Hydrophobic Peptide Research Articles

Gene cloning, recombinant expression, and bioassay of an allatotropin in Spodoptera litura Fabricius\xa0(Lepidoptera: Noctuidae)

BackgroundAllatotropin, a neuropeptide found in several invertebrates indirectly regulates vitellogenesis by stimulating juvenile hormone biosynthesis by the corpora allata. Here, we cloned and expressed the gene encoding allatotropin of Spodoptera litura (tobacco cutworm), a polyphagous pest in the Asian tropics. This study is aimed at studying the effect of recombinant protein on egg-laying in females of S. litura as it could be used as a method to control the pests from a reproductive perspective.ResultsThe protein encodes a full-length open reading frame consisting of 173 amino acids and was rich in arginine (10%) and glutamic acid (9.3%). The theoretical pI of the protein was 5.47 and a hydrophobic signal peptide of 22 residues was predicted. The recombinant allatotropin was expressed in Escherichia coli BL21 (DE3) and purified by nickel exchange chromatography. The molecular weight of the recombinant protein was about 37 kDa and expression levels up to 5.3 mg/ml were achieved. Injection assay in vitro indicated that allatotropin induces egg-laying during the first scotophase after treatment in females of Spodoptera litura.ConclusionAllatotropin induces egg-laying in female moths and could be a potential molecule for the development of control strategies against Spodoptera litura. In this strategy, the protein if delivered to the females before mating may lead to accelerated egg deposition much before she encounters the male moths, thus the population being checked as the eggs deposited by the females are unfertilized. Thus, the present work could lead to the development of a protein based biopesticide resulting into a species-specific and an eco-friendly way of pest control.

Opposing Effects of PhoPQ and PmrAB on the Properties of Salmonella enterica serovar Typhimurium: Implications on Resistance to Antimicrobial Peptides.

The increasing number of resistant bacteria is a major threat worldwide, leading to the search for new antibiotic agents. One of the leading strategies is the use of antimicrobial peptides (AMPs), cationic and hydrophobic innate immune defense peptides. A major target of AMPs is the bacterial membrane. Notably, accumulating data suggest that AMPs can activate the two-component systems (TCSs) of Gram-negative bacteria. These include PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB), responsible for remodeling of the bacterial cell surface. To better understand this mechanism, we utilized bacteria deficient either in one system alone or in both and biophysical tools including fluorescence spectroscopy, single-cell atomic force microscopy, electron microscopy, and mass spectrometry (MoskowitzS. M.;Antimicrob. Agents Chemother.2012, 56, 1019−103022106224; ChengH. Y.;J. Biomed. Sci.2010, 17, 6020653976). Our data suggested that the two systems have opposing effects on the properties of Salmonella enterica. The knockout of PhoPQ made the bacteria more susceptible to AMPs by making the surface less rigid, more polarized, and permeable with a slightly more negatively charged cell wall. In addition, the periplasmic space is thinner. In contrast, the knockout of PmrAB did not affect its susceptibility, while it made the bacterial outer layer very rigid, less polarized, and less permeable than the other two mutants, with a negatively charged cell wall similar to the WT. Overall, the data suggest that the coexistence of systems with opposing effects on the biophysical properties of the bacteria contribute to their membrane flexibility, which, on the one hand, is important to accommodate changing environments and, on the other hand, may inhibit the development of meaningful resistance to AMPs.

Потенційне значення бактеріоцинів при етіологічному лікуванні інфекційних захворювань респіраторного тракту

Бактеріоцини являють собою рибосомно синтезовані гідрофобні антимікробні пептиди, що мають широкий антимікробний спектр дії, основним джерелом яких є грампозитивні бактерії. Бактеріоцини різних класів, діючи на грампозитивні і грамнегативні бактерії, використовують різні молекулярні механізми, функціонування яких призводить до порушення цілісності клітинної стінки або пригнічення синтезу нуклеїнових кислот чи протеїнів таргетних бактерій. На сьогодні описано понад 100 лантибіотичних пептидів, багато з яких використовують для розробки майбутніх лікарських засобів. Кілька лантибіотиків в даний час проходять клінічні та доклінічні випробування, у тому числі біоінженерні лантибіотики, які можуть зіграти ключову роль в подоланні антибіотикорезистентності до бактеріальних патогенів.

Efficient Synthesis of 2'-O-Methoxyethyl Oligonucleotide-Cationic Peptide Conjugates.

Single‐stranded phosphorothioate (PS) oligonucleotide drugs have shown potential for the treatment of several rare diseases. However, a barrier to their widespread use is that they exhibit activity in only a narrow range of tissues. One way to circumvent this constraint is to conjugate them to cationic cell‐penetrating peptides (CPPs). Although there are several examples of morpholino and peptide nucleic acids conjugated with CPPs, there are noticeably few examples of PS oligonucleotide‐CPP conjugates. This is surprising given that PS oligonucleotides presently represent the largest class of approved RNA‐based drugs, including Nusinersen, that bears the 2’‐O‐methoxyethyl (MOE)‐chemistry. In this work, we report a method for in‐solution conjugation of cationic, hydrophobic peptides or human serum albumin to a 22‐nucleotide MOE‐PS oligonucleotide. Conjugates were obtained in high yields and purities. Our findings pave the way for their large‐scale synthesis and testing in vivo.

Direct Target Site Identification of a Sulfonyl-Triazole Covalent Kinase Probe by LC-MS Chemical Proteomics.

Chemical proteomics is widely used for the global investigation of protein activity and binding of small molecule ligands. Covalent probe binding and inhibition are assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to gain molecular information on targeted proteins and probe-modified sites. The identification of amino acid sites modified by large complex probes, however, is particularly challenging because of the increased size, hydrophobicity, and charge state of peptides derived from modified proteins. These studies are important for direct evaluation of proteome-wide selectivity of inhibitor scaffolds used to develop targeted covalent inhibitors. Here, we disclose reverse-phase chromatography and MS dissociation conditions tailored for binding site identification using a clickable covalent kinase inhibitor containing a sulfonyl-triazole reactive group (KY-26). We applied this LC-MS/MS strategy to identify tyrosine and lysine sites modified by KY-26 in functional sites of kinases and other ATP-/NAD-binding proteins (>65 in total) in live cells. Our studies revealed key bioanalytical conditions to guide future chemical proteomic workflows for direct target site identification of complex irreversible probes and inhibitors.

Designing Self-Inhibitory fusion peptide analogous to viral spike protein against novel severe acute respiratory syndrome (SARS-CoV-2)

COVID-19 is a highly contagious viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is declared pandemic by the World Health Organization (WHO). The spike protein of SARS-CoV-2 is a key component playing a pivotal role in facilitating viral fusion as well as release of genome into the host cell. Till date there is no clinically approved vaccine or drug available against Covid-19. We designed four hydrophobic inhibitory peptides (ITPs) based on WWIHS (Wimley and White interfacial hydrophobicity scale) score, targeting the HR1 domain of spike protein. Two inhibitory peptides out of four have a strong affinity to the hydrophobic surface of HR1 domain in pre-fusion spike protein. The MD simulation result showed the strong accommodation of ITPs with HR1 domain surface. These self-inhibitory peptides mimic the function of HR2 by binding to HR1 domain, thus inhibiting the formation of HR1-HR2 post-fusion complex, which is a key structure for virus-host tropism. Communicated by Ramaswamy H. Sarma

Amyloid-like Self-Assembly of a Hydrophobic Cell-Penetrating Peptide and Its Use as a Carrier for Nucleic Acids.

Cell-penetrating peptides (CPPs) are a topical subject potentially exploitable for creating nanotherapeutics for the delivery of bioactive loads. These compounds are often classified into three major categories according to their physicochemical characteristics: cationic, amphiphilic, and hydrophobic. Among them, the group of hydrophobic CPPs has received increasing attention in recent years due to toxicity concerns posed by highly cationic CPPs. The hexapeptide PFVYLI (P, proline; F, phenylalanine; V, valine; Y, tyrosine; L, leucine; and I, isoleucine), a fragment derived from the C-terminal portion of α1-antitrypsin, is a prototypal example of hydrophobic CPP. This sequence shows reduced cytotoxicity and a capacity of nuclear localization, and its small size readily hints at its suitability as a building block to construct nanostructured materials. In this study, we examine the self-assembling properties of PFVYLI and investigate its ability to form noncovalent complexes with nucleic acids. By using a combination of biophysical tools including synchrotron small-angle X-ray scattering and atomic force microscopy-based infrared spectroscopy, we discovered that this CPP self-assembles into discrete nanofibrils with remarkable amyloidogenic features. Over the course of days, these fibrils coalesce into rodlike crystals that easily reach the micrometer range. Despite lacking cationic residues in the composition, PFVYLI forms noncovalent complexes with nucleic acids that retain β-sheet pairing found in amyloid aggregates. In vitro vectorization experiments performed with double-stranded DNA fragments indicate that complexes promote the internalization of nucleic acids, revealing that tropism toward cell membranes is preserved upon complexation. On the other hand, transfection assays with splice-correction oligonucleotides (SCOs) for luciferase expression show limited bioactivity across a narrow concentration window, suggesting that the propensity to form amyloidogenic aggregates may trigger endosomal entrapment. We anticipate that the findings presented here open perspectives for using this archetypical hydrophobic CPP in the fabrication of nanostructured scaffolds, which potentially integrate properties of amyloids and translocation capabilities of CPPs.

Reduction in flavor-intense components in fish protein hydrolysates by membrane filtration.

Enzymatic protein hydrolysates based on side stream materials from the fish-filleting industry are increasingly explored as food ingredients. However, intense sensory properties, and high salt contents, are often a limiting factor. Most of the sensory attributes, such as fish flavor and salty taste, can be ascribed to low-molecular-weight, water-soluble components, whereas bitterness is associated with small hydrophobic peptides. In this study, protein hydrolysates based on head and backbone residuals from Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua) were produced using two different enzymes. The effects of micro- and nanofiltration on the chemical composition, protein recovery, and sensory properties of the final products were investigated. The choice of raw material and enzyme had negligible effects, whereas nanofiltration caused a considerable reduction in metabolites, ash, and the intensity of several sensory attributes. The intensity of bitterness increased after nanofiltration, indicating that small peptides associated with bitter taste were retained by the membrane. Total protein yield after microfiltration was 24%-29%, whereas 19%-24% were recovered in the nanofiltration retentate. PRACTICAL APPLICATION: Enzymatic protein hydrolysates can be included in food products to increase the protein content, and as a nutritional supplement and/or functional ingredient; however, unpalatable and intense flavors limit applications. This study investigated the use of membrane filtration to improve flavor quality and reduce salt content in fish protein hydrolysates. Although some protein loss is unavoidable in micro- and nanofiltration, this study demonstrates the production of fish protein hydrolysates with>90% protein and peptide content, which is suitable for inclusion in foods.

Bacterial Type I Toxins: Folding and Membrane Interactions.

Bacterial type I toxin-antitoxin systems are two-component genetic modules that encode a stable toxic protein whose ectopic overexpression can lead to growth arrest or cell death, and an unstable RNA antitoxin that inhibits toxin translation during growth. These systems are widely spread among bacterial species. Type I antitoxins are cis- or trans-encoded antisense small RNAs that interact with toxin-encoding mRNAs by pairing, thereby inhibiting toxin mRNA translation and/or inducing its degradation. Under environmental stress conditions, the up-regulation of the toxin and/or the antitoxin degradation by specific RNases promote toxin translation. Most type I toxins are small hydrophobic peptides with a predicted α-helical transmembrane domain that induces membrane depolarization and/or permeabilization followed by a decrease of intracellular ATP, leading to plasmid maintenance, growth adaptation to environmental stresses, or persister cell formation. In this review, we describe the current state of the art on the folding and the membrane interactions of these membrane-associated type I toxins from either Gram-negative or Gram-positive bacteria and establish a chronology of their toxic effects on the bacterial cell. This review also includes novel structural results obtained by NMR concerning the sprG1-encoded membrane peptides that belong to the sprG1/SprF1 type I TA system expressed in Staphylococcus aureus and discusses the putative membrane interactions allowing the lysis of competing bacteria and host cells.

Development of protease nanobiocatalysts and their application in hydrolysis of sunflower meal protein isolate

SummaryIn this study, the suitability of fumed silica nanoparticles (FNS) and its derivatives (amino‐modified FNS (AFNS), cyanuric chloride‐activated AFNS (CCAFNS) and epoxy‐modified FNS (GFNS)), for covalent immobilisation of two commercial protease preparations Alcalase® and Flavourzyme® was investigated. The highest hydrolytic activities of immobilised preparations were 25 IU g−1 support (Alcalase‐GFNS) and 2.95 IU g−1 support (Flavourzyme‐CCAFNS). Furthermore, the immobilised preparations showed 43% and 20% of initial specific activities of commercial protease preparations, respectively. Flavourzyme‐CCAFNS also exhibited the highest exopeptidase activity of 22.83 L‐pNAU g−1 support. Finally, these two nanobiocatalysts were successfully applied for hydrolysis of sunflower meal protein isolate (SMPI), providing two times higher hydrolysis yields in comparison to free enzymes, justifying the applied immobilisation process. Namely, the highest hydrolysis yield (30%) was gained by the sequential hydrolysis with Alcalase‐GFNS and Flavourzyme‐CCAFNS, which resulted in the formation of small hydrophobic and hydrophilic peptides, ≤5 kDa, confirmed by HPLC analysis and electrophoretic separation.

Large-Scale Membrane Permeability Prediction of Cyclic Peptides Crossing a Lipid Bilayer Based on Enhanced Sampling Molecular Dynamics Simulations.

Membrane permeability is a significant obstacle facing the development of cyclic peptide drugs. However, membrane permeation mechanisms are poorly understood. To investigate common features of permeable (and nonpermeable) designs, it is necessary to reproduce the membrane permeation process of cyclic peptides through the lipid bilayer. We simulated the membrane permeation process of 100 six-residue cyclic peptides across the lipid bilayer based on steered molecular dynamics (MD) and replica-exchange umbrella sampling simulations and predicted membrane permeability using the inhomogeneous solubility-diffusion model and a modified version of it. Furthermore, we confirmed the effectiveness of this protocol by predicting the membrane permeability of 56 eight-residue cyclic peptides with diverse chemical structures, including some confidential designs from a pharmaceutical company. As a result, a reasonable correlation between experimentally assessed and calculated membrane permeability of cyclic peptides was observed for the peptide libraries, except for strongly hydrophobic peptides. Our analysis of the MD trajectory demonstrated that most peptides were stabilized in the boundary region between bulk water and membrane and that for most peptides, the process of crossing the center of the membrane is the main obstacle to membrane permeation. The height of this barrier is well correlated with the electrostatic interaction between the peptide and the surrounding media. The structural and energetic features of the representative peptide at each vertical position within the membrane were also analyzed, revealing that peptides permeate the membrane by changing their orientation and conformation according to the surrounding environment.

Effect of Hydrophobicity and Charge Separation on the Antifouling Properties of Surface-Tethered Zwitterionic Peptides.

Zwitterionic peptides emerge as a class of highly effective antifouling material in a wide range of applications such as biosensors, biomedical devices, and implants. We incorporated neutral amino acid spacers with different hydrophobicities, including serine (Ser), glycine (Gly), and leucine (Leu), into zwitterionic peptides with lysine-glutamic acid repeating units and investigated the structure and antifouling performance of the zwitterionic peptide brushes by surface plasma resonance, surface force apparatus (SFA), and all-atomistic molecular dynamics (MD) simulation techniques. Our results demonstrate that the hydrophilicity of neutral spacers alters the structure and antifouling performance of the peptide-modified surface. Hydrophilic Ser-inserted peptides reduced the interaction between the peptide monolayer and protein foulants, while hydrophobic Leu significantly increased the protein adhesion. SFA force measurements show that the presence of more spacers would increase the adhesion between the peptide monolayer and the modeling foulant lysozyme, especially for the hydrophobic spacers. MD simulations reveal that hydrophilic Ser spacers retain the hydrophilicity of the peptide monolayer and improve the antifouling performance, and Gly spacers give rise to more interchain cross-links. Leu spacers result in a more hydrophobic peptide monolayer, which leads to dehydration of the peptide monolayer and reduces the antifouling performances.

Dairy-originated digestion-resistant and bioactive peptides increase the risk of hypertension: Tehran Lipid and Glucose Study.

Milk-protein-derived bioactive peptides (BPs) have been proposed as modulators of different regulatory processes involved in blood pressure regulation. Studies on the long-term effects of BPs on blood pressure have not yet been conducted. We aimed to investigate the association of dairy-originated BPs with the risk of hypertension (HTN) in the Tehran Lipid and Glucose Cohort Study (TLGS). In this cohort study, 4378 subjects with a mean follow-up period of 3.1 years were included in the final analysis. Dietary intake, physical activity, demographic, and anthropometric data and blood pressure measurements were obtained for all participants. Various types of dairy-originated BPs were determined by an in silico method. High intake of total digestion-resistant and bioactive peptides (OR: 1.31, CI 95%: 1.01-1.70), dipeptides (OR: 1.33, CI 95%: 1.03-1.73), peptides with more than seven residues (OR: 1.32, CI 95%: 1.01-1.71), glycosylated residues (OR: 1.39, CI 95%: 1.07-1.80), highly hydrophilic peptides (OR: 1.32, CI 95%: 1.01-1.71), and low hydrophobic peptides (OR: 1.32, CI 95%: 1.01-1.71) was associated with an increased risk of HTN in the adjusted model. In addition, subjects in the higher tertile of anti-HTN peptide (OR: 1.33, CI 95%: 1.02-1.72) and antidiabetic peptide (OR: 1.35, CI 95%: 1.04-1.76) intake had a higher risk of HTN than those in the lower tertile. No significant association emerged between calcium intake from dairy and incident risk of HTN. Our results showed that the intake of some forms of digestion-resistant and BPs, such as anti-HTN peptides, dipeptides, and peptides with more than seven residues, can increase the risk of HTN in the TLGS population.

Fusobacterium nucleatum secretes amyloid-like FadA to enhance pathogenicity.

Fusobacterium nucleatum (Fn) is a Gram-negative oral commensal, prevalent in various human diseases. It is unknown how this common commensal converts to a rampant pathogen. We report that Fn secretes an adhesin (FadA) with amyloid properties via a Fap2-like autotransporter to enhance its virulence. The extracellular FadA binds Congo Red, Thioflavin-T, and antibodies raised against human amyloid β42. Fn produces amyloid-like FadA under stress and disease conditions, but not in healthy sites or tissues. It functions as a scaffold for biofilm formation, confers acid tolerance, and mediates Fn binding to host cells. Furthermore, amyloid-like FadA induces periodontal bone loss and promotes CRC progression in mice, with virulence attenuated by amyloid-binding compounds. The uncleaved signal peptide of FadA is required for the formation and stability of mature amyloid FadA fibrils. We propose a model in which hydrophobic signal peptides serve as "hooks" to crosslink neighboring FadA filaments to form a stable amyloid-like structure. Our study provides a potential mechanistic link between periodontal disease and CRC and suggests anti-amyloid therapies as possible interventions for Fn-mediated disease processes.

Looking for a safe haven: tail-anchored proteins and their membrane insertion pathways.

Insertion of membrane proteins into the lipid bilayer is a crucial step during their biosynthesis. Eukaryotic cells face many challenges in directing these proteins to their predestined target membrane. The hydrophobic signal peptide or transmembrane domain (TMD) of the nascent protein must be shielded from the aqueous cytosol and its target membrane identified followed by transport and insertion. Components that evolved to deal with each of these challenging steps range from chaperones to receptors, insertases, and sophisticated translocation complexes. One prominent translocation pathway for most proteins is the signal recognition particle (SRP)-dependent pathway which mediates co-translational translocation of proteins across or into the endoplasmic reticulum (ER) membrane. This textbook example of protein insertion is stretched to its limits when faced with secretory or membrane proteins that lack an amino-terminal signal sequence or TMD. Particularly, a large group of so-called tail-anchored (TA) proteins that harbor a single carboxy-terminal TMD require an alternative, post-translational insertion route into the ER membrane. In this review, we summarize the current research in TA protein insertion with a special focus on plants, address challenges, and highlight future research avenues.

Capacity for increased surface area in the hydrophobic core of β-sheet peptide bilayer nanoribbons.

Amphipathic peptides with amino acids arranged in alternating patterns of hydrophobic and hydrophilic residues efficiently self‐assemble into β‐sheet bilayer nanoribbons. Hydrophobic side chain functionality is effectively buried in the interior of the putative bilayer of these nanoribbons. This study investigates consequences on self‐assembly of increasing the surface area of aromatic side chain groups that reside in the hydrophobic core of nanoribbons derived from Ac‐(XKXE)2‐NH2 peptides (X = hydrophobic residue). A series of Ac‐(XKXE)2‐NH2 peptides incorporating aromatic amino acids of increasing molecular volume and steric profile (X = phenylalanine [Phe], homophenylalanine [Hph], tryptophan [Trp], 1‐naphthylalanine [1‐Nal], 2‐naphthylalanine [2‐Nal], or biphenylalanine [Bip]) were assessed to determine substitution effects on self‐assembly propensity and on morphology of the resulting nanoribbon structures. Additional studies were conducted to determine the effects of incorporating amino acids of differing steric profile in the hydrophobic core (Ac‐X1KFEFKFE‐NH2 and Ac‐(X1,5KFE)‐NH2 peptides, X = Trp or Bip). Spectroscopic analysis by circular dichroism (CD) and Fourier transform infrared (FT‐IR) spectroscopy indicated β‐sheet formation for all variants. Self‐assembly rate increased with peptide hydrophobicity; increased molecular volume of the hydrophobic side chain groups did not appear to induce kinetic penalties on self‐assembly rates. Transmission electron microscopy (TEM) imaging indicated variation in fibril morphology as a function of amino acid in the X positions. This study confirms that hydrophobicity of amphipathic Ac‐(XKXE)2‐NH2 peptides correlates to self‐assembly propensity and that the hydrophobic core of the resulting nanoribbon bilayers has a significant capacity to accommodate sterically demanding functional groups. These findings provide insight that may be used to guide the exploitation of self‐assembled amphipathic peptides as functional biomaterials.

Air/Water Interfacial Monolayer Assembly of Peptide-Conjugated Liquid-Crystalline Molecules

Abstract Potential applications of functional liquid crystals such as biosensors strongly rely on control of the molecular orientation at interfaces. However, little knowledge regarding detailed molecular arrangements at such interfaces is available. In this work, two-dimensional self-assembling behavior at air/water interfaces of two types of amphiphilic mesogens with different peptide chains, arginine-glycine-aspartic acid and glycine-glycine-aspartic acid is investigated. Surface pressure–mean molecular area isotherms indicate that both bioconjugated mesogens are in the liquid expanded state to high surface compression conditions. The monolayer thickness of 16–19 Å derived by atomic force microscopic images is much smaller than the molecular length of ∼50 Å of a completely stretched motif. This implies that the bioconjugated molecules align in an inflected manner where tetraethylene glycol connecting hydrophobic rigid-rod and hydrophilic peptides is the inflection point. Contact angles of water for substrate surface with monolayers remarkably change depending on the surface pressure at the substrate transfer. This can be explained by the varied molecular arrangements with surface compression at the air/water interfaces. Understanding of molecular orientation at air/water interfaces is of fundamental importance for study of the ordering of liquid crystals at various other interfaces, leading to the design and further development of functional liquid-crystalline molecules for attractive sensor platforms.

Sequence-Specific Model for Predicting Peptide Collision Cross Section Values in Proteomic Ion Mobility Spectrometry.

The contribution of peptide amino acid sequence to collision cross section values (CCS) has been investigated using a dataset of ∼134 000 peptides of four different charge states (1+ to 4+). The migration data were acquired using a two-dimensional liquid chromatography (LC)/trapped ion mobility spectrometry/quadrupole/time-of-flight mass spectrometry (MS) analysis of HeLa cell digests created using seven different proteases and was converted to CCS values. Following the previously reported modeling approaches using intrinsic size parameters (ISP), we extended this methodology to encode the position of individual residues within a peptide sequence. A generalized prediction model was built by dividing the dataset into eight groups (four charges for both tryptic/nontryptic peptides). Position-dependent ISPs were independently optimized for the eight subsets of peptides, resulting in prediction accuracy of ∼0.981 for the entire population of peptides. We find that ion mobility is strongly affected by the peptide's ability to solvate the positively charged sites. Internal positioning of polar residues and proline leads to decreased CCS values as they improve charge solvation; conversely, this ability decreases with increasing peptide charge due to electrostatic repulsion. Furthermore, higher helical propensity and peptide hydrophobicity result in a preferential formation of extended structures with higher than predicted CCS values. Finally, acidic/basic residues exhibit position-dependent ISP behavior consistent with electrostatic interaction with the peptide macrodipole, which affects the peptide helicity. The MS raw data files have been deposited with the ProteomeXchange Consortium via the jPOST partner repository (http://jpostdb.org) with the dataset identifiers PXD021440/JPST000959, PXD022800/JPST001017, and PXD026087/ JPST001176.

Design of acid-activated cell-penetrating peptides with nuclear localization capacity for anticancer drug delivery.

Camptothecin (CPT), a DNA-toxin drug, exerts anticancer activity by inhibiting topoisomerase I. Targeted delivery of CPT into the cancer cell nucleus is important for enhancing its therapeutic efficiency. In this study, a new type of acid-activated cell-penetrating peptide (CPP) with nuclear localization capacity was constructed by conjugating six histidine residues and a hydrophobic peptide sequence, PFVYLI, to the nuclear localization sequence (NLS). Our results indicated that HNLS-3 displayed significant pH-dependent cellular uptake efficiency, endosomal escape ability, and nuclear localization activity. More importantly, the HNLS-3-CPT conjugate showed obviously enhanced cytotoxicity and selectivity compared with CPT. Taken together, our findings provide an effective approach to develop efficient CPPs with both cancer- and nucleus-targeting properties.

Thermodynamics-Based Molecular Modeling of α-Helices in Membranes and Micelles.

The Folding of Membrane-Associated Peptides (FMAP) method was developed for modeling α-helix formation by linear peptides in micelles and lipid bilayers. FMAP 2.0 identifies locations of α-helices in the amino acid sequence, generates their three-dimensional models in planar bilayers or spherical micelles, and estimates their thermodynamic stabilities and tilt angles, depending on temperature and pH. The method was tested for 723 peptides (926 data points) experimentally studied in different environments and for 170 single-pass transmembrane (TM) proteins with available crystal structures. FMAP 2.0 detected more than 95% of experimentally observed α-helices with an average error in helix end determination of around 2, 3, 4, and 5 residues per helix for peptides in water, micelles, bilayers, and TM proteins, respectively. Helical and nonhelical residue states were predicted with an accuracy from 0.86 to 0.96, and the Matthews correlation coefficient was from 0.64 to 0.88 depending on the environment. Experimental micelle- and membrane-binding energies and tilt angles of peptides were reproduced with a root-mean-square deviation of around 2 kcal/mol and 7°, respectively. The TM and non-TM states of hydrophobic and pH-triggered α-helical peptides in various lipid bilayers were reproduced in more than 95% of cases. The FMAP 2.0 web server (https://membranome.org/fmap) is publicly available to explore the structural polymorphism of antimicrobial, cell-penetrating, fusion, and other membrane-binding peptides, which is important for understanding the mechanisms of their biological activities.