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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 OSTI2013

Mutational analysis of the rotavirus NSP4 enterotoxic domain that binds to caveolin-1

Background: Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. Our previous data show a direct interaction between RV NSP4 and the structural protein of caveolae, caveolin-1 (cav-1), in yeast and mammalian cells. The binding site of cav-1 mapped to the NSP4 amphipathic helix, and led us to examine which helical face was responsible for the interaction. Methods: A panel of NSP4 mutants were prepared and tested for binding to cav-1 by yeast two hybrid and direct binding assays. The charged residues of the NSP4 amphipathic helix were changed to alanine (NSP4 46-175 -ala6); and three residues in the hydrophobic face were altered to charged amino acids (NSP4 46-175 -HydroMut). In total, twelve mutants of NSP4 were generated to define the cav-1 binding site. Synthetic peptides corresponding to the hydrophobic and charged faces of NSP4 were examined for structural changes by circular dichroism (CD) and diarrhea induction by a neonatal mouse study. Results: Mutations of the hydrophilic face (NSP4 46-175 -Ala6) bound cav-1 akin to wild type NSP4. In contrast, disruption of the hydrophobic face (NSP 446-175 -HydroMut) failed to bind cav-1. These data suggest NSP4 and cav-1 associate via a hydrophobic interaction. Analyses of mutant synthetic peptides in which the hydrophobic residues in the enterotoxic domain of NSP4 were altered suggested a critical hydrophobic residue. Both NSP4 HydroMut112-140 , that contains three charged amino acids (aa113, 124, 131) changed from the original hydrophobic residues and NSP4 AlaAcidic112-140 that contained three alanine residues substituted for negatively charged (aa114, 125, 132) amino acids failed to induce diarrhea. Whereas peptides NSP4wild type 112 -140 and NSP4 AlaBasic112-140 that contained three alanine substituted for positively charged (aa115, 119, 133) amino acids, induced diarrhea. Conclusions: These data show that the cav-1 binding domain is within the hydrophobic face of the NSP4 amphipathic helix. The integrity of the helical structure is important for both cav-1 binding and diarrhea induction implying a connection between NSP4 functional and binding activities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Specific Mutations in H5N1 Mainly Impact the Magnitude and Velocity of the Host Response in Mice

Influenza infection causes respiratory disease that can lead to death. The complex interplay between virus-encoded and host-specific pathogenicity regulators is not well-understood. By analyzing a collection of mouse lung samples infected by A/Vietnam/1203/2004 (H5N1; VN1203) influenza, we characterized a signature of transcripts and proteins associated with the kinetics of the host response. Using a new geometrical representation method and two criteria, we show that infection concentrations and four specific mutations in VN1203 mainly impact on the magnitude and velocity of the host response kinetics, rather than on specific sets of genes up- and down-regulated. We observed similar kinetic effects using A/California/04/2009 (H1N1)-infected samples, and we show that these effects correlate with mice morbidity and viral titer measurements. Speed and extent of changes in the host response between days 1 and 2 post-infection were attenuated for each VN1203 mutant compared to the wild-type, except for PB1-F2 deletion at a high dose, which was associated with high virulence. This indicates that the host response in that time frame is critical and that immunomodulatory therapeutics should specifically be applied during the early days post-infection.

Environmental Molecular Sciences Laboratory↗
DOE OSTI2020

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8 C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Holistic Portfolio Optimization using Directed Mutations

Genetic algorithms provide attractive options for performing nonlinear multi-objective combinatorial design optimization, and they have proven very useful for optimizing individual systems. However, conventional genetic algorithms fall short when performing holistic portfolio optimizations in which the decision variables also include the integer counts of multiple system types over multiple time periods. When objective functions are formulated as analytic functions, we can formally differentiate with respect to system counts and use the resulting gradient information to generate favorable mutations in the count variables. We apply several variations on this basic idea to an idealized hanging chain example to obtain >> 1000x speedups over conventional genetic algorithms in both single - and multi-objective cases. We develop a more complex example of a notional military portfolio that includes combinatorial design variables and dependency constraints between the design choices. In this case, our initial results are mixed, but many variations are still open to further research.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2020

Predicting Drug Resistance Using Deep Mutational Scanning

Drug resistance is a major healthcare challenge, resulting in a continuous need to develop new inhibitors. The development of these inhibitors requires an understanding of the mechanisms of resistance for a critical mass of occurrences. Recent genome editing technologies based on high-throughput DNA synthesis and sequencing may help to predict mutations resulting in resistance by testing large mutagenesis libraries. Here we describe the rationale of this approach, with examples and relevance to drug development and resistance in malaria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Epistatic mutations in PUMA BH3 drive an alternate binding mode to potently and selectively inhibit anti-apoptotic Bfl-1

Overexpression of anti-apoptotic Bcl-2 family proteins contributes to cancer progression and confers resistance to chemotherapy. Small molecules that target Bcl-2 are used in the clinic to treat leukemia, but tight and selective inhibitors are not available for Bcl-2 paralog Bfl-1. Guided by computational analysis, we designed variants of the native BH3 motif PUMA that are > 150-fold selective for Bfl-1 binding. The designed peptides potently trigger disruption of the mitochondrial outer membrane in cells dependent on Bfl-1, but not in cells dependent on other anti-apoptotic homologs. High-resolution crystal structures show that designed peptide FS2 binds Bfl-1 in a shifted geometry, relative to PUMA and other binding partners, due to a set of epistatic mutations. FS2 modified with an electrophile reacts with a cysteine near the peptide-binding groove to augment specificity. Designed Bfl-1 binders provide reagents for cellular profiling and leads for developing enhanced and cell-permeable peptide or small-molecule inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning

The interaction between a bacteriophage and its host is mediated by the phage's receptor binding protein (RBP). Despite its fundamental role in governing phage activity and host range, molecular rules of RBP function remain a mystery. Here, we systematically dissect the functional role of every residue in the tip domain of T7 phage RBP (1660 variants) by developing a high-throughput, locus-specific, phage engineering method. This rich dataset allowed us to cross compare functional profiles across hosts to precisely identify regions of functional importance, many of which were previously unknown. Substitution patterns showed host-specific differences in position and physicochemical properties of mutations, revealing molecular adaptation to individual hosts. We discovered gain-of-function variants against resistant hosts and host-constricting variants that eliminated certain hosts. To demonstrate therapeutic utility, we engineered highly active T7 variants against a urinary tract pathogen. Our approach presents a generalized framework for characterizing sequence–function relationships in many phage–bacterial systems.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1972

Mutations induced in Tradescantia by small doses of X-rays and neutrons - Analysis of dose-response curves.

Dose-response curves for pink somatic mutations in Tradescantia stamen hairs were analyzed after neutron and X-ray irradiation with doses ranging from a fraction of a rad to the region of saturation. The dose-effect relation for neutrons indicates a linear dependence from 0.01 to 8 rads; between 0.25 and 5 rads, a linear dependence is indicated for X-rays also. As a consequence the relative biological effectiveness reaches a constant value (about 50) at low doses. The observations are in good agreement with the predictions of the theory of dual radiation action and support its interpretation of the effects of radiation on higher organisms. The doubling dose of X-rays was found to be nearly 1 rad.

Sparrow, A. H.↗
NASA NTRS1978

The point mutation process in proteins

An optimized scoring matrix for residue-by-residue comparisons of distantly related protein sequences has been developed. The scoring matrix is based on observed exchanges and mutabilities of amino acids in 1572 closely related sequences derived from a cross-section of protein groups. Very few superimposed or parallel mutations are included in the data. The scoring matrix is most useful for demonstrating the relatedness of proteins between 65 and 85% different.

Schwartz, R. M.↗
NASA NTRS1992

Estimation and enhancement of real-time software reliability through mutation analysis

A simulation-based technique for obtaining numerical estimates of the reliability of N-version, real-time software is presented. An extended stochastic Petri net is employed to represent the synchronization structure of N versions of the software, where dependencies among versions are modeled through correlated sampling of module execution times. Test results utilizing specifications for NASA's planetary lander control software indicate that mutation-based testing could hold greater potential for enhancing reliability than the desirable but perhaps unachievable goal of independence among N versions.

Geist, Robert↗
NASA NTRSDate not supplied

Stress-Activated Positive Holes (O− in a Matrix of O2–) Cause DNA Damage in Surface-Dwelling Organisms: Unveiling Mutation-Induced Secrets of Nature

Peroxy defects consist of pairs of tightly bonded oxygen anions in the –1 valence state such as in O3X/OO\YO3 with X, Y = Si4+, Al3+ etc. They commonly occur in igneous, metamorphic and many sedimentary rocks. When such rocks are stressed by tectonic forces, peroxy defects break up, releasing highly mobile electronic charge carriers: defect electrons in the O2– sublattice, i.e. unbound O–, known as “positive holes”, h•. The h• can flow out of stressed rock volumes, spreading far and wide, causing electric currents and electrochemical reactions. This study explores how the h• impact the electron flow in the electron transport chain (ETC) of organisms on the surface of rocks such as gabbro and granite. We found that, by forming hydroxyl radicals and superoxide anions, the h• disrupt the in vivo coordination of reduction-oxidation reactions that are essential for the timing of the redox cycle. Our observations show that stress activation of h• delays the sprouting of certain plant seeds and impedes the growth of yeast cultures, Saccharomyces cerevisiae. The h• induce mutations and affect plant development as evidenced by reduced stem length and leaf size. At the same time, the h• serve as a source of abiotic oxidation, capable of forming various organic compounds in situ. Through the generation of radical species that create new carbon-carbon bonds the h• facilitate the abiotic synthesis of hydrocarbons and other organic molecules essential to life, including porphyrins. Our findings highlight the intricate interplay between positive holes, redox timing, and biological processes, revealing their significant role in influencing the growth and development of organisms in tectonically stressed crustal environments. Understanding these effects enhances our broader comprehension of redox biology and the influence of environmental factors on cellular development in natural settings.

astrobiology↗
DOE OSTI2015

Computational redesign reveals allosteric mutation hotspots of organophosphate hydrolase that enhance organophosphate hydrolysis

Organophosphates are widely used for peaceful (agriculture) and military purposes (chemical warfare agents). The extraordinary toxicity of organophosphates and the risk of deployment, make it critical to develop means for their rapid and efficient deactivation. Organophosphate hydrolase (OPH) already plays an important role in organophosphate remediation, but is insufficient for therapeutic or prophylactic purposes primarily due to low substrate affinity. Current efforts focus on directly modifying the active site to differentiate substrate specificity and increase catalytic activity. Here, we present a novel strategy for enhancing the general catalytic efficiency of OPH through computational redesign of the residues that are allosterically coupled to the active site and validated our design by mutagenesis. Specifically, we identify five such hot-spot residues for allosteric regulation and assay these mutants for hydrolysis activity against paraoxon, a chemical-weapons simulant. A high percentage of the predicted mutants exhibit enhanced activity over wild-type (k cat =16.63 s -1 ), such as T199I/T54I (899.5 s -1 ) and C227V/T199I/T54I (848 s -1 ), while the Km remains relatively unchanged in our high-throughput cell-free expression system. Further computational studies of protein dynamics reveal four distinct distal regions coupled to the active site that display significant changes in conformation dynamics upon these identified mutations. These results validate a computational design method that is both efficient and easily adapted as a general procedure for enzymatic enhancement.

organophosphate hydrolase↗
DOE OSTI2018

Mutations in iron-sulfur cluster proteins that improve xylose utilization

There is provided an engineered host cells comprising (a) one or more mutations in one or more endogenous genes encoding a protein associated with iron metabolism; and (b) at least one gene encoding a polypeptide having xylose isomerase activity, and methods of their use thereof.

59 BASIC BIOLOGICAL SCIENCES↗