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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides

The use of non-standard toxicity models is a hurdle in the early development of antimicrobial peptides towards clinical applications. Herein we report an extensive in vitro and in vivo toxicity study of a library of 24 peptide-based antimicrobials with narrow spectrum activity towards veterinary pathogens. The haemolytic activity of the compounds was evaluated against four different species and the relative sensitivity against the compounds was highest for canine erythrocytes, intermediate for rat and human cells and lowest for bovine cells. Selected peptides were additionally evaluated against HeLa, HaCaT and HepG2 cells which showed increased stability towards the peptides. Therapeutic indexes of 50–500 suggest significant cellular selectivity in comparison to bacterial cells. Three peptides were administered to rats in intravenous acute dose toxicity studies up to 2– 8 × MIC. None of the injected compounds induced any systemic toxic effects in vivo at the concentrations employed illustrating that the correlation between the different assays is not obvious. This work sheds light on the in vitro and in vivo toxicity of this class of promising compounds and provides insights into the relationship between the different toxicity models often employed in different manners to evaluate the toxicity of novel bioactive compounds in general.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

VHH antibody loop guides design of a synthetic macrocyclic peptide that potently blocks influenza virus membrane fusion

Abstract Miniaturizing biologically complex structural motifs to produce synthetic functional mimetics holds significant promise for development of new therapeutic modalities. Here, we demonstrate a unique approach using the key binding loop of the single variable domain of a heavy chain (V H H) llama antibody as a starting point for peptide design. V H H antibodies of camelids and sharks generally have longer, but more ligand-efficient complementarity determining region 3 (CDR3) loops and are relatively stable structures. We harnessed these attributes as templates for design of a series of synthetic macrocyclic peptides. The designed peptides exhibit nanomolar binding to influenza hemagglutinin (HA) and heterosubtypic in vitro neutralization breadth against influenza A viruses by inhibiting the low pH mediated HA conformational changes that lead to membrane fusion. X-ray structures of peptide-HA complexes reveal high structural mimicry with the parent V H H antibody. One such macrocycle peptide candidate is promising for further development of broad protection against influenza A group 1 viruses.

Kadam, Rameshwar U.↗
DOE OSTI2020

Effects of Peptide-Functionalized Surfaces on the Electrochemical Hydrogen Evolution Reaction

Abstract This review outlines the approaches and mechanisms through which peptides and amino acids functionalize electrocatalytically active surfaces to promote or inhibit the electrochemical hydrogen evolution reaction (HER). HER is important in many electrochemical systems. For example, HER is highly desired in water electrolysis, which if driven by renewable energy could serve as a green alternative to the fossil-fuel-driven steam methane-reforming process. However, HER is often an undesired side reaction and thus limits the selectivity of promising electrochemical technologies such as electrochemical nitrogen reduction or carbon dioxide reduction. In pursuing higher product selectivity and yield in emerging and existing electrochemical systems, amino acids and short-chain peptides are promising molecules for the modification of electrochemically active surfaces. Peptides are attractive because they are highly tunable, which allows for versatility in their applications. This short review article summarizes literature that illustrates the mechanisms through which electrode-bound peptides can affect HER including via modulating surface binding and adsorbate coverage, altering the surface composition, and controlling proton transfer rates. Our goal is to motivate additional studies utilizing electrode-bound peptides to modulate electrochemical hydrogen evolution reactions.

Electrochemistry↗
DOE OSTI2020

Francisella novicida Two-Component System Response Regulator BfpR Modulates iglC Gene Expression, Antimicrobial Peptide Resistance, and Biofilm Production

Response regulators are a critical part of the two-component system of gene expression regulation in bacteria, transferring a signal from a sensor kinase into DNA binding activity resulting in alteration of gene expression. In this study, we investigated a previously uncharacterized response regulator in Francisella novicida, FTN_1452 that we have named BfpR (Biofilm-regulating Francisella protein Regulator, FTN_1452). In contrast to another Francisella response regulator, QseB/PmrA, BfpR appears to be a negative regulator of biofilm production, and also a positive regulator of antimicrobial peptide resistance in this bacterium. The protein was crystallized and X-ray crystallography studies produced a 1.8 Å structure of the BfpR N-terminal receiver domain revealing interesting insight into its potential interaction with the sensor kinase. Structural analysis of BfpR places it in the OmpR/PhoP family of bacterial response regulators along with WalR and ResD. Proteomic and transcriptomic analyses suggest that BfpR overexpression affects expression of the critical Francisella virulence factor iglC, as well as other proteins in the bacterium. We demonstrate that mutation of bfpR is associated with an antimicrobial peptide resistance phenotype, a phenotype also associated with other response regulators, for the human cathelicidin peptide LL-37 and a sheep antimicrobial peptide SMAP-29. F. novicida with mutated bfpR replicated better than WT in intracellular infection assays in human-derived macrophages suggesting that the down-regulation of iglC expression in bfpR mutant may enable this intracellular replication to occur. Response regulators have been shown to play important roles in the regulation of bacterial biofilm production. We demonstrate that F. novicida biofilm formation was highly increased in the bfpR mutant, corresponding to altered glycogen synthesis. Waxworm infection experiments suggest a role of BfpR as a negative modulator of iglC expression with de-repression by Mg 2+ . In this study, we find that the response regulator BfpR may be a negative regulator of biofilm formation, and a positive regulator of antimicrobial peptide resistance in F. novicida.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Atomic Force Microscopy to Characterize Antimicrobial Peptide-Induced Defects in Model Supported Lipid Bilayers

Antimicrobial peptides (AMPs) interact with bacterial cell membranes through a variety of mechanisms, causing changes extending from nanopore formation to microscale membrane lysis, eventually leading to cell death. Several AMPs also disrupt mammalian cell membranes, despite their significantly different lipid composition and such collateral hemolytic damage hinders the potential therapeutic applicability of the AMP as an anti-microbial. Elucidating the mechanisms underlying the AMP–membrane interactions is challenging due to the variations in the chemical and structural features of the AMPs, the complex compositional variations of cell membranes and the inadequacy of any single experimental technique to comprehensively probe them. (1) Background: Atomic Force Microscopy (AFM) imaging can be used in combination with other techniques to help understand how AMPs alter the orientation and structural organization of the molecules within cell membranes exposed to AMPs. The structure, size, net charge, hydrophobicity and amphipathicity of the AMPs affect how they interact with cell membranes of differing lipid compositions. (2) Methods: Our study examined two different types of AMPs, a 20-amino acid, neutral, α-helical (amphipathic) peptide, alamethicin, and a 13-amino acid, non-α-helical cationic peptide, indolicidin (which intramolecularly folds, creating a hydrophobic core), for their interactions with supported lipid bilayers (SLBs). Robust SLB model membranes on quartz supports, incorporating predominantly anionic lipids representative of bacterial cells, are currently not available and remain to be developed. Therefore, the SLBs of zwitterionic egg phosphatidylcholine (PC), which represents the composition of a mammalian cell membrane, was utilized as the model membrane. This also allows for a comparison with the results obtained from the Quartz Crystal Microbalance with Dissipation (QCM-D) experiments conducted for these peptides interacting with the same zwitterionic SLBs. Further, in the case of alamethicin, because of its neutrality, the lipid charge may be less relevant for understanding its membrane interactions. (3) Results: Using AFM imaging and roughness analysis, we found that alamethicin produced large, unstable defects in the membrane at 5 µM concentrations, and completely removed the bilayer at 10 µM. Indolicidin produced smaller holes in the bilayer at 5 and 10 µM, although they were able to fill in over time. The root-mean-square (RMS) roughness values for the images showed that the surface roughness caused by visible defects peaked after peptide injection and gradually decreased over time. (4) Conclusions: AFM is useful for helping to uncover the dynamic interactions between different AMPs and cell membranes, which can facilitate the selection and design of more efficient AMPs for use in therapeutics and antimicrobial applications.

Swana, Kathleen W.↗
DOE OSTI2020

Combining Three-Dimensional Modeling with Artificial Intelligence to Increase Specificity and Precision in Peptide–MHC Binding Predictions

The reliable prediction of the affinity of candidate peptides for the MHC is important for predicting their potential antigenicity and thus influences medical applications, such as decisions on their inclusion in T cell–based vaccines. In this study, we present a rapid, predictive computational approach that combines a popular, sequence-based artificial neural network method, NetMHCpan 4.0, with three-dimensional structural modeling. We find that the ensembles of bound peptide conformations generated by the programs MODELLER and Rosetta FlexPepDock are less variable in geometry for strong binders than for low-affinity peptides. In tests on 1271 peptide sequences for which the experimental dissociation constants of binding to the well-characterized murine MHC allele H-2D b are known, by applying thresholds for geometric fluctuations the structure-based approach in a standalone manner drastically improves the statistical specificity, reducing the number of false positives. Furthermore, filtering candidates generated with NetMHCpan 4.0 with the structure-based predictor led to an increase in the positive predictive value (PPV) of the peptides correctly predicted to bind very strongly (i.e., Kd < 100 nM) from 40 to 52% (p = 0.027). The combined method also significantly improved the PPV when tested on five human alleles, including some with limited data for training. Overall, an average increase of 10% in the PPV was found over the standalone sequence-based method. The combined method should be useful in the rapid design of effective T cell–based vaccines.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Meta-analysis of peptides to detect protein significance

Shotgun assays are widely used in biotechnologies to char- acterize large molecules, which are hard to be measured as a whole directly. For instance, in Liquid Chromatography Mass Spectrometry (LC-MS) shotgun experiments, proteins in biological samples are digested into peptides, and then peptides are separated and measured. However, in proteomics study, investigators are usually interested in the performance of the whole proteins instead of those peptide fragments. In light of meta-analysis, we propose an adaptive thresholding method to select informative peptides, and combine peptide-level models to protein-level analysis. Overall, the meta-analysis procedure and modeling rationale can be adapted to data analysis of other types of shotgun assays.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Activation of polycystin-1 signaling by binding of stalk-derived peptide agonists

Polycystin-1 (PC1) is the protein product of thePKD1gene whose mutation causes autosomal dominant Polycystic Kidney Disease (ADPKD). PC1 is an atypical G protein-coupled receptor (GPCR) with an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments. Recently, activation of PC1 CTF signaling was shown to be regulated by a stalk tethered agonist (TA), resembling the mechanism observed for adhesion GPCRs. Here, synthetic peptides of the first 9- (p9), 17- (p17), and 21-residues (p21) of the PC1 stalk TA were shown to re-activate signaling by a stalkless CTF mutant in human cell culture assays. Novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations elucidated binding conformations of p9, p17, and p21 and revealed multiple specific binding regions to the stalkless CTF. Peptide agonists binding to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of stalk TA-mediated PC1 CTF activation. Additional sequence coevolution analyses showed the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. These insights into the structural dynamic mechanism of PC1 activation by TA peptide agonists provide an in-depth understanding that will facilitate the development of therapeutics targeting PC1 for ADPKD treatment.

Life Sciences & Biomedicine - Other Topics↗
NASA NTRS1979

Isoelectric focusing of proteins and peptides

Egg-white solution was chosen as the reference solution in order to assess the effects of operational parameters (voltage, flow rate, ampholine pH range and concentration, and protein concentration) of the RIEF apparatus on protein resolution. Topics of discussion include: (1) comparison of RIEF apparatus to conventional IEF techniques (column and PAG) with respect to resolution and throughput; (2) peptide and protein separation (AHF, Thymosin - Fraction 5, vasoactive peptide, L-asparaginase and ACP); and (3) detection of peptides - dansyl derivatives of amino acids and peptides, post-focusing fluorescent labeling of amino acids, peptides and proteins, and ampholine extraction from focused gels.

Egen, N.↗
NASA NTRS1981

Enhancement of peptide bond formation by polyribonucleotides on clay surfaces in fluctuating environments

The selective effects of polyribonucleotides on the formation of glycine peptide bonds in glycine on clay surfaces are investigated as a model for a template mechanism for the effects of polynucleotides on peptide bond formation. Free oligoglycine yields were determined for the cycling reaction of glycine in the presence and absence of clay and polyribonucleotides or polydeoxyribonucleotides. The polyribonucleotides are observed to lead to increases of up to fourfold increases in oligoglycine formed, with greater enhancements for poly-G nucleotides than for poly-A, poly-U and poly-C, indicating a codonic bias. Polydeoxyribonucleotides are found to provide no enhancement in peptide formation rates, and yields were also greatly reduced in the absence of clay. A mechanism for peptide synthesis is proposed which involves the activation of glycine on the clay surface, followed by the formation of esters between glycine and the 2-prime OH groups of the polyribonucleotide and peptide bonds between adjacent amino acyl esters. It is pointed out that if this mechanism is correct, it may provide a basis for a direct template translation process, which would produce a singlet genetic code.

White, D. H.↗
NASA NTRS1997

Studies of Cell-Mediated Immunity Against Immune Disorders Using Synthetic Peptides and Rotating Bioreactor System

Our proposed experiments included: (1) immunzing mice with synthetic peptides; (2) preparing spleen and lymph node cells; (3) growing them under conventional conditions as well as in the rotatory vessel in appropriate medium reconstituting with synthetic peptides and/or cytokines as needed; and (4) comparing at regular time intervals the specific CTL activity as well as helper T-cell activity (in terms of both proliferative responses and cytokine production) using established procedures in my laboratory. We further proposed that once we demonstrated the merit of rotatory vessel technology to achieve desired results, these studies would be expanded to include immune cells from non-human primates (rhesus monkeys and chimpanzees) and also humans. We conducted a number of experiments to determine CTL induction by the synthetic peptides corresponding to antigenic proteins in HIV and HPV in different mouse strains that express MHC haplotypes H-2b or H-2d. We immunized mice with 100 ug of the synthetic peptide, suspended in sterile water, and emulsified in CFA (1:1). The immune lymph node cells obtained after 7 days were restimulated by culturing in T25 flask, HARV-10, or STLV-50, in the presence of the peptide at 20 ug/ml. The results from the 5'Cr-release assay consistently revealed complete abrogation of CTL activity of cells grown in the bioreactors (both HARV and STLV), while significant antigen-specific CTL activity was observed with cells cultured in tissue culture flasks. Thus, overall the data we generated in this study proved the usefulness of the NASA-developed developed technology for understanding the known immune deficiency during space travel. Additionally, this ex vivo microgravity technology since it mimics effectively the in vivo situation, it is also useful in understanding immune disorders in general. Thus, our proposed studies in TMC-NASA contract round II application benefit from data generated in this TMC-NASA contract round I study.

Sastry, Jagannadha K.↗
NASA NTRS1996

Template-Directed Ligation of Peptides to Oligonucleotides

Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

Bruick, Richard K.↗
DOE OSTI2026

Peptide‐Induced Ferroelectricity in Charge‐Transfer Supramolecular Materials

Organic ferroelectrics are of great interest in sustainable energy conversion, information storage, flexible electronics, and potential biomedical applications as soft implants, among many other applications. Despite their broad potential, the development of organic ferroelectrics has remained limited, with only a few known examples in solid-state systems, primarily due to the lack of well-established design strategies compared to inorganic systems. Bio-inspired supramolecular chemistry offers a path to create functional nanostructures that are water-processable and biocompatible. We report here on supramolecular charge transfer (CT) systems in which peptides are covalently linked to dyads of electron-donating and electron-accepting moieties, creating amphiphiles that self-assemble into nanoscale ribbons in water. The peptide chirality-induced symmetry breaking in these crystalline nanostructures not only results in second harmonic activity but also generates ferroelectric behavior across multiple CT systems, demonstrating a versatile supramolecular approach to the design of new organic ferroelectrics. Furthermore, culturing primary neuron cells on coatings of the ferroelectric materials promoted axonal growth and enhanced action potentials, indicating improved neuronal maturity facilitated by the polar structure of the ferroelectric nanomaterials. The supramolecular strategy used here holds promise to create new water-processable ferroelectric biomaterials, opening avenues for innovative applications in cell charge transfer, neuronal axon growth, peptide symmetry breaking, self-assembling peptides, supramolecular ferroelectrics, proliferation, and bioelectronics.

charge transfer↗
DOE OSTI2024

Polyproline peptide targets Klebsiella pneumoniae polysaccharides to collapse biofilms

Hypervirulent Klebsiella pneumoniae is known for its increased extracellular polysaccharide production. Biofilm matrices of hyper- virulent K. pneumoniae have increased polysaccharide abundance and are uniquely susceptible to disruption by peptide bactenecin 7 (bac7 (1–35)). Here, using confocal microscopy, we show that poly- saccharides within the biofilm matrix collapse following bac7 (1–35) treatment. This collapse led to the release of cells from the biofilm, which were then killed by the peptide. Characterization of truncated peptide analogs revealed that their interactions with polysaccharide were responsible for the biofilm matrix changes that accompany bac7 (1–35) treatment. Ultraviolet photodissociation mass spec- trometry with the parental peptide or a truncated analog bac7 (10–35) reveal the important regions for bac7 (1–35) complexing with polysaccharides. Finally, we tested bac7 (1–35) using a murine skin abscess model and observed a significant decrease in the bacte- rial burden. These findings unveil the potential of bac7 (1–35) poly- saccharide interactions to collapse K. pneumoniae biofilms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evaluation of Differential Peptide Loading on Tandem Mass Tag-Based Proteomic and Phosphoproteomic Data Quality

Global and phosphoproteome profiling has demonstrated great utility for the analysis of clinical specimens. One major barrier to the broad clinical application of proteomic profiling is the large amount of biological material required, particularly for phosphoproteomics—currently on the order of 25 mg wet tissue weight, depending on tissue type. For hematopoietic cancers such as acute myeloid leukemia (AML), the sample requirement is in excess of 10 million (1E7) peripheral blood mononuclear cells (PBMCs). Throughout the course of a prospective study, this requirement will certainly exceed what is obtainable from many of the individual patients/timepoints. For this reason, we were interested in examining the impact of differential peptide loading across multiplex channels on proteomic data quality. Methods: To achieve this, we tested a range of channel loading amounts (20, 40, 100, 200, and 400 µg of tryptic peptides, or approximately the material obtainable from 5E5, 1E6, 2.5E6, 5E6, and 1E7 AML patient cells) to assess proteome coverage, quantification reproducibility and accuracy in experiments utilizing isobaric tandem mass tag (TMT) labeling. As expected, we found that fewer missing values are observed in TMT channels with higher peptide loading amounts compared to those with lower loading. Moreover, channels with lower loading amounts have greater quantitative variability than channels with higher loading amounts. Statistical analysis of the differences in means among the five loading groups showed that the 20 µg loading group was significantly different from the 400 µg loading group. However, no significant differences were detected among the 40, 100, 200 and 400 µg loading groups. Conclusions: These assessment data demonstrate the practical limits of loading differential quantities of peptides across channels in TMT multiplexes, and provide a basis for designing the optimal clinical proteomics study when specimen quantities are limited.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Enhanced rare earth element recovery with cross-linked glutaraldehyde-lanthanide binding peptides in foam-based separations

Lanthanide Binding Tag (LBT) peptides that coordinate selectively with lanthanide ions can be used to replace the energy intensive processes used for the separation of rare earth elements (REEs). These surface-active biomolecules, once selectively complexed with the trivalent REE cations, can adsorb to air/aqueous interfaces of bubbles for foam-based REEs recovery. Glutaraldehyde, an organic compound that is a homobifunctional crosslinker for proteins and peptides, can be used to enhance the adsorption and interfacial stabilization of lanthanide-bound peptides films. The stability of the interfacial cross-linked films was tested by measuring their dilational and shear surface rheological properties. Surface activity of the adsorbed species was analyzed using pendant drop tensiometry, while surface density and molecular arrangement were determined using x-ray reflectivity and x-ray fluorescence near total reflection. Glutaraldehyde cross-linked REE-peptide complexes enhance the adsorption of lanthanides to air-water interfaces, resulting in thicker interfacial structures. Subsequently, these thicker layers enhance the dilational and shear interfacial rheological properties. The interfacial film stabilization and REEs extraction promoted by the cross-linker presented in this work provides an approach to integrate glutaraldehyde as a substitute of common foam stabilizers such as polymers, surfactants, and particles to optimize the recovery of REEs when using biomolecules as extractants.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Pushing and Pulling: A Dual pH Trigger Controlled by Varying the Alkyl Tail Length in Heme Coordinating Peptide Amphiphiles

Some organisms in nature that undergo anaerobic respiration utilize 1D nanoscale arrays of densely packed cytochromes containing the molecule heme. The assemblies can be mimicked with 1D nanoscale fibrils composed of peptide amphiphiles designed to coordinate heme in dense arrays. To create such materials and assemblies it is critical to understand the assembly process and what controls the various aspects of hierarchical assembly. MD simulations suggest that shorter alkyl chains on the peptide lead to more dynamic structures than the peptides with longer chains that yield kinetically trapped states. The hydration parameters manifest themselves experimentally through the observation of a dual pH trigger which controls the peptide assembly rate, the heme binding affinity, and heme organization kinetics. Great strides in understanding the relative complexity of the self-assembly process in relation to incorporating a functional moiety like heme opens up many possibilities in developing abiotic assemblies for bioelectronic devices and assemblies

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Dueling Backbones: Comparing Peptoid and Peptide Analogues of a Mussel Adhesive Protein

Ensembles of amino acid side chains often dominate the interfacial interactions of intrinsically disordered proteins; however, backbone contributions are far from negligible. Using a combination of nanoscale force measurements and molecular dynamics simulations, we demonstrated with analogous mussel-mimetic adhesive peptides and peptoids both 34 residues long that highly divergent adhesive/cohesive outcomes can be achieved on mica surfaces by altering backbone chemistry only. The Phe, Tyr, and Dopa containing peptoid variants used in this study deposited as dehydrated and incompressible films that facilitated analysis of peptoid side chain contributions to adhesion and cohesion. For example, whereas Phe and Dopa peptoids exhibited similar cohesion, Dopa peptoids were ~3 times more adhesive than Phe peptoids on mica. Compared with the peptides, Phe peptoid achieved only ~20% of Phe containing peptide adhesion, but the Dopa peptoids were >2-fold more adhesive than the Dopa peptides. Cation-π interactions accounted for some but not all of the cohesive interactions. Finally, our results were corroborated by molecular dynamics simulations and highlight the importance of backbone chemistry and the potential of peptoids or peptoid/peptide hybrids as wet adhesives and primers.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗