Α-helical Structure Research Articles

Analysis of the structure and interactions of the SARS-CoV-2 ORF7b accessory protein

SARS-CoV-2 carries a sizeable number of proteins that are accessory to replication but may be essential for virus-host interactions and modulation of the host immune response. Here, we investigated the structure and interactions of the largely unknown ORF7b, a small membranous accessory membrane protein of SARS-CoV-2. We show that structural predictions indicate a transmembrane (TM) leucine zipper for ORF7b, and experimentally confirm the predominantly α-helical secondary structure within a phospholipid membrane mimetic by solid-state NMR. We also show that ORF7b forms heterogeneous higher-order multimers. We determined ORF7b interactions with cellular TM leucine zipper proteins using both biochemical and NMR approaches, providing evidence for ORF7b interaction with the TM domains of E-cadherin, as well as phospholamban. Our results place ORF7b as a hypothetical interferer in cellular processes that utilize leucine zipper motifs in transmembrane multimerization domains.

Identification and synthesis of a long-chain antimicrobial peptide from the venom of the Liocheles australasiae scorpion.

Scorpion venom contains linear peptides without disulfide bonds in addition to peptides with disulfide bonds. Many such linear peptides have an amphiphilic α-helical structure, often with antimicrobial activity and can be classified into three groups based on their molecular size. Among them, long-chain antimicrobial peptides consisting of more than 40 residues have not been thoroughly studied due to the difficulty of synthesizing them. We have previously reported a transcriptome analysis of the venom gland of Liocheles australasiae that revealed precursor sequences of long-chain antimicrobial peptides. In the study reported here, we identified the mature structure of one such long-chain antimicrobial peptide, LaCT1, which we synthesized using chemical ligation to confirm its structure and evaluate its biological activities. The result showed that LaCT1 exhibited significant antimicrobial activity. In addition, we identified its partial peptides consisting of an N- or C-terminal region, which may be generated by enzymatic cleavage in the venom. Among them, only the peptide containing the N-terminal half region was active. LaCT1 also not only showed insecticidal activity but also synergistically enhanced the effects of another insecticidal peptide identified in L. australasiae venom as well. These results provide insights into the role of antimicrobial peptides in scorpion venom.

Development of α-Helical Antimicrobial Peptides with Imperfect Amphipathicity for Superior Activity and Selectivity.

The advancement of antimicrobial peptides (AMPs) as therapeutic agents is hindered by their poor selectivity. Recent evidence indicates that controlled disruption of the amphipathicity of α-helical AMPs may increase the selectivity. This study investigated the role of imperfect amphipathicity in optimizing AMPs with varied sequences to enhance their activity and selectivity. Among these, the lead peptide RI-18, characterized by an imperfectly amphipathic α-helical structure, demonstrated potent and broad-spectrum antibacterial activity without inducing hemolytic or cytotoxic effects. RI-18 effectively eliminated planktonic and biofilm-associated bacteria as well as persister cells and exhibited high bacterial plasma membrane affinity, inducing rapid membrane permeabilization and rupture. Notably, RI-18 significantly reduced bacterial loads without promoting bacterial resistance, highlighting its therapeutic potential. Overall, this study identified RI-18 as a promising antimicrobial candidate. The rational strategy of tuning imperfect amphipathicity to enhance the AMP activity and selectivity may facilitate the design and development of AMPs.

The Anti-Flyaway/Frizz Effect by Inducing the α-Helical Structure Transition of Hair

In order to reduce chronic hair flyaways/frizz, both reducing and oxidizing agents have to be used, leaving aside the hair damage issues. This study presents changes in hair morphology caused by treatment with a shampoo containing only reducing agents, excluding oxidizing agents that affect critical hair damages. As a result of flyaway/frizz improvement rates calculated through monitoring of the area of light transmittance in the hair tresses, reducing agents, such as ammonium thioglycolate (ATG), L-cysteine, and sodium sulfite were found to be effective in decreasing hair flyaway/frizz. Additionally, the methods to maintain homeostasis and control damage caused by oxidation during washing were also used to see flyaway/frizz improvement rates. Measurements using electrostatic force microscopy (EFM) showed that the surface charge of hair tresses treated with shampoo containing reducing agents was lowered. Using Raman spectroscopic analysis, it has been suggested that these treatments with reducing agents induced a 3D structural transition of the hair from an α-helix to a random coil. In addition, this structural release was confirmed, identifying the reduction in the enthalpy of the α-helix using differential scanning calorimetry (DSC). Furthermore, we verified that this change causes no hair damage through a tensile strength test. Therefore, the formulation of shampoo with reducing agents can be used as an effective strategy to care for hair flyaway/frizz without hair damage issues.

The Wheel of Fortune: Helical Wheel Alanine Scanning of a Spider Venom Antimicrobial Peptide Reveals Residues Involved in Antimicrobial and Cytotoxic Activity.

A preference for several amino acids is observed to occur at particular positions of cationic α-helical antimicrobial peptides (AMPs), which ensures the formation of amphipathic regions once they assume their correct secondary structure in membranes or membrane-mimicking environments and makes them active against pathogens. This study determined the effect of alanine mutations on the secondary structure and bioactivity of lyp1987 (GRLQAFLAKMKEIAAQTL-NH2), a cationic α-helical AMP obtained from the venom of Lycosa poonaensis which exhibits broad range activity against Gram-positive and Gram-negative bacteria with micromolar minimum inhibitory concentrations (MIC). CD spectroscopy revealed no significant difference in the secondary structure, with all alanine-substituted analogs exhibiting predominantly α-helical structure in buffered 2,2,2-trifluoroethanol solution. Alanine substitution at Glu12 and Thr17 increased the activity of lyp1987 against Gram-positive and -negative bacteria, while alanine substitution at Lys9 increased its selectivity against Gram-positive bacteria. Further investigation can be done to determine positions and substitutions that will give less cytotoxic analogs.

Interactions of Li+ ions with NCS1: A potential mechanism of Li+ neuroprotective action against psychotic disorders

Li+ based drugs have been used for the treatment of psychiatric disorders due to their mood stabilizing role for decades. Recently, several studies reported the protective effect of Li+ against severe neuropathologies such as Parkinson's, Alzheimer's, and Huntington's disease. Surprisingly, despite a broad range of Li+ effects on neurological conditions, little is known about its molecular mechanism. In this study, we propose that neuronal calcium sensor 1 (NCS1), can be an effective molecular target for Li+ action. Here we show that the EF-hands in ApoNCS1 have submillimolar affinity for Li+ with Kd = 223 ± 19 μM. Li+ binding to ApoNCS1 quenches Trp emission intensity, suggesting distinct Trp sidechains environment in Li+NCS1 compared to ApoNCS1 and Ca2+NCS1. Li+ association also stabilizes the protein α-helical structure, in a similar way to Ca2+. Li+ association does not promote NCS1 dimerization. Association of Li+ increases NCS1 affinity for the D2R receptor binding peptide, in a similar way to Ca2+, however, the affinity of NCS1 for chlorpromazine is reduced with respect to Ca2+NCS1, possibly due to a decrease in solvent exposed hydrophobic area on the NCS1 surface in the presence of Li+. MD simulation data suggests that Li+ ions are coordinated by four oxygens from Asp and Glu sidechains and one carbonyl oxygen, in a similar way as reported previously for Li+ binding to DREAM. Overall, the data shows that Li+ binds to EF-hands of NCS1 and Li+NCS1 interactions may be involved in the potential neuroprotective role of Li+ against psychotic disorders.

Raman Microspectroscopy of Hair: Low-Frequency Markers of Protein Secondary Structure.

Low-frequency intervals that can be used to study the secondary structure of proteins are determined. Compared are Ramanspectra of keratins from unpigmented human hair, measured in two experimental configurations: with excitation radiation coaxial with the hair and perpendicular to it. Based on the polarization sensitivity, the bands peaked at 150 and 221 cm-1 are assigned to vibrations of α-helical structures. The comparison of Raman spectra of hair fragments with different contents of secondary structure elements shows that the vibrations of β-structure are manifested in a spectral interval of 270-340 cm-1. The results obtained for a particular object (hair keratin) can be used in the study of the secondary structure of proteins.

Effect of ultrasound-assisted Maillard reaction on functional properties and flavor characteristics of Oyster protein enzymatic hydrolysates

To address the delamination phenomenon during storage and flavor characteristics of Oyster protein hydrolysates (OPH). In this study, xylo-oligosaccharides (XOS) were selected to covalently graft with OPH through ultrasound-assisted Maillard reaction, and the effect of ultrasound-assisted Maillard reaction on the structure, functional properties, and flavor characteristics of OPH were investigated. The results revealed that the ultrasound treatment led to a 1.46-fold increase in the degree of grafting compared with the conventional wet-heat Maillard reaction methods. Structural analyses at various levels indicated substantial alterations in the OPH structure following the ultrasound-assisted Maillard reaction. More ordered α-helical secondary structures were shifting to random coiling, the tertiary structure showed more stretching changes, and the surface structure was characterized by loose and porous features. Compared with OPH, the solubility of the ultrasound-assisted Maillard reaction products (OPH-U-M) increased from 54.67% to 70.14%, leading to a notable enhancement in storage stability. Flavor profile analysis demonstrated a decrease in unsaturated aldehydes and ketones presenting fishy and bitter aromas, while an increase in presenting meat aroma compounds was observed in OPH-U-M. Furthermore, OPH-U-M exhibited superior antioxidant properties with DPPH and ABTS radical scavenging abilities enhancing 46.05% and 42.09% in comparison with OPH, respectively. The results demonstrated that covalently binding with XOS under ultrasonication pretreatment endowed OPH with superior functional properties (including solubility, storage stability, and antioxidant activity), and the improvement of flavor profile. This study can provide theoretical guidance and practical implications for promoting the processing applications of oyster protein.

A novel antimicrobial peptide WBp-1 from wheat bran: Purification, characterization and antibacterial potential against Listeria monocytogenes

This study introduces a novel antimicrobial peptide (AMP), WBp-1, isolated from wheat bran and purified via reversed-phase high-performance liquid chromatography. The amino acid sequence, determined as IITGASSGIGKAIAKHFI by LC–MS/MS, was composed predominantly of alkaline and hydrophobic residues. WBp-1 was predicted to be a stable, hydrophobic, cationic peptide with an α-helical structure. Moreover, it displayed significant antibacterial efficacy against Listeria monocytogenes, with a minimum inhibitory concentration of 150 μg/mL. Further mechanistic studies suggest that WBp-1 exerts its bactericidal activity by disrupting cell membrane integrity, impeding peptidoglycan synthesis by binding to penicillin-binding protein 4 via hydrogen bonding, increasing cell permeability, altering membrane potential and fluidity, and altering surface hydrophobicity. Interestingly, WBp-1 showed minimal hemolytic activity and cytotoxicity against LO2 cells, even at 16× MIC. These findings highlight the strong potential of WBp-1 as a novel antibacterial agent and food preservative against Listeria monocytogenes.

Molecular Mechanics Simulation and Ftir Spectrocopy Optimization of Secondary Structure of Aβ (25-35) Peptide

Abstract In this study, we probed the secondary structure of a soluble Aβ (25-35) peptide in water solution by applying a molecular mechanics method as well as infrared spectroscopy. We have used Fourier Transformed Infrared Spectroscopy (FTIR) to examine the secondary structure of this peptide. The amide I bands of Ab (25-35) obtained from transmission-FTIR spectra consists of one main band at 1658 cm-1, at both concentrations used (200 mM and 1 mM). This band shows us that Ab (25-35) in aqueous solution was mostly organized into a a-helical structure (48 %). The contribution of the unordered structure was found to be about 12 %. The proportion of b-sheet and b-turn structures are slightly lower, 12-15 % and 13-14 %, respectively for both concentrations. FTIR spectra of the peptide in water solution (100 µM) after incubation for 12h showed Aβ peptide conformation is critically dependent on the environmental conditions used. The simulation approach reveals that the C-terminal octapeptide of this molecule preferably adopts an α-helical structure, while the N-terminal tripeptide may exist as a β-turn and unordered structures.

Sequential application of cold plasma in chicken breast: Improvement of meat tenderness and bactericidal mechanism against histamine-producing Raoultella sp.

The feasibility of plasma-activated lactic acid solution (PALA) combined with dielectric barrier discharge (DBD) plasma treatment in chicken breast was evaluated. Results indicated that sequential plasma treatments slowed down the increase in the pH value and TVB-N value of meat during storage. Moreover, PALA treatment weakened the lipid oxidation triggered by DBD plasma treatment. Furthermore, centrifugal loss and shear force of chicken breast in the PALA-DBD group were reduced by 2.88% and 19.4%, respectively, and the myofibril fragmentation index was significantly increased compared to the control group, and PALA treatment increased free water content. Microbiological analyses showed that PALA-DBD treatment disrupted the cell membrane integrity of Raoultella sp., induced oxidative stress and reduced the activity of antioxidant enzymes (catalase and superoxide dismutase), led to the degradation of bacterial proteins and the disruption of α-helical structure.

Effect of citrus fiber on physicochemical quality of Frankfurter sausages: A study on lipid oxidation and protein gel characteristics

Citrus fibers (CF) are frequently used to enhance the water-holding capacity and nutritional profile of meat products. However, their impact on the lipid oxidation and gel properties of emulsified meat products remains incompletely elucidated. This study aims to comprehensively assess the physicochemical and sensory attributes of frankfurter sausages enriched with CF and elucidate the underlying mechanisms. The properties of lipid oxidation, protein intermolecular interactions, and microstructural changes in CF-enriched frankfurter sausages were investigated. The results indicate the potential role of flavonoids contained in CF in preventing lipid oxidation in frankfurters sausages. Furthermore, the incorporation of CF contributes to the formation of a denser microstructure, achieved through the exposure of protein hydrophobic groups and the transformation of α-helical structures into β-turn configurations. These findings advance the understanding of how CF impacts the quality of frankfurter sausages, offering insights for improving the utilization of CF resources and developing higher-value functional meat products.

Impact of lipidation site on the activity of α-helical antimicrobial peptides

Antimicrobial peptides (AMPs) display advantages over traditional antibiotics due to their broad spectrum of activity against various pathogens, and may even overcome bacterial drug resistance. However, despite their potential therapeutic benefits, widespread application of AMPs is limited by their instability, sensitivity to high salt concentrations, toxicity, and immunogenicity. Lipidation is a promising strategy in overcoming these drawbacks and potential problems for drug candidates. While N-terminal lipidation is a well-studied form of acylation of biologically active peptides, fatty acylation of the lysine side chain has still been poorly explored. In this study, we examined systematic introduction of octanoic (C8) or decanoic (C10) acid into the sequences of three antimicrobial α-helical peptides, namely LL-I, LK6, and ATRA-1, by acylation of subsequent lysine residues, resulting in 17 lipopeptides. Fatty acid lengths optimal for antimicrobial activity were selected based on a previous study on the N-terminal lipidated counterparts of these peptides. Shuffling the position of the fatty acid tails in the sequences of the peptides preserved high activity against Gram-positive bacteria, increased activity against Gram-negative strains and reduced cytotoxicity, compared to the N-terminal acylated counterparts. In the case of the LL-I and LK6 conjugates, the interactions with artificial negatively charged membranes induced formation of an α-helical structure but without a direct correlation between helicity and amphipathicity. Unexpectedly, the ATRA-1 derivatives showed only a small tendency, if any, to adopt a helical structure upon binding to POPG vesicles, which may indicate a non-helical active conformation. A more detailed study of the selected analogues, namely LL-I-4C8, LK6-7C8, and ATRA-1-11C10, provided evidence of a tendency to self-assemble into clumped and/or isolated fibrils, micelles or clusters of micelles, and proved that the lipid bilayer is the main target of action of the tested lipopeptides. In summary, the results of the present study highlight that alternative conjugation sites for lipid modification of AMPs, rather than the commonly applied N-terminal conjugation site, may improve the selectivity of action and be feasible in testing for the development of new lipid-peptide conjugates.

Effect of chickpea protein modified with combined heating and high-pressure homogenization on enhancing the gelation of reduced phosphate myofibrillar protein

The effects of chickpea protein (CP) modified by heating and/or high-pressure homogenization (HPH) on the gelling properties of myofibrillar protein under reduced phosphate conditions (5 mM sodium triphosphate, STPP) were investigated. The results showed that heating and HPH dual-modified CP could decrease the cooking loss by 29.57 %, elevate the water holding capacity by 17.08 %, and increase the gel strength by 126.88 %, which conferred myofibrillar protein with gelation performance comparable with, or even surpassing, that of the high-phosphate (10 mM STPP) control. This gelation behavior improvement could be attributed to enhanced myosin tail–tail interactions, decreased myosin thermal stability, elevated trans–gauche–trans disulfide conformation, strengthened hydrophobic interactions and hydrogen bonding, the uncoiling of α-helical structures, the formation of well-networked myofibrillar protein gel, and the disulfide linkages between the myosin heavy chain, actin, and CP subunits. Therefore, the dual-modified CP could be a promising phosphate alternative to develop healthier meat products.

Mixed Stereochemistry Macrocycle Acts as a Helix-Stabilizing Peptide N-Cap.

Interactions between proteins and α-helical peptides have been the focus of drug discovery campaigns. However, the large interfaces formed between multiple turns of an α-helix and a binding protein represent a significant challenge to inhibitor discovery. Modified peptides featuring helix-stabilizing macrocycles have shown promise as inhibitors of these interactions. Here, we tested the ability of N-terminal to side-chain thioether-cyclized peptides to inhibit the α-helix binding protein Mcl-1, by screening a trillion-scale library. The enriched peptides were lariats featuring a small, four-amino-acid N-terminal macrocycle followed by a short linear sequence that resembled the natural α-helical Mcl-1 ligands. These "Heliats" (helical lariats) bound Mcl-1 with tens of nM affinity, and inhibited the interaction between Mcl-1 and a natural peptide ligand. Macrocyclization was found to stabilize α-helical structures and significantly contribute to affinity and potency. Yet, the 2nd and 3rd positions within the macrocycle were permissible to sequence variation, so that a minimal macrocyclic motif, of an N-acetylated d-phenylalanine at the 1st position thioether connected to a cysteine at the 4th, could be grafted into a range of peptides and stabilize helical conformations. We found that d-stereochemistry is more helix-stabilizing than l- at the 1st position in the motif, as the d-amino acid can utilize polyproline II torsional angles that allow for more optimal intrachain hydrogen bonding. This mixed stereochemistry macrocyclic N-cap is synthetically accessible, requiring only minor modifications to standard solid-phase peptide synthesis, and its compatibility with peptide screening can provide ready access to helix-focused peptide libraries for de novo inhibitor discovery.

The fate and behavior mechanism of antibiotic resistance genes in different-sized aerobic granular sludge under antibiotics pressure

Aerobic granular sludge (AGS) is a promising wastewater treatment technology. Studying the distribution of ARGs in different-sized AGS under antibiotic pressure is crucial for ARGs risk assessment and control technology development. Herein, the risk of intracellular ARGs (iARGs) in viable/dead bacteria within small (0–1 mm), moderate (1–2 mm), and big (2–3 mm) AGS (S/M/B-AGS) were clarified under tetracycline and sulfadiazine stress. Besides, the potential mechanism of ARGs enrichment differences in S/M/B-AGS was revealed. M−AGS had the lowest iARGs risk (30–60 days) in both viable (3.3E+08 copies/g-dw) and dead (6.7E+08 copies/g-dw) bacteria, averaging 29.8 % lower than B-AGS and 72.1 % lower than S-AGS, respectively. The average changes in abundance of the top 20 genera in S/M/B-AGS were 3.0 %, 2.6 %, and 2.9 % from day 30 to day 60, respectively. The complexity between ARGs and bacterial communities was highest in S-AGS, followed by B-AGS and M−AGS. Among the 19–20 identified hosts in S/M/B-AGS, only 11 hosts were shared. Extracellular polymeric substances (EPS) and protein, particularly those with α-helical structure, adsorbed antibiotics and envelop bacteria, enhancing mass transfer resistance and effectively reducing antibiotic stress on bacteria. M−AGS exhibited the highest contents of EPS and protein compared to S/B-AGS, potentially explaining the lowest iARGs risk observed. Overall, this study discovered the different behaviors of iARGs in viable/dead bacteria in S/M/B-AGS, offering a novel perspective on granular size modulation strategies to reduce ARGs risk.

Molecular characterisation of interactions between 11S glycinin and hexanal – An off flavour compound

This work examines the binding characteristics between the 11S glycinin from soy flour with the aldehyde compound hexanal, at near neutral pH and ambient temperature. FTIR and CD spectroscopy determined that secondary structure changes were evident upon complexation with observed increases in α-helical and unordered structures along with reductions in β-sheets and β-turn structures upon complexation. UV–Vis and MALDI-TOF-MS analysis confirmed the physical nature of hexanal stabilisation, with in silico analysis and fluorescence spectroscopy revealed a low to medium binding strength with a KA range of 3.1 × 102 to 3.1 × 104 M−1 along with a 1:1 binding stoichiometry between the compound and 11S. Molecular docking simulations determined the binding location is situated between chain A and B of the trimer and is largely stabilised by pi-alkyl, hydrogen and van der Waals forces. Findings from this study will provide increased knowledge in the field of flavour compound and protein interactions thus advancing our knowledge of flavour binding and release in plant-based beverage formulations.

Activity of Membrane-Permeabilizing Lpt Peptides.

Herein, we investigated the toxicity and membrane-permeabilizing capabilities of Lpt and Lpt-like peptides, belonging to type I toxin-antitoxin systems carried by plasmid DNA of Lacticaseibacillus strains. These 29 amino acid peptides are predicted to form α-helical structures with a conserved central hydrophobic sequence and differently charged hydrophilic termini. Like Lpt, the expression of Lpt-like in E. coli induced growth arrest, nucleoid condensation, and cell membrane damage, suggesting membrane interaction as the mode of action. The membrane permeabilization activity of both peptides was evaluated by using liposome leakage assays, dynamic light scattering, and CD spectroscopy. Lpt and Lpt-like showed liposome leakage activity, which did not lead to liposome disruption but depended on peptide concentration. Lpt was generally more effective than Lpt-like, probably due to different physical chemical properties. Leakage was significantly reduced in larger liposomes and increased with negatively charged PCPS liposomes, indicating that electrostatic interactions and membrane curvature influence peptide activity. Contrary to most membrane-active peptides, Lpt an Lpt-like progressively lost their α-helical structure upon interaction with liposomes. Our data are inconsistent with the formation of membrane-spanning peptide pores but support a mechanism relying on the transient failure of the membrane permeability barrier possibly through the formation of "lipid pores".

Photothermally Heated Asymmetric Thin Nanopores Suggest the Influence of Temperature on the Intermediate Conformational State of Cytochrome c in an Electric Field.

Nanopore sensing is a label-free single-molecule technique that enables the study of the dynamical structural properties of proteins. Here, we detect the translocation of cytochrome c (Cyt c) through an asymmetric thin nanopore with photothermal heating to evaluate the influence of temperature on Cyt c conformation during its translocation in an electric field. Before Cyt c translocates through an asymmetric thin SiNx nanopore, ∼1 ms trapping events occur due to electric field-induced denaturation. These trapping events were corroborated by a control analysis with a transmission electron microscopy-drilled pore and denaturant buffer. Cyt c translocation events exhibited markedly greater broad current blockade when the pores were photothermally heated. Collectively, our molecular dynamics simulation predicted that an increased temperature facilitates denaturation of the α-helical structure of Cyt c, resulting in greater blockade current during Cyt c trapping. Our photothermal heating method can be used to study the influence of temperature on protein conformation at the single-molecule level in a label-free manner.

SCN as a local probe of protein structural dynamics.

The dynamics of lysozyme is probed by attaching -SCN to all alanine residues. The one-dimensional infrared spectra exhibit frequency shifts in the position of the maximum absorption of 4 cm-1, which is consistent with experiments in different solvents and indicates moderately strong interactions of the vibrational probe with its environment. Isotopic substitution 12C → 13C leads to a redshift by -47 cm-1, which agrees quantitatively with experiments for CN-substituted copper complexes in solution. The low-frequency, far-infrared part of the protein spectra contains label-specific information in the difference spectra when compared with the wild type protein. Depending on the position of the labels, local structural changes are observed. For example, introducing the -SCN label at Ala129 leads to breaking of the α-helical structure with concomitant change in the far-infrared spectrum. Finally, changes in the local hydration of SCN-labeled alanine residues as a function of time can be related to the reorientation of the label. It is concluded that -SCN is potentially useful for probing protein dynamics, both in the high-frequency part (CN-stretch) and in the far-infrared part of the spectrum.