Search NASA⌕ Search

SEARCH · Search NASA

Results for “Protein Science”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Design principles for site-selective hydroxylation by a Rieske oxygenase

Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C–H bond functionalization reactions. Thus far, only a handful of Rieske oxygenases have been structurally characterized and remarkably little information exists regarding how these enzymes use a common architecture and set of metallocenters to facilitate a diverse range of reactions. Herein, we detail how two Rieske oxygenases SxtT and GxtA use different protein regions to influence the site-selectivity of their catalyzed monohydroxylation reactions. We present high resolution crystal structures of SxtT and GxtA with the native β-saxitoxinol and saxitoxin substrates bound in addition to a Xenon-pressurized structure of GxtA that reveals the location of a substrate access tunnel to the active site. Ultimately, this structural information allowed for the identification of six residues distributed between three regions of SxtT that together control the selectivity of the C–H hydroxylation event. Substitution of these residues produces a SxtT variant that is fully adapted to exhibit the non-native site-selectivity and substrate scope of GxtA. Importantly, we also found that these selectivity regions are conserved in other structurally characterized Rieske oxygenases, providing a framework for predictively repurposing and manipulating Rieske oxygenases as biocatalysts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Novel Mutant Alleles Reveal a Role of the Extra-Large G Protein in Rice Grain Filling, Panicle Architecture, Plant Growth, and Disease Resistance

Plant growth and grain filling are the key agronomical traits for grain weight and yield of rice. The continuous improvement in rice yield is required for a future sustainable global economy and food security. The heterotrimeric G protein complex containing a canonical α subunit (RGA1) couples extracellular signals perceived by receptors to modulate cell function including plant development and grain weight. We hypothesized that, besides RGA1, three atypical, extra-large GTP-binding protein (XLG) subunits also regulate panicle architecture, plant growth, development, grain weight, and disease resistance. Here, we identified a role of XLGs in agronomic traits and stress tolerance by genetically ablating all three rice XLGs individually and in combination using the CRISPR/Cas9 genome editing in rice. For this study, eight (three single, two double, and three triple) null mutants were selected. Three XLG proteins combinatorically regulate seed filling, because loss confers a decrease in grain weight from 14% with loss of one XLG and loss of three to 32% decrease in grain weight. Null mutations in XLG2 and XLG4 increase grain size. The mutants showed significantly reduced panicle length and number per plant including lesser number of grains per panicle compared to the controls. Loss-of-function of all individual XLGs contributed to 9% more aerial biomass compared to wild type (WT). The double mutant showed improved salinity tolerance. Moreover, loss of the XLG gene family confers hypersensitivity to pathogens. Our findings suggest that the non-canonical XLGs play important roles in regulating rice plant growth, grain filling, panicle phenotype, stress tolerance, and disease resistance. Genetic manipulation of XLG s has the potential to improve agronomic properties in rice.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Monomer-scale design of functional protein polymers using consensus repeat sequences

Protein-based polymers possess chemically defined sequences that can encode diverse properties and functions into a new class of biopolymeric materials. However, sequence variation that emerges from evolution can obscure the sequence–function relationships of naturally derived polymers. One strategy to clarify these relationships is to identify common sequences between proteins with similar functions. These conserved sequences often emerge from repeat proteins, and “consensus repeat sequences” provide a convenient platform for systematic investigations of biopolymer sequence–property relationships. In this review, we highlight recent approaches to engineer tunable polymeric materials using monomer-scale design of consensus repeat proteins. Here, we explore established and emerging protein-based materials with mechanical resilience, thermodynamic phase behavior, chemical responsiveness, biomolecular transport, and hierarchical structure. Overall, recent advances in the monomer-scale design of repetitive protein polymers present exciting fundamental and translational opportunities for polymer scientists and engineers.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Construction, characterization and crystal structure of a fluorescent single-chain Fv chimera [Slides]

Conclusions of the presentation were: The scTGPs are recombinant intrinsically fluorescent antibodies, ready for use in traditional fluorescent antibody based assays; The scFPs can be constructed as vL-FP-vH format, TGP an extremely stable monomeric fluorescent protein is suitable FP; The scFPs can be effectively displayed on yeast, where they function similarly to their corresponding scFv counterparts, recognize specific antigen similar affinity; Functional expression levels of scFPs in the bacterial cytoplasm are relatively high and can rescue some scFvs otherwise expressed at low levels in the periplasm; Straightforward assessment of expression levels and monitoring of protein purification steps; Antigen recognition in one step binding assays, novel assays such as epitope binning TGP is a monomeric fluorescent protein amenable to crystallization. These features provide unique and valuable characteristics to scTGP molecules for their use in protein chemistry; and, We present unique antibody format with wide variety of use in research and clinical setting

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV-cofactor

Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N 2 binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor. Fe-only nitrogenase is a poor catalyst for N 2 fixation, when compared to the Modependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N 2 . These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth.

nitrogenase↗
DOE OSTI2022

Design of protein-binding proteins from the target structure alone

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains a challenge. Here we describe a general solution to this problem that starts with a broad exploration of the vast space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and, following experimental optimization, bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder–target complexes, and all five closely match the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein–protein interactions, and should guide improvements of both. Our approach enables the targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A generalized platform for artificial intelligence-powered autonomous enzyme engineering

Proteins are the molecular machines of life with numerous applications in energy, health, and sustainability. However, engineering proteins with desired functions for practical applications remains slow, expensive, and specialist-dependent. Here we report a generally applicable platform for autonomous enzyme engineering that integrates machine learning and large language models with biofoundry automation to eliminate the need for human intervention, judgement, and domain expertise. Requiring only an input protein sequence and a quantifiable way to measure fitness, this automated platform can be applied to engineer a wide array of proteins. As a proof of concept, we engineer Arabidopsis thaliana halide methyltransferase (AtHMT) for a 90-fold improvement in substrate preference and 16-fold improvement in ethyltransferase activity, along with developing a Yersinia mollaretii phytase (YmPhytase) variant with 26-fold improvement in activity at neutral pH. This is accomplished in four rounds over 4 weeks, while requiring construction and characterization of fewer than 500 variants for each enzyme. This platform for autonomous experimentation paves the way for rapid advancements across diverse industries, from medicine and biotechnology to renewable energy and sustainable chemistry.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Rice NIN-LIKE PROTEIN 4 plays a pivotal role in nitrogen use efficiency

Nitrogen (N) is one of the key essential macronutrients that affects rice growth and yield. Inorganic N fertilizers are excessively used to boost yield and generate serious collateral environmental pollution. Therefore, improving crop N use efficiency (NUE) is highly desirable and has been a major endeavour in crop improvement. However, only a few regulators have been identified that can be used to improve NUE in rice to date. Here we show that the rice NIN-like protein 4 (OsNLP4) significantly improves the rice NUE and yield. Field trials consistently showed that loss-of-OsNLP4 dramatically reduced yield and NUE compared with wild type under different N regimes. In contrast, the OsNLP4 overexpression lines remarkably increased yield by 30% and NUE by 47% under moderate N level compared with wild type. Transcriptomic analyses revealed that OsNLP4 orchestrates the expression of a majority of known N uptake, assimilation and signalling genes by directly binding to the nitrate-responsive cis-element in their promoters to regulate their expression. Moreover, overexpression of OsNLP4 can recover the phenotype of Arabidopsis nlp7 mutant and enhance its biomass. Our results demonstrate that OsNLP4 plays a pivotal role in rice NUE and sheds light on crop NUE improvement.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

A simple technique to classify diffraction data from dynamic proteins according to individual polymorphs

One often observes small but measurable differences in the diffraction data measured from different crystals of a single protein. These differences might reflect structural differences in the protein and may reveal the natural dynamism of the molecule in solution. Partitioning these mixed-state data into single-state clusters is a critical step that could extract information about the dynamic behavior of proteins from hundreds or thousands of single-crystal data sets. Mixed-state data can be obtained deliberately (through intentional perturbation) or inadvertently (while attempting to measure highly redundant single-crystal data). To the extent that different states adopt different molecular structures, one expects to observe differences in the crystals; each of the polystates will create a polymorph of the crystals. After mixed-state diffraction data have been measured, deliberately or inadvertently, the challenge is to sort the data into clusters that may represent relevant biological polystates. Here, this problem is addressed using a simple multi-factor clustering approach that classifies each data set using independent observables, thereby assigning each data set to the correct location in conformational space. This procedure is illustrated using two independent observables, unit-cell parameters and intensities, to cluster mixed-state data from chymotrypsinogen (ChTg) crystals. It is observed that the data populate an arc of the reaction trajectory as ChTg is converted into chymotrypsin.

36 MATERIALS SCIENCE↗
DOE OSTI2023

rRNA methylation by Spb1 regulates the GTPase activity of Nog2 during 60S ribosomal subunit assembly

Biogenesis of the large ribosomal (60S) subunit involves the assembly of three rRNAs and 46 proteins, a process requiring approximately 70 ribosome biogenesis factors (RBFs) that bind and release the pre-60S at specific steps along the assembly pathway. The methyltransferase Spb1 and the K-loop GTPase Nog2 are essential RBFs that engage the rRNA A-loop during sequential steps in 60S maturation. Spb1 methylates the A-loop nucleotide G2922 and a catalytically deficient mutant strain ( spb 1 D52A ) has a severe 60S biogenesis defect. However, the assembly function of this modification is currently unknown. Here, we present cryo-EM reconstructions that reveal that unmethylated G2922 leads to the premature activation of Nog2 GTPase activity and capture a Nog2-GDP-AlF 4 - transition state structure that implicates the direct involvement of unmodified G2922 in Nog2 GTPase activation. Genetic suppressors and in vivo imaging indicate that premature GTP hydrolysis prevents the efficient binding of Nog2 to early nucleoplasmic 60S intermediates. We propose that G2922 methylation levels regulate Nog2 recruitment to the pre-60S near the nucleolar/nucleoplasmic phase boundary, forming a kinetic checkpoint to regulate 60S production. Our approach and findings provide a template to study the GTPase cycles and regulatory factor interactions of the other K-loop GTPases involved in ribosome assembly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Assembly of JAZ–JAZ and JAZ–NINJA complexes in jasmonate signaling

Jasmonates (JAs) are plant hormones with crucial roles in development and stress resilience. They activate MYC transcription factors by mediating the proteolysis of MYC inhibitors called JAZ proteins. In the absence of JA, JAZ proteins bind and inhibit MYC through the assembly of MYC–JAZ–Novel Interactor of JAZ (NINJA)–TPL repressor complexes. However, JAZ and NINJA are predicted to be largely intrinsically unstructured, which has precluded their experimental structure determination. Through a combination of biochemical, mutational, and biophysical analyses and AlphaFold-derived ColabFold modeling, we characterized JAZ–JAZ and JAZ–NINJA interactions and generated models with detailed, high-confidence domain interfaces. We demonstrate that JAZ, NINJA, and MYC interface domains are dynamic in isolation and become stabilized in a stepwise order upon complex assembly. By contrast, most JAZ and NINJA regions outside of the interfaces remain highly dynamic and cannot be modeled in a single conformation. Our data indicate that the small JAZ Zinc finger expressed in Inflorescence Meristem (ZIM) motif mediates JAZ–JAZ and JAZ–NINJA interactions through separate surfaces, and our data further suggest that NINJA modulates JAZ dimerization. This study advances our understanding of JA signaling by providing insights into the dynamics, interactions, and structure of the JAZ–NINJA core of the JA repressor complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product (Final Report)

The goal is to achieve a comprehensive understanding of the nature, mechanism of function and biogenesis of c-type cytochromes and their oligomeric complexes. These cytochrome are ubiquitous heme proteins that are required for cellular bioenergetics, including photosynthesis (Ps) and respiration (Res), which are central to chemical energy (ATP) production by oxidative- and photophosphorylation. We contribute to the molecular characterization of individual and membrane-integral cytochromes as parts of Ps and Res electron transport (ET) pathways, using the facultative phototrophic bacteria of as excellent models for multidisciplinary investigations. Supported by this grant we discovered novel membrane-bound cytochromes and cyts c complexes that are important for Ps- and Res-ET. We worked out their biogenesis including maturation (covalent heme addition) and assembly of mature subunits into complexes. These involve fundamental process found virtually in all organisms and are fundamental processes with major significance to the “Photosynthetic Systems” program of DOE-BES, because the cytochromes are integral components of natural energy production systems. Elucidation of basic principles, and the knowledge gained by their study greatly impact on our ability to design, engineer, and create new natural, bio-hybrid, bio-inspired and artificial systems to better capture light, transduce and store its energy with improved efficacy and are of broad significance.

14 SOLAR ENERGY↗
DOE OSTI2022

RCSB Protein Data bank: Tools for visualizing and understanding biological macromolecules in 3D

Abstract Now in its 52nd year of continuous operations, the Protein Data Bank (PDB) is the premiere open‐access global archive housing three‐dimensional (3D) biomolecular structure data. It is jointly managed by the Worldwide Protein Data Bank (wwPDB) partnership. The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) is funded by the National Science Foundation, National Institutes of Health, and US Department of Energy and serves as the US data center for the wwPDB. RCSB PDB is also responsible for the security of PDB data in its role as wwPDB‐designated Archive Keeper. Every year, RCSB PDB serves tens of thousands of depositors of 3D macromolecular structure data (coming from macromolecular crystallography, nuclear magnetic resonance spectroscopy, electron microscopy, and micro‐electron diffraction). The RCSB PDB research‐focused web portal ( RCSB.org ) makes PDB data available at no charge and without usage restrictions to many millions of PDB data consumers around the world. The RCSB PDB training, outreach, and education web portal ( PDB101.RCSB.org ) serves nearly 700 K educators, students, and members of the public worldwide. This invited Tools Issue contribution describes how RCSB PDB (i) is organized; (ii) works with wwPDB partners to process new depositions; (iii) serves as the wwPDB‐designated Archive Keeper; (iv) enables exploration and 3D visualization of PDB data via RCSB.org ; and (v) supports training, outreach, and education via PDB101.RCSB.org . New tools and features at RCSB.org are presented using examples drawn from high‐resolution structural studies of proteins relevant to treatment of human cancers by targeting immune checkpoints.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Nitric oxide delivery and heme-assisted S-nitrosation by the bedbug nitrophorin

Here, nitrophorins are heme proteins used by blood feeding insects to deliver nitric oxide (NO) to a victim, leading to vasodilation and antiplatelet activity. Cimex lectularius (bedbug) nitrophorin (cNP) accomplishes this with a cysteine ligated ferric (Fe(III)) heme. In the acidic environment of the insect's salivary glands, NO binds tightly to cNP. During a blood meal, cNP-NO is delivered to the feeding site where dilution and increased pH lead to NO release. In a previous study, cNP was shown to not only bind heme, but to also nitrosate the proximal cysteine, leading to Cys-NO (SNO) formation. SNO formation requires oxidation of the proximal cysteine, which was proposed to be metal-assisted through accompanying reduction of ferric heme and formation of Fe(II)-NO. Here, we report the 1.6 Å crystal structure of cNP first chemically reduced and then exposed to NO, and show that Fe(II)-NO is formed but SNO is not, supporting a metal-assisted SNO formation mechanism. Crystallographic and spectroscopic studies of mutated cNP show that steric crowding of the proximal site inhibits SNO formation while a sterically relaxed proximal site enhances SNO formation, providing insight into specificity for this poorly understood modification. Experiments examining the pH dependence for NO implicate direct protonation of the proximal cysteine as the underlying mechanism. At lower pH, thiol heme ligation predominates, leading to a smaller trans effect and 60-fold enhanced NO affinity (K d = 70 nM). Unexpectedly, we find that thiol formation interferes with SNO formation, suggesting cNP-SNO is unlikely to form in the insect salivary glands.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

A Prodrug Strategy to Conditionally Trap Therapeutic Payloads for Improved Tumor Retention

Altered extracellular proteolysis has been exploited to selectively activate therapeutics in diseases such as cancer; however, once activated, extracellular drugs can diffuse away, limiting efficacy. We address this challenge by coupling proteolytic activation with membrane tethering to retain drugs within diseased tissue. To accomplish this, we developed “restricted interaction peptides” (RIPs), a delivery platform that leverages elevated proteolytic activity to activate membrane-interacting peptides, localizing cargos near the site of proteolysis. We demonstrate that RIPs can deliver diverse therapeutic cargos, including cytotoxins and radioisotopes. As proof of concept, we engineered “FRIP,” a RIP designed for cleavage by fibroblast activation protein (FAP), an endoprotease upregulated in solid tumors and fibrosis. Efficient P4–P4’ substrate sequences were identified and incorporated into FRIPs. Cell-based studies showed that, upon activation, the peptide adhered to membranes rapidly internalized and successfully delivered therapeutic cargos. Consistent with this, FRIPs delivering MMAE inhibited proliferation in an FAP-dependent manner. Imaging studies confirmed tumor targeting with minimal uptake in normal tissues. Finally, FRIPs delivering MMAE or Cu-67 exhibited potent antitumor effects. These findings establish membrane tethering as a strategy to enhance drug retention.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Linking secretion and cytoskeleton in immunity– a case for Arabidopsis TGNap1

In plants, robust defense depends on the efficient and resilient trafficking supply chains to the site of pathogen attack. Though the importance of intracellular trafficking in plant immunity has been well established, a lack of clarity remains regarding the contribution of the various trafficking pathways in transporting immune-related proteins. We have recently identified a trans-Golgi network protein, TGN-ASSOCIATED PROTEIN 1 (TGNap1), which functionally links post-Golgi vesicles with the cytoskeleton to transport immunity-related proteins in the model plant species Arabidopsis thaliana. We propose new hypotheses on the various functional implications of TGNap1 and then elaborate on the surprising heterogeneity of TGN vesicles during immunity revealed by the discovery of TGNap1 and other TGN-associated proteins in recent years.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Spatial top-down proteomics for the functional characterization of human kidney

Background: The Human Proteome Project has credibly detected nearly 93% of the roughly 20,000 proteins which are predicted by the human genome. However, the proteome is enigmatic, where alterations in amino acid sequences from polymorphisms and alternative splicing, errors in translation, and post-translational modifications result in a proteome depth estimated at several million unique proteoforms. Recently mass spectrometry has been demonstrated in several landmark efforts mapping the human proteoform landscape in bulk analyses. Herein, we developed an integrated workflow for characterizing proteoforms from human tissue in a spatially resolved manner by coupling laser capture microdissection, nanoliter-scale sample preparation, and mass spectrometry imaging. Results: Using healthy human kidney sections as the case study, we focused our analyses on the major functional tissue units including glomeruli, tubules, and medullary rays. After laser capture microdissection, these isolated functional tissue units were processed with microPOTS (microdroplet processing in one-pot for trace samples) for sensitive top-down proteomics measurement. This provided a quantitative database of 616 proteoforms that was further leveraged as a library for mass spectrometry imaging with near-cellular spatial resolution over the entire section. Notably, several mitochondrial proteoforms were found to be differentially abundant between glomeruli and convoluted tubules, and further spatial contextualization was provided by mass spectrometry imaging confirming unique differences identified by microPOTS, and further expanding the field-of-view for unique distributions such as enhanced abundance of a truncated form (1-74) of ubiquitin within cortical regions. Conclusions: We developed an integrated workflow to directly identify proteoforms and reveal their spatial distributions. Where of the 20 differentially abundant proteoforms identified as discriminate between tubules and glomeruli by microPOTS, the vast majority of tubular proteoforms were of mitochondrial origin (8 of 10) where discriminate proteoforms in glomeruli were primarily hemoglobin subunits (9 of 10). These trends were also identified within ion images demonstrating spatially resolved characterization of proteoforms that has the potential to reshape discovery-based proteomics because the proteoforms are the ultimate effector of cellular functions. Applications of this technology have the potential to unravel etiology and pathophysiology of disease states, informing on biologically active proteoforms, which remodel the proteomic landscape in chronic and acute disorders.

59 BASIC BIOLOGICAL SCIENCES↗