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All sourcesNASA NTRSDOE OSTI
NASA NTRS2015

Martian Superoxide and Peroxide O2 Release (OR) Assay: A New Technology for Terrestrial and Planetary Applications

This study presents an assay for the detection and quantification of soil metal superoxides and peroxides in regolith and soil. The O2 release (OR) assay is based on the enzymatic conversion of the hydrolysis products of metal oxides to O2, and their quantification by an O2 electrode based on the stoichiometry of the involved reactions: The intermediate product O2 from the hydrolysis of metal superoxides is converted by cytochrome c to O2, and also by superoxide dismutase (SOD) to 1/2 mol O2 and 1/2 mol H2O2, which is then converted by catalase (CAT) to 1/2 mol O2. The product H2O2 from the hydrolysis of metal peroxides and hydroperoxides is converted to 1/2 mol O2 by CAT. The assay-method was validated in a sealed sample chamber using a liquid-phase Clark-type O2 electrode with known concentrations of O2 and H2O2, and with commercial metal superoxide and peroxide mixed with Mars analogue Mojave and Atacama Desert soils. Carbonates and perchlorates, both present on Mars, do not interfere with the assay. The assay lower limit of detection, using luminescence quenching/optical sensing O2-electrodes, is 1 nmol O2 cm(exp. -3) or better. The activity of the assay enzymes SOD and cytochrome c was unaffected up to 6 Gy exposure by gamma-radiation, while CAT retained 100% and 40% of its activity at 3 and 6 Gy, respectively, demonstrating the suitability of these enzymes for planetary missions, e.g., in Mars or Europa.

soil metal superoxides and peroxides↗
NASA NTRS1989

Influence of performance on gene expression in skeletal muscle: effects of forced inactivity

Joint immobilization and hindlimb suspension are used to examine muscle protein expression and mRNA quantities in rats. A decrease in protein synthesis was not associated with alteration in alpha-actin mRNA, cytochrome c mRNA, or beta-myosin heavy chain mRNA early in treatment. Percentage declines after seven days are compared with early treatment quantities to determine acute and chronic response to muscular atrophy.

Non-NASA Center↗
DOE OSTI2021

Transient absorption spectroscopy to explore cellular pathways to photobiomodulation

Photobiomodulation (PBM) describes the use of low irradiance light in the red to near-infrared wavelength range to stimulate biological effects in tissue, and many biological and spectroscopic techniques are used to study PBM. However, these techniques focus on the products or downstream effects rather than the electronic transitions that initiate the PBM processes. This study presents a novel approach to studying low irradiance light exposures on individual proteins and/or protein complexes by combining a continuous wave (CW) laser diode with femtosecond transient absorption spectroscopy (TAS), coined here as CW-TAS, and tests the system on reduced cytochrome c (Cyt c) for proof of principle. TAS was conducted using a 532-nm excitation pump beam and a 350-600 nm supercontinuum probe. In this work, CW laser diodes with wavelengths of 450 nm, 635 nm, and 808 nm were interchangeably fiber coupled into the HELIOS Fire. Samples of Cyt c were tested by TAS using a pump power of 15 µW, both with and without CW exposure. CW exposures were carried out with irradiances of 1.60 and 3.20 mW/cm 2 , except for 808 nm, which was only tested at 1.60 mW/cm 2 . Both kinetic and global analyses were performed on the TAS data and the time constants for sets with and without CW exposures were compared. The TAS data for Cyt c with the full dosage of CW exposures did not alter the TAS data distinguishably from the control data. No new electronic transient signals were observed beyond the background when testing Cyt c with the CW exposures. Kinetic analysis confirmed that existing transients did not deviate beyond uncertainty. Global time constants for Cyt c were calculated to be 0.25 ± 0.03 ps and 5.1 ± 0.3 ps for the control study, and the time constants for the CW exposed Cyt c were not significantly different. This study concludes that CW irradiation, at doses delivered, does not alter the transient absorption data of Cyt c. The CW-TAS method provides a new tool for studying PBM effects in other proteins and protein complexes, such as Complex IV, in future studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Protein folding from heterogeneous unfolded state revealed by time-resolved X-ray solution scattering

One of the most challenging tasks in biological science is to understand how a protein folds. In theoretical studies, the hypothesis adopting a funnel-like free-energy landscape has been recognized as a prominent scheme for explaining protein folding in views of both internal energy and conformational heterogeneity of a protein. Despite numerous experimental efforts, however, comprehensively studying protein folding with respect to its global conformational changes in conjunction with the heterogeneity has been elusive. Here we investigate the redox-coupled folding dynamics of equine heart cytochrome c (cyt-c) induced by external electron injection by using time-resolved X-ray solution scattering. A systematic kinetic analysis unveils a kinetic model for its folding with a stretched exponential behavior during the transition toward the folded state. With the aid of the ensemble optimization method combined with molecular dynamics simulations, we found that during the folding the heterogeneously populated ensemble of the unfolded state is converted to a narrowly populated ensemble of folded conformations. These observations obtained from the kinetic and the structural analyses of X-ray scattering data reveal that the folding dynamics of cyt-c accompanies many parallel pathways associated with the heterogeneously populated ensemble of unfolded conformations, resulting in the stretched exponential kinetics at room temperature. This finding provides direct evidence with a view to microscopic protein conformations that the cyt-c folding initiates from a highly heterogeneous unfolded state, passes through still diverse intermediate structures, and reaches structural homogeneity by arriving at the folded state.

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

Thiol-based pathways in the thylakoid lumen and their role in photoprotection

The long-term goal of this research is to elaborate the catalysis of thiol-disulfide transactions in the thylakoid lumen, a compartment required for transducing energy via photophosphorylation. The molecular identity of the redox players catalyzing thiol-disulfide exchanges, their relevant targets of action in vivo and how thiol-disulfide chemistry in general controls photosynthesis are the experimental questions this research aims to address. Through previous work, we documented the requirement of catalyzed disulfide formation and reduction for the biogenesis of two photosynthetic enzymes, namely Photosystem II and the cytochrome b6f complex. We established that dedicated trans-thylakoid pathways operate in these processes by delivering reducing and oxidizing power to cysteine-containing subunits of the photosynthetic complexes, which are localized in the lumen compartment. Here we focused on CCDA, CCS4 and CCS5/HCF164, the components of the disulfide-reducing pathway which maintains the heme binding cysteines of apoforms of plastid cytochrome c in the reduced form prior to covalent heme ligation. We showed that CCS4 and CCS5 operate in functionally redundant pathways for the supply of reducing power and postulate a role for CCS4 in recruiting the source of reductants to CCDA or controlling the CCDA-dependent transduction of reducing equivalents. We also demonstrate the disulfide reducing pathway operates to counter thiol oxidation of the heme-binding cysteines by disulfide forming enzyme LTO1 in the thylakoid lumen.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Protein Motifs for Proton Transfers That Build the Transmembrane Proton Gradient

Biological membranes are barriers to polar molecules, so membrane embedded proteins control the transfers between cellular compartments. Protein controlled transport moves substrates and activates cellular signaling cascades. In addition, the electrochemical gradient across mitochondrial, bacterial and chloroplast membranes, is a key source of stored cellular energy. This is generated by electron, proton and ion transfers through proteins. The gradient is used to fuel ATP synthesis and to drive active transport. Here the mechanisms by which protons move into the buried active sites of Photosystem II (PSII), bacterial RCs (bRCs) and through the proton pumps, Bacteriorhodopsin (bR), Complex I and Cytochrome c oxidase (CcO), are reviewed. These proteins all use water filled proton transfer paths. The proton pumps, that move protons uphill from low to high concentration compartments, also utilize Proton Loading Sites (PLS), that transiently load and unload protons and gates, which block backflow of protons. PLS and gates should be synchronized so PLS proton affinity is high when the gate opens to the side with few protons and low when the path is open to the high concentration side. Proton transfer paths in the proteins we describe have different design features. Linear paths are seen with a unique entry and exit and a relatively straight path between them. Alternatively, paths can be complex with a tangle of possible routes. Likewise, PLS can be a single residue that changes protonation state or a cluster of residues with multiple charge and tautomer states.

cytochrome c oxidase↗
DOE OSTI2022

The mitochondrial Cu + transporter PiC2 (SLC25A3) is a target of MTF1 and contributes to the development of skeletal muscle in vitro

The loading of copper (Cu) into cytochrome c oxidase (COX) in mitochondria is essential for energy production in cells. Extensive studies have been performed to characterize mitochondrial cuproenzymes that contribute to the metallation of COX, such as Sco1, Sco2, and Cox17. However, limited information is available on the upstream mechanism of Cu transport and delivery to mitochondria, especially through Cu-impermeable membranes, in mammalian cells. The mitochondrial phosphate transporter SLC25A3, also known as PiC2, binds Cu + and transports the ion through these membranes in eukaryotic cells, ultimately aiding in the metallation of COX. We used the well-established differentiation model of primary myoblasts derived from mouse satellite cells, wherein Cu availability is necessary for growth and maturation, and showed that PiC2 is a target of MTF1, and its expression is both induced during myogenesis and favored by Cu supplementation. PiC2 deletion using CRISPR/Cas9 showed that the transporter is required for proliferation and differentiation of primary myoblasts, as both processes are delayed upon PiC2 knock-out. The effects of PiC2 deletion were rescued by the addition of Cu to the growth medium, implying the deleterious effects of PiC2 knockout in myoblasts may be in part due to a failure to deliver sufficient Cu to the mitochondria, which can be compensated by other mitochondrial cuproproteins. Co-localization and co-immunoprecipitation of PiC2 and COX also suggest that PiC2 may participate upstream in the copper delivery chain into COX, as verified by in vitro Cu + -transfer experiments. These data indicate an important role for PiC2 in both the delivery of Cu to the mitochondria and COX, favoring the differentiation of primary myoblasts.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1984

Development of a new class of chemical and biological ultrasensors: Ribonuclease contamination and control

In order to define ribonuclease contamination, an assay for ribonuclease having picogram level sensitivity was established. In this assay, polycytidylic acid is digested by ribonuclease leading to smaller fragments of poly C that remain soluble after treatment of the sample with perchloric acid and lanthanum acetate. An absorbance measurement at 260 nm of the supernatant from the centrifuged sample measures the ribonuclease. A standard curve is shown. Using this assay procedure, ribonuclease contamination was found to be significant in routine laboratory proteins, in particular, bovine serum albumin, lysozyme, catalase, and cytochrome C. This was confirmed by demonstrating a considerable reduction in this activity in the presence of phosphate buffer since phosphate inhibits ribonuclease. Ribonuclease contamination was not significantly encountered in routine laboratory glassware, plasticware, column surfaces, chromatographic particles, and buffer reagents, including airborne contamination. Some contamination could be introduced by fingerprints, however.

Source record↗
NASA NTRS1976

Stability Characteristics of "Aerobic" Acetyl-CoA Synthetase of Yeast

During the purification of the "aerobic" acetyl-CoA synthetase (ACS) of Saccharomyces cerevisiae, strain LK2Gl2, it was noted that stronge at 4 C resulted in the loss of enzyme activity within 24 hr. Similar losses were observed during column chromatography. Addition of boiled extracts from either aerobic or anerobic cells completely prevents this. The stabilizing factor (SF) in these extracts is non-dialyzable and organic in nature. SF is excluded on G-25 and G-50 Sephadex columns and is slightly retarded on G-75 columns. On G-100 columns, SF elutes as a peak exactly coincident with that of cytochrome c, indicating a molecular weight of 13,000. SF activity was not destroyed by Pronase treatment, was adsorbed onto Norite, and absorbed in the UV with a single maximum at 260 nm. The action of SF could be replaced by a number of nucleotides. At 0.01 M, the order of effectiveness was: ATP>ADP>AMP>GTP>CTP>/=UTP>XTP. Even at 2 x 10(exp -4) M, ATP and ADP, but not AMP, cyclic AMP, adenosine or adenine, were effective in stabilizing this ACS. The mechanism of stabilization by ATP and AMP appears to be the same, since AMP competitively inhibited the ACS with respect to ATP in in vitro assays, while ADP gave a mixed type of inhibition, thus indicating a different mechanism. ACS from nonaerobic cells is also unstable in the absence of SF but, unlike aerobic ACS, is not affected by ATP or other nucleotides.

Satyanarayana, T.↗
DOE OSTI2021

Upregulation of PPPDE1 contributes to anorectal malformations via the mitochondrial apoptosis pathway during hindgut development in rats

Congenital anorectal malformations (ARMs) are among the most prominent deformities of the gastrointestinal tract; however, their precise aetiology remains obscure. Immunohistochemistry demonstrated that, in the ARM group, the PPPDE1-positive cells were widely distributed in the hindgut epithelial tissue from GD13 to GD16. Immunofluorescence revealed that most TUNEL-, Bax-, and Cytochrome C (Cyt C)-positive cells overlapped with PPPDE1-positive cells in the urorectal septum (URS). Western blotting and quantitative real-time RT-PCR revealed that PPPDE1 levels were significantly higher in the ARM group from GD13 to GD14 (p < 0.05). IEC-6 cells were transfected with PPPDE1 overexpression plasmid/NC (negative control) or si-PPPDE1/si-NC. Flow cytometry analysis and CCK-8 assay (used to detect apoptosis and proliferation, respectively), as well as western blotting, showed that the levels of PPPDE1 were positively correlated with the pro-apoptotic molecules Bax and Cyt C. Accordingly, aberrantly high expression of PPPDE1 caused a spatiotemporal imbalance in foetal rats with ARMs during hindgut development. Therefore, the upregulation of PPPDE1 may promote epithelial apoptosis and reduce proliferation in the hindgut via the mitochondrial apoptotic pathway. This could affect the fusion of the URS and cloacal membrane, ultimately inhibiting the hindgut development and resulting in ARMs.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1992

Isolation, characterization, and amino acid sequences of auracyanins, blue copper proteins from the green photosynthetic bacterium Chloroflexus aurantiacus

Three small blue copper proteins designated auracyanin A, auracyanin B-1, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus aurantiacus. All three auracyanins are peripheral membrane proteins. Auracyanin A was described previously (Trost, J. T., McManus, J. D., Freeman, J. C., Ramakrishna, B. L., and Blankenship, R. E. (1988) Biochemistry 27, 7858-7863) and is not glycosylated. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparent monomer molecular masses are 14 (A), 18 (B-2), and 22 (B-1) kDa. The amino acid sequences of the B forms are presented. All three proteins have similar absorbance, circular dichroism, and resonance Raman spectra, but the electron spin resonance signals are quite different. Laser flash photolysis kinetic analysis of the reactions of the three forms of auracyanin with lumiflavin and flavin mononucleotide semiquinones indicates that the site of electron transfer is negatively charged and has an accessibility similar to that found in other blue copper proteins. Copper analysis indicates that all three proteins contain 1 mol of copper per mol of protein. All three auracyanins exhibit a midpoint redox potential of +240 mV. Light-induced absorbance changes and electron spin resonance signals suggest that auracyanin A may play a role in photosynthetic electron transfer. Kinetic data indicate that all three proteins can donate electrons to cytochrome c-554, the electron donor to the photosynthetic reaction center.

NASA Discipline Exobiology↗
DOE OSTI2021

Aerobic Respiration and Its Regulation in the Metal Reducer Shewanella oneidensis

Shewanella oneidensis MR-1 is a facultative anaerobe known for its ability to reduce metal oxides. Anaerobic respiration, especially metal reduction, has been the subject of extensive research. In contrast, S. oneidensis aerobic respiration has received less attention. S. oneidensis expresses cbb3- and aa3-type cytochrome c oxidases and a bd-type quinol oxidase. The aa3-type oxidase, which in other bacteria is the major oxygen reductase under oxygen replete conditions, does not appear to contribute to aerobic respiration and growth in S. oneidensis. Our results indicated that although the aa3-type oxidase does not play a role in aerobic growth on lactate, the preferred carbon source for S. oneidensis, it is involved in growth on pyruvate or acetate. These results highlight the importance of testing multiple carbon and energy sources when attempting to identify enzyme activities and mutant phenotypes. Several regulatory proteins contribute to the regulation of aerobic growth in S. oneidensis including CRP and ArcA. The 3',5'-cAMP phosphodiesterase (CpdA) appears to play a more significant role in aerobic growth than either CRP or ArcA, yet the deficiency does not appear to be the result of reduced oxidase genes expression. Interestingly, the ΔcpdA mutant was more deficient in aerobic respiration with several carbon sources tested compared to Δcrp, which was moderately deficient only in the presence of lactate. To identify the reason for ΔcpdA aerobic growth deficiency, we isolated a suppressor mutant with transposon insertion in SO_3550. Inactivation of this gene, which encodes an anti-sigma factor, restored aerobic growth in the cpdA mutant to wild-type levels. Inactivation of SO_3550 in wild-type cells, however, did not affect aerobic growth. The S. oneidensis genome encodes two additional CRP-like proteins that we designated CrpB and CrpC. Mutants that lack crpB and crpC were deficient in aerobic growth, but this deficiency was not due to the loss of oxidase gene expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Single‐Cell Nanodroplet Processing Proteomics Pipeline for Analysis of Human‐Derived Microglia

Single-cell omics tools provide unique insights into heterogeneous cell populations and their responses to stimuli. For example, single-cell RNA sequencing has identified several transcriptionally distinct populations of microglia, which are resident immune cells of the central nervous system (CNS) that are responsive to CNS injury, infection, and neurodegeneration. To date, single-cell studies of microglia have focused on RNA-sequencing or cytometry by time of flight (CyTOF), which provide indirect readouts of protein abundance or quantification of a limited number of targets. Herein, we present a workflow based on FACS-assisted isolation, cryopreservation, and nanodroplet-based processing for single-cell mass spectrometry proteomics analysis of the postmortem human brain cortex-derived microglia. From a single microglial cell, 1039 proteins could be identified on average. As a proof-of-principle, we applied single-cell proteomics for exploring the heterogeneity of brain microglia at the cellular level. This pilot proteomics data partially recapitulates the prior microglia subtypes. Specifically, we determined that mitochondrial proteins, in particular members of NADH dehydrogenase (Complex I), cytochrome b-c1 (Complex III), cytochrome c oxidase (Complex IV), F1-ATPase (Complex V), and Na+/K+-ATPase complex, drive variation across microglia. This pipeline offers the potential for identifying functionally and analytically relevant protein targets for microglia in Alzheimer's disease and other neurological disorders.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Computational analysis of the tryptophan cation radical energetics in peroxidase Compound $\mathrm{I}$

Three well-characterized heme peroxidases (cytochrome c peroxidase = CCP, ascorbate peroxidase = APX, and Leishmania major peroxidase = LMP) all have a Trp residue tucked under the heme stacked against the proximal His heme ligand. The reaction of peroxidases with H 2 O 2 to give Compound I results in the oxidation of this Trp to a cationic radical in CCP and LMP but not in APX. Considerable experimental data indicate that the local electrostatic environment controls whether this Trp or the porphyrin is oxidized in Compound I. Attempts have been made to place the differences between these peroxidases on a quantitative basis using computational methods. These efforts have been somewhat limited by the approximations required owing to the computational cost of using fully solvated atomistic models with well-developed forcefields. This now has changed with available GPU computing power and the associated development of software. Here we employ thermodynamic integration and multistate Bennett acceptance ratio methods to help fine-tune our understanding on the energetic differences in Trp radical stabilization in all three peroxidases. These results indicate that the local solvent structure near the redox active Trp plays a significant role in stabilization of the cationic Trp radical.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Peptides from human BNIP5 and PXT1 and non-native binders of pro-apoptotic BAK can directly activate or inhibit BAK-mediated membrane permeabilization

Apoptosis is important for development and tissue homeostasis, and its dysregulation can lead to diseases, including cancer. As an apoptotic effector, BAK undergoes conformational changes that promote mitochondrial outer membrane disruption, leading to cell death. This is termed “activation” and can be induced by peptides from the human proteins BID, BIM, and PUMA. To identify additional peptides that can regulate BAK, we used computational protein design, yeast surface display screening, and structure-based energy scoring to identify 10 diverse new binders. We discovered peptides from the human proteins BNIP5 and PXT1 and three non-native peptides that activate BAK in liposome assays and induce cytochrome c release from mitochondria. Crystal structures and binding studies reveal a high degree of similarity among peptide activators and inhibitors, ruling out a simple function-determining property. Our results shed light on the vast peptide sequence space that can regulate BAK function and will guide the design of BAK-modulating tools and therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Anatomy of Protein Electrospray Ionization Mass Spectra by Superconducting Tunnel Junction Mass and Energy Spectrometry

Cryogenic superconducting tunnel junction (STJ) detectors have the advantage of single-particle sensitivity, high quantum efficiency, low noise, and the ability to detect the time and relative impact energy of deposited ions. This makes them attractive for use in mass spectrometry (MS) and as a form of energy spectrometry. STJ cryodetectors have been coupled to time-offlight (TOF) mass spectrometers equipped with a matrix-assisted laser desorption ionization (MALDI) source and to an electrospray ionization (ESI) TOF mass spectrometer. Here, a lab-made linear ion trap (LIT) mass spectrometer system was coupled to an ESI source and a 16-channel Nb-STJ array with improved readout electronics. The goal was to investigate fundamentals of ESI-generated protein ions by further exploiting the advantage of resolving these ions in a third dimension of the relative energy deposited into the STJs. The proteins equine cytochrome c, bovine carbonic anhydrase, bovine serum albumin, and murine immunoglobulin G were studied using this ESI-LIT-STJ-MS instrument. Multiply charged monomers, multimers, and fragments from metastable ions were resolved from monomer peaks by differences in ion deposition energy even when these ions have the same mass-to-charge ratio as the corresponding monomer. The determination of fragment mass from metastable decomposition is accomplished without knowing the charge state of the fragment. The average charge state of the multimers is reduced with each addition of a protein which is a direct reflection of the surface area available for charging. Multiply charged in-source fragments have also been observed and distinguished in the mass spectrum of carbonic anhydrase by using the differences in the energy deposited in the STJs.

42 ENGINEERING↗