its reaction rate constants increase considerably with concentration and temperature. The reaction order is found to be an average value of 1.58 with respect to LysK. The forward second-order kinetic rate constant, k2 0 , are obtained as 31615 and 84822 m3 kmol−1 s−1 at 298 and 313 K, respectively with activation energy of 51.0 kJ mol−1. The contribution of water to the zwitterion deprotonation seems to be more significant than that of LysK for the above-mentioned kinetic conditions