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At least 739 records · Page 41
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Protein Factory on a Chip for Rapid Therapeutics

The potential for new, re-emerging, or engineered pandemic threats could challenge our nation's biosecurity, requiring rapid and flexible countermeasure development platforms. In healthcare, one of the main hindrances to the timely development of protein-based therapeutics for cancer, infectious disease, and other diseases has been a slow and expensive quality-controlled production stage. We present a protein-based technology that has the potential to address these crucial problems in biosecurity and healthcare by maximizing protein production and system portability, while minimizing production latency and cost. Specifically, we have developed a reusable, portable first-generation platform that leverages the advantages of cell-free protein synthesis (CFPS) and flow cells to create practical amounts of clinically relevant therapeutics. We have demonstrated that this technology is reusable and programmable, enabling a single platform that can synthesize multiple dosages of various therapeutics and vaccine in the field. Implementing this technology could mitigate pandemic threats by providing a rapidly-deployable (just-in-time), versatile (precision medicine) production of biologic therapeutics to the infected patient’s locale (point-of-care). In drug development, once promising drug variations are identified, the platform can be scaled to quickly produce practical amounts of protein for patient testing in the matter of days (vs. months) at a fraction of the cost of existing technology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Characterizing proteases in sorghum drought response using activity-based protein profiling (ABPP)

Sorghum (Sorghum bicolor) is a C4 grass species that is a major food and bioenergy crop grown worldwide. Understanding the response of sorghum to climate change is therefore critical to future sustainable food and bioenergy production. Major advancements in genome sequencing have provided a wealth of genomic data for DOE-relevant plant species, but the functions of many of these genes remain unknown. Genotype to phenotype characterization of sorghum and other bioenergy crops is needed to identify key genes responsible for plant resilience to drought and other environmental stressors. Proteases, which modulate protein degradation and cellular signaling processes by catalyzing the hydrolysis of proteins into smaller proteins, peptides, or amino acids, are highly implicated in plant drought stress responses. We developed a method for using activity-based protein profiling (ABPP) of selected protease activities (serine and cysteine proteases) to characterize drought stress responses in sorghum roots and leaves. Analysis of these ABPP data identified several known serine and cysteine proteases as well as uncharacterized sorghum proteins which may have protease activities that can be explored in future validation studies.

09 BIOMASS FUELS↗
DOE OSTI2022

Thermodynamic Constraints on Electromicrobial Protein Production

Global consumption of protein is projected to double by the middle of the 21st century. However, protein production is one of the most energy intensive and environmentally damaging parts of the food supply system today. Electromicrobial production technologies that combine renewable electricity and CO 2 -fixing microbial metabolism could dramatically increase the energy efficiency of commodity chemical production. Here we present a molecular-scale model that sets an upper limit on the performance of any organism performing electromicrobial protein production. We show that engineered microbes that fix CO 2 and N 2 using reducing equivalents produced by H 2 -oxidation or extracellular electron uptake could produce amino acids with energy inputs as low as 64 MJ kg −1 , approximately one order of magnitude higher than any previous estimate of the efficiency of electromicrobial protein production. This work provides a roadmap for development of engineered microbes that could significantly expand access to proteins produced with a low environmental footprint.

60 APPLIED LIFE 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

Implementation of a Practical Teaching Course on Protein Engineering

Protein Engineering is a highly evolved field of engineering aimed at developing proteins for specific industrial, medical, and research applications. Here, we present a practical teaching course to demonstrate fundamental techniques used to express, purify and analyze a recombinant protein produced in Escherichia coli—the enhanced green fluorescent protein (eGFP). The methodologies used for eGFP production were introduced sequentially over six laboratory sessions and included (i) bacterial growth, (ii) sonication (for cell lysis), (iii) affinity chromatography and dialysis (for eGFP purification), (iv) bicinchoninic acid (BCA) and fluorometry assays for total protein and eGFP quantification, respectively, and (v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative analysis. All groups were able to isolate the eGFP from the cell lysate with purity levels up to 72%. Additionally, a mass balance analysis performed by the students showed that eGFP yields up to 46% were achieved at the end of the purification process following the adopted procedures. A sensitivity analysis was performed to pinpoint the most critical steps of the downstream processing.

Gomes, Luciana (ORCID:0000000289921097)↗
DOE OSTI2020

Distinct Preflowering Drought Tolerance Strategies of Sorghum bicolor Genotype RTx430 Revealed by Subcellular Protein Profiling

Drought is the largest stress affecting agricultural crops, resulting in substantial reductions in yield. Plant adaptation to water stress is a complex trait involving changes in hormone signaling, physiology, and morphology. Sorghum (Sorghum bicolor (L.) Moench) is a C4 cereal grass; it is an agricultural staple, and it is particularly drought-tolerant. To better understand drought adaptation strategies, we compared the cytosolic- and organelle-enriched protein profiles of leaves from two Sorghum bicolor genotypes, RTx430 and BTx642, with differing preflowering drought tolerances after 8 weeks of growth under water limitation in the field. In agreement with previous findings, we observed significant drought-induced changes in the abundance of multiple heat shock proteins and dehydrins in both genotypes. Interestingly, our data suggest a larger genotype-specific drought response in protein profiles of organelles, while cytosolic responses are largely similar between genotypes. Organelle-enriched proteins whose abundance significantly changed exclusively in the preflowering drought-tolerant genotype RTx430 upon drought stress suggest multiple mechanisms of drought tolerance. These include an RTx430-specific change in proteins associated with ABA metabolism and signal transduction, Rubisco activation, reactive oxygen species scavenging, flowering time regulation, and epicuticular wax production. We discuss the current understanding of these processes in relation to drought tolerance and their potential implications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Targeted Quantification of Protein Phosphorylation and Its Contributions towards Mathematical Modeling of Signaling Pathways

Post-translational modifications (PTMs) are key regulatory mechanisms that can control protein function. Of these, phosphorylation is the most common and widely studied. Because of its importance in regulating cell signaling, precise and accurate measurements of protein phosphorylation across wide dynamic ranges are crucial to understanding how signaling pathways function. Although immunological assays are commonly used to detect phosphoproteins, their lack of sensitivity, specificity, and selectivity often make them unreliable for quantitative measurements of complex biological samples. Recent advances in Mass Spectrometry (MS)-based targeted proteomics have made it a more useful approach than immunoassays for studying the dynamics of protein phosphorylation. Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—and parallel reaction monitoring (PRM) can quantify relative and absolute abundances of protein phosphorylation in multiplexed fashions targeting specific pathways. In addition, the refinement of these tools by enrichment and fractionation strategies has improved measurement of phosphorylation of low-abundance proteins. The quantitative data generated are particularly useful for building and parameterizing mathematical models of complex phospho-signaling pathways. Potentially, these models can provide a framework for linking analytical measurements of clinical samples to better diagnosis and treatment of disease.

mathematical modeling↗
DOE OSTI2021

A bacterial membrane sculpting protein with BAR domain-like activity

Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here, we report a bacterial protein with BAR domain-like activity, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and influences scaffolding of OMEs into a consistent diameter and curvature. Cryo-TEM reveals that a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubules or narrow chains produced by the wild-type strain. Overexpression of BdpA promotes OME formation during planktonic growth of S. oneidensis where they are not typically observed. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli . Based on the ability of BdpA to alter membrane architecture in vivo, we propose that BdpA and its homologs comprise a newly identified class of bacterial BAR domain-like proteins.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1981

Protein synthesis rates in atrophied gastrocnemius muscles after limb immobilization

Noting that protein synthesis declines in the gastrocnemius 6 hr after immobilization, the study sought to detect an increase of protein synthesis when the limb was freed, and to examine the effects of exercise on the rate of increase. Rats were used as subjects, with their hind legs in plaster of Paris in plantar flexion to eliminate strain on the gastrocnemius. Periods of immobilization were varied and samples of blood from the muscle were taken to track protein synthesis rates for different groups in immobilization and exercise regimens (running and weightlifting). Synthesis rates declined 3.6% during time in the cast, then increased 6.3%/day after the casts were removed. Both running and weightlifting were found to increase the fractional rate of protein formation in the gastrocnemius muscle when compared with contralateral muscles that were not exercised and were used as controls, suggesting that the mechanism controlling protein synthesis in skeletal muscles is rapidly responsive to changes in muscular contractile activity.

Tucker, K. R.↗
NASA NTRS1983

Information contained in protein shapes

The sequence of local conformations at C-alpha atoms of a protein has been considered as an informational message string. The total self-information contents and self-information per letter have been evaluated for 83 globular proteins whose structures are known from X-ray crystallography. The derived information contents provide a method of quantitating structural specificity of proteins. This method of analysis enables repeating, intricate structural features to be recognized. Among the globular proteins whose structures have been solved, high potential iron protein stands out with the largest three-letter dependence.

Sundaram, K.↗
NASA NTRS1985

Changes in muscle protein composition induced by disuse atrophy - Analysis by two-dimensional electrophoresis

Using 320 g rats, a two-dimensional electrophoretic analysis of muscle proteins in the soleus and EDL muscles from hindlimbs maintained load-free for 10 days is performed. Statistical analysis of the two-dimensional patterns of control and suspended groups reveals more protein alteration in the soleus muscle, with 25 protein differences, than the EDL muscle, with 9 protein differences, as a result of atrophy. Most of the soleus differences reside in minor components. It is suggested that the EDL may also show alteration in its two-dimensional protein map, even though no significant atrophy occurred in muscle wet weight. It is cautioned that strict interpretation of data must take into account possible endocrine perturbations.

Ellis, S.↗
NASA NTRS1990

Nucleation and growth control in protein crystallization

The five topics summarized in this final report are as follows: (1) a technique for the expedient, semi-automated determination of protein solubilities as a function of temperature and application of this technique to proteins other than lysozyme; (2) a small solution cell with adjustable temperature gradients for the growth of proteins at a predetermined location through temperature programming; (3) a microscopy system with image storage and processing capability for high resolution optical studies of temperature controlled protein growth and etching kinetics; (4) growth experiments with lysozyme in thermosyphon flow ; and (5) a mathematical model for the evolution of evaporation/diffusion induced concentration gradients in the hanging drop protein crystallization technique.

Rosenberger, Franz↗
NASA NTRS1991

Cyclic AMP-receptor proteins in heart muscle of rats flown on Cosmos 1887

The cellular compartmentalization of the cyclic AMP-receptor proteins in heart ventricular tissue obtained from rats flown on the Cosmos 1887 is determined. Photoaffinity labeling of soluble and particular cell fractions with a (32P)-8-azido analog of cyclic AMP is followed by electrophoretic separation of the proteins and by autoradiographic identification of the labeled isoforms of cAPK R subunits. It is shown that RII in the particulate subcellular fraction was significantly decreased in heart cells from rats in the flight group when compared to controls. Protein banding patterns in both the cytoplasmic fraction and in a fraction enriched in chromatin-bound proteins exhibited some variability in tissues of individual animals, but showed no changes that could be directly attributed to flight conditions. No significant change was apparent in the distribution of RI or RII cyclic AMP binding in the soluble fractions. It is inferred that the cardiac cell integrity or its protein content is not compromised under flight conditions.

Mednieks, Maija I.↗
NASA NTRS1991

Use of T7 RNA polymerase to direct expression of outer Surface Protein A (OspA) from the Lyme disease Spirochete, Borrelia burgdorferi

The OspA gene from a North American strain of the Lyme disease Spirochete, Borrelia burgdorferi, was cloned under the control of transciption and translation signals from bacteriophage T7. Full-length OspA protein, a 273 amino acid (31kD) lipoprotein, is expressed poorly in Escherichia coli and is associated with the insoluble membrane fraction. In contrast, a truncated form of OspA lacking the amino-terminal signal sequence which normally would direct localization of the protein to the outer membrane is expressed at very high levels (less than or equal to 100 mg/liter) and is soluble. The truncated protein was purified to homogeneity and is being tested to see if it will be useful as an immunogen in a vaccine against Lyme disease. Circular dichroism and fluorescence spectroscopy was used to characterize the secondary structure and study conformational changes in the protein. Studies underway with other surface proteins from B burgdorferi and a related spirochete, B. hermsii, which causes relapsing fever, leads us to conclude that a strategy similar to that used to express the truncated OspA can provide a facile method for producing variations of Borrelia lipoproteins which are highly expressed in E. coli and soluble without exposure to detergents.

Dunn, John J.↗
NASA NTRS1994

Convective flow effects on protein crystal growth

A high-resolution microscopic interferometric setup for the monitoring of protein morphologies has been developed. Growth or dissolution of a crystal can be resolved with a long-term depth resolution of 200 A and a lateral resolution of 2 microns. This capability of simultaneously monitoring the interfacial displacement with high local depth resolution has yielded several novel results. We have found with lysozyme that (1) the normal growth rate is oscillatory, and (2) depending on the impurity content of the solution, the growth step density is either greater or lower at the periphery of a facet than in its center. The repartitioning of Na plus and Cl minus ions between lysozyme solutions and crystals was studied for a wide range of crystallization conditions. A nucleation-growth-repartitioning model was developed, to interpret the large body of data in unified way. The results strongly suggest that (1) the ion to lysozyne ratio in the crystal depends mostly on kinetic rather than crystallographic parameters, and (2) lysozyme crystals possess a salt-rich core with a diameter electron microscopy results appear to confirm this finding, which could have far-reaching consequences for x-ray diffraction studies. A computational model for diffusive-convective transport in protein crystallization has been applied to a realistic growth cell geometry, taking into account the findings of the above repartitioning studies and our kinetics data for the growth of lysozyme. The results show that even in the small cell employed, protein concentration nonuniformities and gravity-driven solutal convection can be significant. The calculated convection velocities are of the same order to magnitude as those found in earlier experiments. As expected, convective transport, i.e., at Og, lysozyme crystal growth remains kinetically limited. The salt distribution in the crystal is predicted to be non-uniform at both 1g and 0g, as a consequence of protein depletion in the solution. Static and dynamic light scattering studies in undersaturated and supersaturated solutions have been performed. Diffusivities in undersaturated solutions, were found to vary with lysozyme concentrations. Depending on the salt concentration, the diffusivities either increase or decrease. Interestingly, the corresponding static scattering intensities behave oppositely, Our current analysis indicates that these changes are inconsistent with aggregation in undersaturated solutions. However, the data are compatible with concentration-dependent changes of the interactions between protein and salt.

Rosenberger, Franz↗
NASA NTRS1995

Sizing of colloidal particle and protein molecules in a hanging fluid drop

We report non-invasive particle size measurements of polystyrene latex colloidal particles and bovine serum albumin (BSA) protein molecules suspended in tiny hanging fluid drops of 30 micro-Liter volume using a newly designed fiber optic probe. The probe is based upon the principles of the technique of dynamic light scattering (DLS). The motivation for this work comes from growing protein crystals in outer space. Protein crystals have been grown previously in hanging drops in microgravity experiments on-board the space shuttle orbiter. However, obtaining quantitative information on nucleation and growth of the protein crystals in real time has always been a desired goal, but hitherto not achieved. Several protein researchers have shown interest in using DLS to monitor crystal growth process in a droplet, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. We demonstrate that such an experiment is now possible. Our system offers fast (5 seconds) determination of particle size, utilize safe levels of very low laser power (less than or equal to 0.2 mW), a small scattering volume (approximately 2 x 10(exp -5) cu mm) and high spatial coherence (Beta) values. This is a major step forward when compared to currently available DLS systems.

Ansari, Rafat R.↗
NASA NTRS1997

Characterization of Proteins in Filtrate from Biodegradation of Crop Residue

Biodegradation of plant biomass is a feasible path for transformation of crop residue and recycling of nutrients for crop growth. The need to model the effects of factors associated with recycling of plant biomass resulting from hydroponic sweet potato production has led to investigation of natural soil isolates with the capacity for starch hydrolysis. This study sought to use nondenaturing gel electrophoresis to characterize the proteins present in filtered effluent from bioreactors seeded with starch hydrolyzing bacterial culture used in the biodegradation of senesced sweet potato biomass. The study determined the relative molecular weight of proteins in sampled effluent and the protein banding pattern was characterized. The protein profiles of effluent were similar for samples taken from independent runs under similar conditions of starch hydrolysis. The method can be used as a quality control tool for confirmation of starch hydrolysis of crop biomass. In addition, this method will allow monitoring for presence of contaminants within the system-protein profiles indicative of new enzymes in the bioreactors.

Horton, Wileatha↗
NASA NTRS1993

The RNA World: Life Before DNA and Protein

All of the life that is known, all organisms that exist on Earth today or are known to have existed on Earth in the past, are of the same life form: a life form based on DNA and protein. It does not necessarily have to be that way. Why not have two competing life forms on this planet? Why not have biology as we know it and some other biology that occupies its own distinct niche? Yet that is not how evolution has played out. From microbes living on the surface of antarctic ice to tube worms lying near the deep-sea hydrothermal vents, all known organisms on this planet are of the same biology. Looking at the single known biology on Earth, it is clear that this biology could not have simply sprung forth from the primordial soup. The biological system that is the basis for all known. life is far too complicated to have arisen spontaneously. This brings us to the notion that something else something simpler, must have preceded life based on DNA and protein. One suggestion that has gained considerable acceptance over the past decade is that DNA and protein-based life was preceded by RNA-based life in a period referred to as the 'RNA world'. Even an RNA-based life form would have been fairly complicated - not as complicated as our own DNA- and protein-based life form - but far too complicated, according to prevailing scientific thinking, to have arisen spontaneously from the primordial soup. Thus, it has been argued that something else must have preceded RNA-based life, or even that there was a succession of life forms leading from the primordial soup to RNA-based life. The experimental evidence to support this conjecture is not strong because, after all, the origin of life was a historical event that left no direct physical record. However, based on indirect evidence in both the geological record and the phylogenetic record of evolutionary history on earth, it is possible to reconstruct a rough picture of what life was like before DNA and protein.

Joyce, Gerald F.↗