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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 433 records · Page 24
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Computational design of closely related proteins that adopt two well-defined but structurally divergent folds

The plasticity of naturally occurring protein structures, which can change shape considerably in response to changes in environmental conditions, is critical to biological function. While computational methods have been used for de novo design of proteins that fold to a single state with a deep free-energy minimum [P.-S. Huang, S. E. Boyken, D. Baker, Nature 537, 320–327 (2016)], and to reengineer natural proteins to alter their dynamics [J. A. Davey, A. M. Damry, N. K. Goto, R. A. Chica, Nat. Chem. Biol.13, 1280–1285 (2017)] or fold [P. A. Alexander, Y. He, Y. Chen, J. Orban, P. N. Bryan, Proc. Natl. Acad. Sci. U.S.A. 106, 21149–21154 (2009)], the de novo design of closely related sequences which adopt well-defined but structurally divergent structures remains an outstanding challenge. We designed closely related sequences (over 94% identity) that can adopt two very different homotrimeric helical bundle conformations—one short (~66 Å height) and the other long (~100 Å height)—reminiscent of the conformational transition of viral fusion proteins. Crystallographic and NMR spectroscopic characterization shows that both the short- and long-state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large-scale conformational switches between structurally divergent forms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural delineation and phase-dependent activation of the costimulatory CD27:CD70 complex

CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2003

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

NASA Discipline Cell Biology↗
NASA NTRS2002

A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

Non-NASA Center↗
DOE OSTI2020

Structure of the Receptor Binding Domain of EnvP(b)1, an Endogenous Retroviral Envelope Protein Expressed in Human Tissues

EnvP(b)1 is an endogenous retroviral envelope gene found in human and other primate genomes. We report EnvP(b)1 sequences in primate genomes consistent with an integration event between 40 and 71 million years ago. Using a highly specific polyclonal antiserum raised against the putative receptor binding domain (RBD) of human EnvP(b)1, we detected expression in human placenta, ovaries, and thymus. We found that EnvP(b)1 is proteolytically processed, and using cell-cell fusion assays in multiple primate cell lines, we demonstrated that extant EnvP(b)1 proteins from a variety of primate genomes are fusogenic. This work supports the idea that EnvP(b)1 is under purifying selection and its fusogenic activity has been maintained for over 40 million years. We determined the structure of the RBD of human EnvP(b)1, which defines structural similarities with extant leukemia viruses, despite little sequence conservation. This structure highlights a common scaffold from which novel receptor binding specificities likely evolved. The evolutionary plasticity of this domain may underlie the diversity of related Envs in circulating viruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

RCSB Protein Data Bank: supporting research and education worldwide through explorations of experimentally determined and computationally predicted atomic level 3D biostructures

The Protein Data Bank (PDB) was established as the first open-access digital data resource in biology and medicine in 1971 with seven X-ray crystal structures of proteins. Today, the PDB houses >210 000 experimentally determined, atomic level, 3D structures of proteins and nucleic acids as well as their complexes with one another and small molecules ( e.g. approved drugs, enzyme cofactors). These data provide insights into fundamental biology, biomedicine, bioenergy and biotechnology. They proved particularly important for understanding the SARS-CoV-2 global pandemic. The US-funded Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) and other members of the Worldwide Protein Data Bank (wwPDB) partnership jointly manage the PDB archive and support >60 000 `data depositors' (structural biologists) around the world. wwPDB ensures the quality and integrity of the data in the ever-expanding PDB archive and supports global open access without limitations on data usage. The RCSB PDB research-focused web portal at https://www.rcsb.org/ (RCSB.org) supports millions of users worldwide, representing a broad range of expertise and interests. In addition to retrieving 3D structure data, PDB `data consumers' access comparative data and external annotations, such as information about disease-causing point mutations and genetic variations. RCSB.org also provides access to >1 000 000 computed structure models (CSMs) generated using artificial intelligence/machine-learning methods. To avoid doubt, the provenance and reliability of experimentally determined PDB structures and CSMs are identified. Related training materials are available to support users in their RCSB.org explorations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Cryo-EM Structure of the Mnx Protein Complex Reveals a Tunnel Framework for the Mechanism of Manganese Biomineralization

The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O 2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The ß-sheet–rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxE 3 F 3 cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Furthermore, comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.

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

Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication

For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter “crown” complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 Å. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the ~19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at ~35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (-) and (+)RNA synthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Extended experimental inferential structure determination method in determining the structural ensembles of disordered protein states

Proteins with intrinsic or unfolded state disorder comprise a new frontier in structural biology, requiring the characterization of diverse and dynamic structural ensembles. Here we introduce a comprehensive Bayesian framework, the Extended Experimental Inferential Structure Determination (X-EISD) method, which calculates the maximum log-likelihood of a disordered protein ensemble. X-EISD accounts for the uncertainties of a range of experimental data and back-calculation models from structures, including NMR chemical shifts, J-couplings, Nuclear Overhauser Effects (NOEs), paramagnetic relaxation enhancements (PREs), residual dipolar couplings (RDCs), hydrodynamic radii (R h ), single molecule fluorescence Förster resonance energy transfer (smFRET) and small angle X-ray scattering (SAXS). We apply X-EISD to the joint optimization against experimental data for the unfolded drkN SH3 domain and find that combining a local data type, such as chemical shifts or J-couplings, paired with long-ranged restraints such as NOEs, PREs or smFRET, yields structural ensembles in good agreement with all other data types if combined with representative IDP conformers.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Exploring the fragmentation efficiency of proteins analyzed by MALDI-TOF-TOF tandem mass spectrometry using computational and statistical analyses

Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) is a rapid technique for identifying intact proteins from unfractionated mixtures by top-down proteomic analysis. MS/MS allows isolation of specific intact protein ions prior to fragmentation, allowing fragment ion attribution to a specific precursor ion. However, the fragmentation efficiency of mature, intact protein ions by MS/MS post-source decay (PSD) varies widely, and the biochemical and structural factors of the protein that contribute to it are poorly understood. With the advent of protein structure prediction algorithms such as Alphafold2, we have wider access to protein structures for which no crystal structure exists. In this work, we use a statistical approach to explore the properties of bacterial proteins that can affect their gas phase dissociation via PSD. We extract various protein properties from Alphafold2 predictions and analyze their effect on fragmentation efficiency. Our results show that the fragmentation efficiency from cleavage of the polypeptide backbone on the C-terminal side of glutamic acid (E) and asparagine (N) residues were nearly equal. In addition, we found that the rearrangement and cleavage on the C-terminal side of aspartic acid (D) residues that result from the aspartic acid effect (AAE) were higher than for E- and N-residues. From residue interaction network analysis, we identified several local centrality measures and discussed their implications regarding the AAE. We also confirmed the selective cleavage of the backbone at D-proline bonds in proteins and further extend it to N-proline bonds. Finally, we note an enhancement of the AAE mechanism when the residue on the C-terminal side of D-, E- and N-residues is glycine. To the best of our knowledge, this is the first report of this phenomenon. Our study demonstrates the value of using statistical analyses of protein sequences and their predicted structures to better understand the fragmentation of the intact protein ions in the gas phase.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1992

Growing protein crystals in microgravity - The NASA Microgravity Science and Applications Division (MSAD) Protein Crystal Growth (PCG) program

In collaboration with a medical researcher at the University of Alabama at Birmingham, NASA's Marshall Space Flight Center in Huntsville, Alabama, under the sponsorship of the Microgravity Science and Applications Division (MSAD) at NASA Headquarters, is continuing a series of space experiments in protein crystal growth which could lead to innovative new drugs as well as basic science data on protein molecular structures. From 1985 through 1992, Protein Crystal Growth (PCG) experiments will have been flown on the Space Shuttle a total of 14 times. The first four hand-held experiments were used to test hardware concepts; later flights incorporated these concepts for vapor diffusion protein crystal growth with temperature control. This article provides an overview of the PCG program: its evolution, objectives, and plans for future experiments on NASA's Space Shuttle and Space Station Freedom.

Herren, B.↗
NASA NTRS1995

Microgravity sciences application visiting scientist program

Marshall Space Flight Center pursues scientific research in the area of low-gravity effects on materials and processes. To facilitate these Government performed research responsibilities, a number of supplementary research tasks were accomplished by a group of specialized visiting scientists. They participated in work on contemporary research problems with specific objectives related to current or future space flight experiments and defined and established independent programs of research which were based on scientific peer review and the relevance of the defined research to NASA microgravity for implementing a portion of the national program. The programs included research in the following areas: protein crystal growth, X-ray crystallography and computer analysis of protein crystal structure, optimization and analysis of protein crystal growth techniques, and design and testing of flight hardware.

Glicksman, Martin↗