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At least 145 records · Page 8

All sources NASA NTRS DOE OSTI

NASA NTRS2000

Ion channels and the transduction of light signals

Studies of biological light-sensing mechanisms are revealing important roles for ion channels. Photosensory transduction in plants is no exception. In this article, the evidence that ion channels perform such signal-transducing functions in the complex array of mechanisms that bring about plant photomorphogenesis will be reviewed and discussed. The examples selected for discussion range from light-gradient detection in unicellular algae to the photocontrol of stem growth in Arabidopsis. Also included is some discussion of the technical aspects of studies that combine electrophysiology and photobiology.

NASA Discipline Plant Biology↗

NASA NTRS2001

Long-distance signaling within Coleus x hybridus leaves; mediated by changes in intra-leaf CO2?

Rapid long-distance signaling in plants can occur via several mechanisms, including symplastic electric coupling and pressure waves. We show here in variegated Coleus leaves a rapid propagation of electrical signals that appears to be caused by changes in intra-leaf CO2 concentrations. Green leaf cells, when illuminated, undergo a rapid depolarization of their membrane potential (Vm) and an increase in their apoplastic pH (pHa) by a process that requires photosynthesis. This is followed by a slower hyperpolarization of Vm and apoplastic acidification, which do not require photosynthesis. White (chlorophyll-lacking) leaf cells, when in isolated white leaf segments, show only the slow response, but when in mixed (i.e. green and white) segments, the rapid Vm depolarization and increase in pHa propagate over more than 10 mm from the green to the white cells. Similarly, these responses propagate 12-20 mm from illuminated to unilluminated green cells. The fact that the propagation of these responses is eliminated when the leaf air spaces are infiltrated with solution indicates that the signal moves in the apoplast rather than the symplast. A depolarization of the mesophyll cells is induced in the dark by a decrease in apoplastic CO2 but not by an increase in pHa. These results support the hypothesis that the propagating signal for the depolarization of the white mesophyll cells is a photosynthetically induced decrease in the CO2 level of the air spaces throughout the leaf.

NASA Discipline Plant Biology↗

NASA NTRS1995

Lipopolysaccharide modulation of a CD14-like molecule on porcine alveolar macrophages

Cluster of differentiation antigen 14 (CD14) functions as a receptor for lipopolysaccharide (LPS) LPS-binding protein (LBP) complexes. Because LPS has varying effects on CD14 expression in vitro, we evaluated CD14 expression in response to LPS with a fully differentiated macrophage phenotype, the alveolar macrophage. By using flow microfluorometric analysis and a radioimmunoassay with an anti-human CD14 monoclonal antibody (My4) that cross-reacts with porcine CD14, we found that macrophages stimulated with LPS for 24 h exhibited a two- to fivefold increase in CD14-like antigen compared with unstimulated cells. At low concentrations of LPS, up-regulation of the CD14-like antigen was dependent on serum; at higher concentrations of LPS, serum was not required. In the absence of serum a 10-fold higher dose of LPS (10 ng/ml) was required to increase CD14-like expression. In addition, LPS-induced CD14-like up-regulation correlated with secretion of tumor necrosis factor-alpha, regardless of serum concentration. Blockade with My4 antibody significantly inhibited LPS-induced tumor necrosis factor-alpha secretion at 1 ng/ml of LPS. However, inhibition decreased as we increased the LPS concentration, suggesting the existence of CD14-independent pathways of macrophage activation in response to LPS. Alternatively, My4 may have a lower affinity for the porcine CD14 antigen than LPS, which may have only partially blocked the LPS-LBP binding site at high concentrations of LPS. Therefore, these data suggest that LPS activation of porcine alveolar macrophages for 24 h increased CD14-like receptor expression. The degree of CD14-like up-regulation was related to LPS concentration, however, activation did not require the presence of serum at high concentrations of LPS.

Non-NASA Center↗

NASA NTRS2009

Modular and Stochastic Approaches to Molecular Pathway Models of ATM, TGF beta, and WNT Signaling

Deterministic pathway models that describe the biochemical interactions of a group of related proteins, their complexes, activation through kinase, etc. are often the basis for many systems biology models. Low dose radiation effects present a unique set of challenges to these models including the importance of stochastic effects due to the nature of radiation tracks and small number of molecules activated, and the search for infrequent events that contribute to cancer risks. We have been studying models of the ATM, TGF -Smad and WNT signaling pathways with the goal of applying pathway models to the investigation of low dose radiation cancer risks. Modeling challenges include introduction of stochastic models of radiation tracks, their relationships to more than one substrate species that perturb pathways, and the identification of a representative set of enzymes that act on the dominant substrates. Because several pathways are activated concurrently by radiation the development of modular pathway approach is of interest.

Cucinotta, Francis A.↗

NASA NTRS2007

Detergent-Specific Membrane Protein Crystallization Screens

A suite of reagents has been developed for three-dimensional crystallization of integral membranes present in solution as protein-detergent complexes (PDCs). The compositions of these reagents have been determined in part by proximity to the phase boundaries (lower consolute boundaries) of the detergents present in the PDCs. The acquisition of some of the requisite phase-boundary data and the preliminary design of several of the detergent- specific screens was supported by a NASA contract. At the time of expiration of the contract, a partial set of preliminary screens had been developed. This work has since been extended under non-NASA sponsorship, leading to near completion of a set of 20 to 30 different and unique detergent- specific 96-condition screens.

Wiener, Michael↗

NASA NTRS2019

Carbon Nanostructures in Biological Sensors for Space and Terrestrial Applications

Biosensing devices comprised of carbon nanotubes and nanofibers have been developed for astronaut crew point-of-care. Their inherent nanometer scale, high conductivity, wide potential window, good biocompatibility and well-defined surface chemistry make them ideal candidates as biosensor electrodes. Here, we report two studies using carbon nanotube and carbon nanofiber electrodes for biomedical applications. First, a 3x3 electrode device, with each electrode containing 40,000 carbon nanofiber nanoelectrodes was fabricated on silicon using traditional microfabrication processing. The device was demonstrated as a multiplexed immunosensor for simultaneous, label-free detection of cardiac troponin-I, C-reactive protein and myoglobin. Antibodies specific to cardiac troponin-I, C-reactive protein and myoglobin were covalently bound to the CNF surface and were characterized using electrochemical impedance spectroscopy and differential pulse voltammetry. Each step of the modification process resulted in changes in resistance to charge transfer due to the changes at the electrode surface upon antibody immobilization and binding to the specific cardiac protein. The real-time label free detection of the three cardiac markers from pure components and mixtures was demonstrated with high sensitivity, down to 0.2 ng/mL, and good selectivity. Detection in human blood serum did not present false positives from non-specific protein adsorption. More recently, this detection scheme has been applied to inkjet printed carbon nanotube electrodes on Kapton and paper. Printed devices have several unique advantages including simple and inexpensive fabrication. The results demonstrate that these sensors can serve a miniaturized, low cost device for detection of proteins in complex mixtures making this platform a good candidate for early stage diagnosis of myocardial infarction. Future inkjet printed devices can be fabricated have the added advantage in their suitability to be manufactured in an in-space, microgravity environment.

carbon nanotubes↗

NASA NTRS2019

Carbon Nanomaterials for Biosensing Applications

carbon nanotubes↗

DOE OSTI2025

Electrosome assembly: Structural insights from high voltage-activated calcium channel (CaV)–chaperone interactions

Ion channels are multicomponent complexes (termed here as“electrosomes”) that conduct the bioelectrical signals required for life. It has been appreciated for decades that assembly is critical for proper channel function, but knowledge of the factors that undergird this important process has been lacking. Although there are now exemplar structures of representatives of most major ion channel classes, there has been no direct structural information to inform how these complicated, multipart complexes are put together or whether they interact with chaperone proteins that aid in their assembly. Recent structural characterization of a complex of the endoplasmic membrane protein complex (EMC) chaperone and a voltage-gated calcium channel (CaV) assembly intermediate comprising the pore-forming CaVα1 and cytoplasmic CaVβ subunits offers the first structural view into the assembly of a member of the largest ion channel class, the voltagegated ion channel (VGIC) superfamily. The structure shows how the EMC remodels the CaVα1/CaVβ complex through a set of rigid body movements for handoff to the extracellular CaVα2δ subunit to complete channel assembly in a process that involves intersubunit coordination of a divalent cation and ordering of CaVα1 elements. These findings set a new framework for deciphering the structural underpinnings of ion channel biogenesis that has implications for understanding channel function, how drugs and disease mutations act, and for investigating how other membrane proteins may engage the ubiquitous EMC chaperone.

Biochemistry & Molecular Biology↗

DOE OSTI2025

Sequence Programmable Order–Disorder Transitions in Supramolecular Assembly of Peptide Nanofibers

Protein–protein interactions determine the assembly of complexes that are responsible for numerous key biological processes. The assembly of many natural protein complexes is mediated by post-translational structural changes and environmental stimuli. In this study, we show that incorporation of adjacent lysine residues results in the pH-tunable stability of peptide secondary structure and assembly, allowing for the incorporation of complementary order-inducing motifs. The strategic placement of cysteine pairs in the same peptide sequence results in redox-dependent disulfide staple formation, inducing a transition from random coil to β-sheet conformation and subsequent supramolecular nanofiber assembly from otherwise disordered peptide monomers. Spectroscopic, imaging, molecular dynamics, and kinetic studies highlight the critical role of sequence motif location, oligomerization, and the competitive interplay between intra- and interpeptide disulfide bonding in determining assembly outcomes. We extend this approach to demonstrate phosphorylation-dependent assembly from the design of the same parent peptide sequence, suggesting a general approach to the design of diverse stimulus-responsive peptide sequences for supramolecular assembly. Furthermore, these findings also provide a framework for investigating sequence-dependent pathways in amyloid fiber formation with potential implications for neurodegenerative disease research.

Disulfides↗

DOE OSTI2025

A conserved chaperone protein is required for the formation of a noncanonical type VI secretion system spike tip complex

Type VI secretion systems (T6SSs) are dynamic protein nanomachines found in Gram-negative bacteria that deliver toxic effector proteins into target cells in a contact-dependent manner. Prior to secretion, many T6SS effector proteins require chaperones and/or accessory proteins for proper loading onto the structural components of the T6SS apparatus. However, despite their established importance, the precise molecular function of several T6SS accessory protein families remains unclear. In this study, we set out to characterize the DUF2169 family of T6SS accessory proteins. Using gene co-occurrence analyses, we find that DUF2169-encoding genes strictly co-occur with genes encoding T6SS spike complexes formed by valine-glycine repeat protein G (VgrG) and DUF4150 domains. Although structurally similar to Pro-Ala-Ala-Arg (PAAR) domains, “PAAR-like” DUF4150 domains lack PAAR motifs and instead contain a conserved PIPY motif, leading us to designate them PIPY domains. Next, we present both genetic and biochemical evidence that PIPY domains require a cognate DUF2169 protein to form a functional T6SS spike complex with VgrG. This contrasts with canonical PAAR proteins, which bind VgrG on their own to form functional spike complexes. By solving the first crystal structure of a DUF2169 protein, we show that this T6SS accessory protein adopts a novel protein fold. Furthermore, biophysical and structural modeling data suggest that DUF2169 contains a dynamic loop that physically interacts with a hydrophobic patch on the surface of its cognate PIPY domain. Based on these findings, we propose a model whereby DUF2169 proteins function as molecular chaperones that maintain VgrG–PIPY spike complexes in a secretion-competent state prior to their export by the T6SS apparatus.

DUF2169↗

DOE OSTI2024

Distinct binding conformations of epinephrine with α- and β-adrenergic receptors

Abstract Agonists targeting α 2 -adrenergic receptors (ARs) are used to treat diverse conditions, including hypertension, attention-deficit/hyperactivity disorder, pain, panic disorders, opioid and alcohol withdrawal symptoms, and cigarette cravings. These receptors transduce signals through heterotrimeric Gi proteins. Here, we elucidated cryo-EM structures that depict α 2A -AR in complex with Gi proteins, along with the endogenous agonist epinephrine or the synthetic agonist dexmedetomidine. Molecular dynamics simulations and functional studies reinforce the results of the structural revelations. Our investigation revealed that epinephrine exhibits different conformations when engaging with α-ARs and β-ARs. Furthermore, α 2A -AR and β 1 -AR (primarily coupled to Gs, with secondary associations to Gi) were compared and found to exhibit different interactions with Gi proteins. Notably, the stability of the epinephrine–α 2A -AR–Gi complex is greater than that of the dexmedetomidine–α 2A -AR–Gi complex. These findings substantiate and improve our knowledge on the intricate signaling mechanisms orchestrated by ARs and concurrently shed light on the regulation of α-ARs and β-ARs by epinephrine.

Biochemistry & Molecular Biology↗

DOE OSTI2024

Unveiling the nanoscale architectures and dynamics of protein assembly with in situ atomic force microscopy

Proteins play a vital role in different biological processes by forming complexes through precise folding with exclusive inter- and intra-molecular interactions. Understanding the structural and regulatory mechanisms underlying protein complex formation provides insights into biophysical processes. Furthermore, the principle of protein assembly gives guidelines for new biomimetic materials with potential applications in medicine, energy, and nanotechnology. Atomic force microscopy (AFM) is a powerful tool for investigating protein assembly and interactions across spatial scales (single molecules to cells) and temporal scales (milliseconds to days). It has significantly contributed to understanding nanoscale architectures, inter- and intra-molecular interactions, and regulatory elements that determine protein structures, assemblies, and functions. This review describes recent advancements in elucidating protein assemblies with in situ AFM. We discuss the structures, diffusions, interactions, and assembly dynamics of proteins captured by conventional and high-speed AFM in near-native environments and recent AFM developments in the multimodal high-resolution imaging, bimodal imaging, live cell imaging, and machine-learning-enhanced data analysis. These approaches show the significance of broadening the horizons of AFM and enable unprecedented explorations of protein assembly for biomaterial design and biomedical research.

36 MATERIALS SCIENCE↗

NASA NTRS1999

A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

Oligodeoxyribonucleotides/chemistry/metabolism↗

NASA NTRS1999

Protein machines and self assembly in muscle organization

The remarkable order of striated muscle is the result of a complex series of protein interactions at different levels of organization. Within muscle, the thick filament and its major protein myosin are classical examples of functioning protein machines. Our understanding of the structure and assembly of thick filaments and their organization into the regular arrays of the A-band has recently been enhanced by the application of biochemical, genetic, and structural approaches. Detailed studies of the thick filament backbone have shown that the myosins are organized into a tubular structure. Additional protein machines and specific myosin rod sequences have been identified that play significant roles in thick filament structure, assembly, and organization. These include intrinsic filament components, cross-linking molecules of the M-band and constituents of the membrane-cytoskeleton system. Muscle organization is directed by the multistep actions of protein machines that take advantage of well-established self-assembly relationships. Copyright 1999 John Wiley & Sons, Inc.

NASA Discipline Musculoskeletal↗

NASA NTRS1981

Microdisc gel electrophoresis in sodium dodecyl sulfate of organic material from rat otoconial complexes

The gravity receptors of all vertebrates utilize a 'test mass' consisting of a complex arrangement of mineral and organic substance that lies over the sensory receptor areas. In most vertebrates, the mineral is a polymorph of calcium carbonate in the form of minute, single crystals called otoconia. An investigation is conducted to determine the number of proteins in otoconial complexes and their molecular weights. The investigation makes use of a microdisk gel electrophoresis method reported by Gainer (1971). The most important finding of the reported research is that analysis of the proteins of the organic material of the otoconial complexes is possible when sensitive microanalytical methods are employed. Further modification of the basic technique employed and the inclusion of other sensitive staining methods should mean that, in the future, protein separation by molecular weight will be possible in sample pools containing only two otoconial masses.

Ross, M. D.↗

NASA NTRS2000

Integrin activation and focal complex formation in cardiac hypertrophy

Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

NASA Discipline Cardiopulmonary↗

DOE OSTI2025

Selective deuteration of an RNA:RNA complex for structural analysis using small-angle scattering

The structures of RNA:RNA complexes regulate many biological processes. Despite their importance, protein-free RNA:RNA complexes represent a tiny fraction of experimentally determined structures. Here, we describe a joint small-angle X-ray and neutron scattering (SAXS/SANS) approach to structurally interrogate conformational changes in a model RNA:RNA complex. Using SAXS, we measured the solution structures of the individual RNAs and of the overall RNA:RNA complex. With SANS, we demonstrate, as a proof of principle, that isotope labeling and contrast matching (CM) can be combined to probe the bound state structure of an RNA within a selectively deuterated RNA:RNA complex. Furthermore, we show that experimental scattering data can validate and improve predicted AlphaFold 3 RNA:RNA complex structures to reflect its solution structure. In conclusion, our work demonstrates that in silico modeling, SAXS, and CM-SANS can be used in concert to directly analyze conformational changes within RNAs when in complex, enhancing our understanding of RNA structure in functional assemblies.

HIV-1 dimerization initiation site↗

DOE OSTI2024

SEC ‐ SAXS / MC Ensemble Structural Studies of the Microtubule Binding Protein Cdt1 Show Monomeric, Folded‐Over Conformations

ABSTRACT Cdt1 is a mixed folded protein critical for DNA replication licensing and it also has a “moonlighting” role at the kinetochore via direct binding to microtubules and the Ndc80 complex. However, it is unknown how the structure and conformations of Cdt1 could allow it to participate in these multiple, unique sets of protein complexes. While robust methods exist to study entirely folded or unfolded proteins, structure–function studies of combined, mixed folded/disordered proteins remain challenging. In this work, we employ orthogonal biophysical and computational techniques to provide structural characterization of mitosis‐competent human Cdt1. Thermal stability analyses shows that both folded winged helix domains1 are unstable. CD and NMR show that the N‐terminal and linker regions are intrinsically disordered. DLS shows that Cdt1 is monomeric and polydisperse, while SEC‐MALS confirms that it is monomeric at high concentrations, but without any apparent inter‐molecular self‐association. SEC‐SAXS enabled computational modeling of the protein structures. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble of structures. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with SAXS. The best‐fit models have the N‐terminal and linker disordered regions extended into the solution and the two folded domains close to each other in apparent “folded over” conformations. We hypothesize the best‐fit Cdt1 conformations could be consistent with a function as a scaffold protein that may be sterically blocked without binding partners. Our study also provides a template for combining experimental and computational techniques to study mixed‐folded proteins.

Cell Biology↗