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Results for “peptides and proteins”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
NASA NTRS2012

Apparatus and methods for manipulation and optimization of biological systems

The invention provides systems and methods for manipulating, e.g., optimizing and controlling, biological systems, e.g., for eliciting a more desired biological response of biological sample, such as a tissue, organ, and/or a cell. In one aspect, systems and methods of the invention operate by efficiently searching through a large parametric space of stimuli and system parameters to manipulate, control, and optimize the response of biological samples sustained in the system, e.g., a bioreactor. In alternative aspects, systems include a device for sustaining cells or tissue samples, one or more actuators for stimulating the samples via biochemical, electromagnetic, thermal, mechanical, and/or optical stimulation, one or more sensors for measuring a biological response signal of the samples resulting from the stimulation of the sample. In one aspect, the systems and methods of the invention use at least one optimization algorithm to modify the actuator's control inputs for stimulation, responsive to the sensor's output of response signals. The compositions and methods of the invention can be used, e.g., to for systems optimization of any biological manufacturing or experimental system, e.g., bioreactors for proteins, e.g., therapeutic proteins, polypeptides or peptides for vaccines, and the like, small molecules (e.g., antibiotics), polysaccharides, lipids, and the like. Another use of the apparatus and methods includes combination drug therapy, e.g. optimal drug cocktail, directed cell proliferations and differentiations, e.g. in tissue engineering, e.g. neural progenitor cells differentiation, and discovery of key parameters in complex biological systems.

Ho, Chih-Ming↗
DOE OSTI2023

Harnessing Aromatic‐Histidine Interactions through Synergistic Backbone Extension and Side Chain Modification

Abstract Peptide engineering efforts have delivered drugs for diverse human diseases. Side chain alteration is among the most common approaches to designing new peptides for specific applications. The peptide backbone can be modified as well, but this strategy has received relatively little attention. Here we show that new and favorable contacts between a His side chain on a target protein and an aromatic side chain on a synthetic peptide ligand can be engineered by rational and coordinated side chain modification and backbone extension. Side chain modification alone was unsuccessful. Binding measurements, high‐resolution structural studies and pharmacological outcomes all support the synergy between backbone and side chain modification in engineered ligands of the parathyroid hormone receptor‐1, which is targeted by osteoporosis drugs. These results should motivate other structure‐based designs featuring coordinated side chain modification and backbone extension to enhance the engagement of peptide ligands with target proteins.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Structural, Electronic, and Electrostatic Determinants for Inhibitor Binding to Subsites S1 and S2 in SARS-CoV-2 Main Protease

Creating small-molecule antivirals specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins is crucial to battle coronavirus disease 2019 (COVID-19). SARS-CoV-2 main protease (Mpro) is an established drug target for the design of protease inhibitors. We performed a structure–activity relationship (SAR) study of noncovalent compounds that bind in the enzyme’s substrate-binding subsites S1 and S2, revealing structural, electronic, and electrostatic determinants of these sites. The study was guided by the X-ray/neutron structure of Mpro complexed with Mcule-5948770040 (compound 1), in which protonation states were directly visualized. Virtual reality-assisted structure analysis and small-molecule building were employed to generate analogues of 1. In vitro enzyme inhibition assays and room-temperature X-ray structures demonstrated the effect of chemical modifications on Mpro inhibition, showing that (1) maintaining correct geometry of an inhibitor’s P1 group is essential to preserve the hydrogen bond with the protonated His163; (2) a positively charged linker is preferred; and (3) subsite S2 prefers nonbulky modestly electronegative groups.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1992

Development and use of domain-specific antibodies in a characterization of the large subunits of soybean photosystem 1

The molecular architecture of the soybean photosystem 1 reaction center complex was examined using a combination of surface labeling and immunological methodology on isolated thylakoid membranes. Synthetic peptides (12 to 14 amino acids in length) were prepared which correspond to the N-terminal regions of the 83 and 82.4 kDa subunits of photosystem 1 (the PsaA and PsaB proteins, respectively). Similarly, a synthetic peptide was prepared corresponding to the C-terminal region of the PsaB subunit. These peptides were conjugated to a carrier protein, and were used for the production of polyclonal antibodies in rabbits. The resulting sera could distinguish between the PsaA and PsaB photosystem 1 subunits by Western blot analysis, and could identify appropriate size classes of cyanogen bromide cleavage fragments as predicted from the primary sequences of these two subunits. When soybean thylakoid membranes were surface-labeled with N-hydroxysuccinimidobiotin, several subunits of the complete photosystem 1 lipid/protein complex incorporated label. These included the light harvesting chlorophyll proteins of photosystem 1, and peptides thought to aid in the docking of ferredoxin to the complex during photosynthetic electron transport. However, the PsaA and PsaB subunits showed very little biotinylation. When these subunits were examined for the domains to which biotin did attach, most of the observed label was associated with the N-terminal domain of the PsaA subunit, as identified using a domain-specific polyclonal antisera.

NASA Program NSCORT↗
DOE OSTI2020

Proteomic Characterization of Damaged Single Hairs Recovered after an Explosion for Protein-Based Human Identification

Evidence recovery is challenging where an explosion has occurred. Though hair evidence may be sufficiently robust to be recovered at the site, forensic analysis underutilizes the matrix by relying on morphological analysis. Where DNA is compromised, particularly in hair, protein-based human identification presents a promising alternative. Detection of amino acid polymorphisms in hair proteins as genetically variant peptides (GVPs) permits the inference of individualizing single nucleotide polymorphisms for identification. However, an explosive blast may damage hair proteins and compromise GVP identification. This work evaluates effects of an explosive blast on the hair proteome and GVP identification, investigates microscopy as a predictor of proteome profiling success in recovered hairs to improve analysis throughput, and quantifies discriminative power in damaged hairs. The proteomics dataset has been deposited into the ProteomeXchange Consortium (PXD017427). With the exception of degradation in keratins K75, K80, K40, and keratin-associated protein KAP10-11 as markers of hair cuticular damage, corroborated by scanning electron microscopic analysis, minimal hair proteome degradation following explosion allowed successful proteome profiling of single hairs regardless of morphological damage. Finally, GVP identification remained independent of explosion conditions, permitting similar discriminative power between exploded and undamaged hairs. These findings lend greater confidence to GVP analysis in one-inch hairs for forensic identification and provide information about hair protein localization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure of Diisobutylene Maleic Acid Copolymer (DIBMA) and Its Lipid Particle as a “Stealth” Membrane-Mimetic for Membrane Protein Research

The study of membrane proteins remains challenging, especially in a native membrane environment. Recently, major progress has been made using maleic acid copolymers, such as styrene maleic acid, to purify membrane proteins and study them directly with native lipids associated with the membrane. Additional maleic acid copolymers, such as diisobutylene maleic acid (DIBMA) membrane-mimetic systems, are being developed and found to have improved spectroscopic properties and pH stability. We studied DIBMA and its lipid particles in solution to better understand its assembly, without and with the lipids, to provide an insight regarding how to use it in solution for better membrane extraction. Using small-angle neutron and X-ray scattering (SANS/SAXS), we show that DIBMA organizes into structures of different size scales at various concentrations and ionic strengths. The polymer performed reasonably well under most solvent conditions except in very low concentrations and high-salt conditions that could result in limited interaction with lipids. To explore DIBMA lipid particles as a suitable membrane-mimetic system for neutron scattering studies of membrane proteins, we measured and determined the contrast-matching point of DIBMA to be ~12% (v/v) D 2 O — similar to that of most protiated lipid molecules but distinct from that of regular protiated proteins — providing a natural contrast for separating their neutron scattering signals. Using SANS contrast variation, we demonstrated that the scattering from the whole lipid particle can be annihilated. Further, we determined that a well-defined lipid nanodisc structure with DIBMA was contrast-matched. These results demonstrate that the DIBMA lipid particle is an outstanding “stealth” membrane-mimetic for membrane proteins. The results provide a structural framework for understanding the organization and assembly process of the polymer itself and the lipid molecules. Such an understanding is imperative for structural techniques such as cryo-electron microscopy, nuclear magnetic resonance, small-angle scattering, and other biophysical techniques.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition

Presequence protease (PreP), a 117kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3–4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structurally silent peptide anchor modifications allosterically modulate T cell recognition in a receptor-dependent manner

Presentation of peptides by class I MHC proteins underlies T cell immune responses to pathogens and cancer. The association between peptide binding affinity and immunogenicity has led to the engineering of modified peptides with improved MHC binding, with the hope that these peptides would be useful for eliciting cross-reactive immune responses directed toward their weak binding, unmodified counterparts. Increasing evidence, however, indicates that T cell receptors (TCRs) can perceive such anchor-modified peptides differently than wild-type (WT) peptides, although the scope of discrimination is unclear. We show here that even modifications at primary anchors that have no discernible structural impact can lead to substantially stronger or weaker T cell recognition depending on the TCR. Surprisingly, the effect of peptide anchor modification can be sensed by a TCR at regions distant from the site of modification, indicating a through-protein mechanism in which the anchor residue serves as an allosteric modulator for TCR binding. Our findings emphasize caution in the use and interpretation of results from anchor-modified peptides and have implications for how anchor modifications are accounted for in other circumstances, such as predicting the immunogenicity of tumor neoantigens. Our data also highlight an important need to better understand the highly tunable dynamic nature of class I MHC proteins and the impact this has on various forms of immune recognition.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

In vitro enhancement of Zika virus infection by preexisting West Nile virus antibodies in human plasma-derived immunoglobulins revealed after P2 binding site-specific enrichment

ABSTRACT Human immunoglobulin preparations contain a diverse range of polyclonal antibodies that reflect past immune responses against pathogens encountered by the blood donor population. In this study, we examined a panel of intravenous immunoglobulins (IGIVs) manufactured over the past two decades (1998–2020) for their capacity to neutralize or enhance Zika virus (ZIKV) infectionin vitro. These IGIVs were selected specifically based on their production dates in relation to the occurrences of two flavivirus outbreaks in the U.S.: the West Nile virus (WNV) outbreak in 1999 and the ZIKV outbreak in 2015. As demonstrated by enzyme-linked immunosorbent assay (ELISA) experiments, IGIVs made before the ZIKV outbreak already harbored antibodies that bind to various peptides across the envelope protein of ZIKV because of the WNV outbreak. Using phage display, the most dominant binding site was mapped precisely to the P2 peptide between residues 211 and 230 within domain II, where BF1176-56, an anti-ZIKV monoclonal antibody, also binds. When tested in permissive Vero E6 cells for ZIKV neutralization, the IGIVs, even after undergoing rigorous enrichment for P2 binding specificity, failed, as did BF1176-56. Meanwhile, BF1176-56 enhanced ZIKV infection in both FcγRII-expressing K562 cells and human peripheral blood mononuclear cells. However, for enhancement by the IGIVs to be detected in these cells, a substantial increase in their P2 binding specificity was required, thus linking the P2 site with ZIKV enhancementin vitro. Our findings warrant further study of the significance of elevated levels of anti-WNV antibodies in IGIVs, considering that various mechanisms operatingin vivomay modulate ZIKV infection outcomes. IMPORTANCE We investigated the capacity of intravenous immunoglobulins manufactured previously over two decades (1998–2020) to neutralize or enhance Zika virus infectionin vitro. West Nile virus antibodies in IGIVs could not neutralize Zika virus initially; however, once the IGIVs were concentrated further, they enhanced its infection. These findings lay the groundwork for exploring how preexisting WNV antibodies in IGIVs could impact Zika infection, bothin vitroandin vivo. Our observations are historically significant, since we tested a panel of IGIV lots that were carefully selected based on their production dates which covered two major flavivirus outbreaks in the U.S.: the WNV outbreak in 1999 and the ZIKV outbreak in 2015. These findings will facilitate our understanding of the interplay among closely related viral pathogens, particularly from a historical perspective regarding large blood donor populations. They should remain relevant for future outbreaks of emerging flaviviruses that may potentially affect vulnerable populations.

Microbiology↗
NASA NTRS2002

Isolation and characterization of a novel calmodulin-binding protein from potato

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.

NASA Discipline Plant Biology↗
DOE OSTI2023

Snekmer: a scalable pipeline for protein sequence fingerprinting based on amino acid recoding

Abstract Motivation The vast expansion of sequence data generated from single organisms and microbiomes has precipitated the need for faster and more sensitive methods to assess evolutionary and functional relationships between proteins. Representing proteins as sets of short peptide sequences (kmers) has been used for rapid, accurate classification of proteins into functional categories; however, this approach employs an exact-match methodology and thus may be limited in terms of sensitivity and coverage. We have previously used similarity groupings, based on the chemical properties of amino acids, to form reduced character sets and recode proteins. This amino acid recoding (AAR) approach simplifies the construction of protein representations in the form of kmer vectors, which can link sequences with distant sequence similarity and provide accurate classification of problematic protein families. Results Here, we describe Snekmer, a software tool for recoding proteins into AAR kmer vectors and performing either (i) construction of supervised classification models trained on input protein families or (ii) clustering for de novo determination of protein families. We provide examples of the operation of the tool against a set of nitrogen cycling families originally collected using both standard hidden Markov models and a larger set of proteins from Uniprot and demonstrate that our method accurately differentiates these sequences in both operation modes. Availability and implementation Snekmer is written in Python using Snakemake. Code and data used in this article, along with tutorial notebooks, are available at http://github.com/PNNL-CompBio/Snekmer under an open-source BSD-3 license. Supplementary information Supplementary data are available at Bioinformatics Advances online.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Results from a multi-laboratory ocean metaproteomic intercomparison: effects of LC-MS acquisition and data analysis procedures

Metaproteomics is an increasingly popular methodology that provides information regarding the metabolic functions of specific microbial taxa and has potential for contributing to ocean ecology and biogeochemical studies. A blinded multi-laboratory intercomparison was conducted to assess comparability and reproducibility of taxonomic and functional results and their sensitivity to methodological variables. Euphotic zone samples from the Bermuda Atlantic Time-series Study (BATS) in the North Atlantic Ocean collected by in situ pumps and the autonomous underwater vehicle (AUV) Clio were distributed with a paired metagenome, and one-dimensional (1D) liquid chromatographic data-dependent acquisition mass spectrometry analysis was stipulated. Analysis of mass spectra from seven laboratories through a common bioinformatic pipeline identified a shared set of 1056 proteins from 1395 shared peptide constituents. Quantitative analyses showed good reproducibility: pairwise regressions of spectral counts between laboratories yielded R 2 values averaged 0.62±0.11, and a Sørensen similarity analysis of the top 1000 proteins revealed 70 %–80 % similarity between laboratory groups. Taxonomic and functional assignments showed good coherence between technical replicates and different laboratories. A bioinformatic intercomparison study, involving 10 laboratories using eight software packages, successfully identified thousands of peptides within the complex metaproteomic datasets, demonstrating the utility of these software tools for ocean metaproteomic research. Lessons learned and potential improvements in methods were described. Future efforts could examine reproducibility in deeper metaproteomes, examine accuracy in targeted absolute quantitation analyses, and develop standards for data output formats to improve data interoperability. Together, these results demonstrate the reproducibility of metaproteomic analyses and their suitability for microbial oceanography research, including integration into global-scale ocean surveys and ocean biogeochemical models.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

Non-NASA Center↗
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.↗