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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1996

A Fiber Optic Probe for Monitoring Protein Aggregation, Nucleation, and Crystallization

Protein crystals are experimentally grown in hanging drops in microgravity experiments on-board the Space Shuttle orbiter. The technique of dynamic light scattering (DLS) can be used to monitor crystal growth process in hanging droplets (approx. 30 (L)) in microgravity experiments, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. In this paper we demonstrate that such experiments are now feasible. We apply a newly developed fiber optic probe to various earth and space (micro- gravity) bound protein crystallization system configurations to test its capability. These include conventional batch (cuvette or capillary) systems, hanging drop method in a six-pack hanging drop vapor diffusion apparatus (HDVDA), a modified HDVDA for temperature- induced nucleation and aggregation studies, and a newly envisioned dynamically controlled vapor diffusion system (DCVDS) configuration. Our compact system exploits the principles of DLS and offers a fast (within a few seconds) means of quantitatively and non-invasively monitoring the various growth stages of protein crystallization. In addition to DLS capability, the probe can also be used for performing single-angle static light scattering measurements. It utilizes extremely low levels of laser power (approx. few (W)) without a need of having any optical alignment and vibration isolation. The compact probe is also equipped with a miniaturized microscope for visualization of macroscopic protein crystals. This new optical diagnostic system opens up enormous opportunity for exploring new ways to grow good quality crystals suitable for x-ray crystallographic analysis and may help develop a concrete scientific basis for understanding the process of crystallization.

Ansari, Rafat R.↗
NASA NTRS1992

Size effects in models for mechanically-stressed protein crystals and aggregates

As protein aggregates increase in size, they become easier to disrupt mechanically. Using the scaling properties of models proposed to govern protein aggregation, the effect of thermal vibrations and gravity are investigated as deforming forces. For typical protein assemblies made of 30 A proteins, the assembled diameter must remain less than 100-10,000 times the molecular radius to survive in finite thermal and gravity fields. The analysis predicts the following experimental outcomes: (1) reductions in gravitational strain should favor larger protein aggregates; (2) in comparing the aggregate stability of different proteins, the addition of peptide chains should stabilize against thermal strain, but should not affect gravitational strain; (3) critical aggregate sizes should show significant (exponential) sensitivity to cluster geometry, solution preparation and growth conditions. The analysis is extended to consider qualitative size effects in crystal damage during X-ray exposure.

Noever, David A.↗
NASA NTRS1982

Protein folding, protein structure and the origin of life: Theoretical methods and solutions of dynamical problems

Theoretical methods and solutions of the dynamics of protein folding, protein aggregation, protein structure, and the origin of life are discussed. The elements of a dynamic model representing the initial stages of protein folding are presented. The calculation and experimental determination of the model parameters are discussed. The use of computer simulation for modeling protein folding is considered.

Weaver, D. L.↗
NASA NTRS2014

Protein Colloidal Aggregation Project

To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

Technology Portfolio System↗
NASA NTRS2006

Efficient Agent-Based Models for Non-Genomic Evolution

Modeling dynamical systems composed of aggregations of primitive proteins is critical to the field of astrobiological science involving early evolutionary structures and the origins of life. Unfortunately traditional non-multi-agent methods either require oversimplified models or are slow to converge to adequate solutions. This paper shows how to address these deficiencies by modeling the protein aggregations through a utility based multi-agent system. In this method each agent controls the properties of a set of proteins assigned to that agent. Some of these properties determine the dynamics of the system, such as the ability for some proteins to join or split other proteins, while additional properties determine the aggregation s fitness as a viable primitive cell. We show that over a wide range of starting conditions, there are mechanisins that allow protein aggregations to achieve high values of overall fitness. In addition through the use of agent-specific utilities that remain aligned with the overall global utility, we are able to reach these conclusions with 50 times fewer learning steps.

Gupta, Nachi↗
NASA NTRS2018

Light Microscopy Module Biophysics - 4 (LMM-B4)

Compare incorporation of protein aggregates into growing protein crystals on ISS (International Space Station) and on Earth. Measure growth rates in 1g (1 gravity) versus microgravity (micro-g) for different size aggregates of proteins. Compare the defect density and crystal quality via fluorescent-based atomic force microscopy and X-ray diffraction quality of crystals grown at different rates in a 1g environment.

Penn, Benjamin G.↗
NASA NTRS2001

TGF-beta1 release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function

BACKGROUND: Controlled release of transforming growth factor-beta1 (TGF-beta1) to a bone defect may be beneficial for the induction of a bone regeneration cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF-beta1 delivery and the effects of released TGF-beta1 on the proliferation and differentiation of marrow stromal cells in vitro. METHODS: Recombinant human TGF-beta1 was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) was co-encapsulated as a porogen. The effects of PEG content (0, 1, or 5% by weight [wt%]) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for as long as 28 days. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of released TGF-beta1 was determined after 3 days in culture. The effects of TGF-beta1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period. RESULTS: TGF-beta1 was encapsulated along with FITC-BSA into PLGA/PEG blend microparticles and released in a multiphasic fashion including an initial burst for as long as 28 days in vitro. Increasing the initial PEG content resulted in a decreased cumulative mass of released proteins. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. The degradation of PLGA was increased at higher PEG content and significantly accelerated at acidic pH conditions. Rat marrow stromal cells cultured on PPF substrates showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating that the activity of TGF-beta1 was retained during microparticle fabrication and after growth factor release. At an optimal TGF-beta1 dosage of 1.0 ng/ml after 3 days, the released TGF-beta1 enhanced the proliferation and osteoblastic differentiation of marrow stromal cells over 21 days of culture, with increased total cell number, alkaline phosphatase activity, and osteocalcin production. CONCLUSIONS: PLGA/PEG blend microparticles can serve as delivery vehicles for controlled release of TGF-beta1, and the released growth factor enhances marrow stromal cell proliferation and osteoblastic differentiation in vitro. CLINICAL RELEVANCE: Controlled release of TGF-beta1 from PLGA/PEG microparticles is representative of emerging tissue engineering technologies that may modulate cellular responses to encourage bone regeneration at a skeletal defect site.

Non-NASA Center↗
NASA NTRS2000

Applications in Bioastronautics and Bioinformatics: Early Radiation Cataracts Detected by Noninvasive, Quantitative, and Remote Means

Human exploration of Mars is a key goal in NASA's exploration planning in the next 20 years. Maintaining crew health and good vision is certainly an important aspect of achieving a successful mission. Continuous radiation exposure is a risk factor for radiation-induced cataracts in astronauts because radiation exposure in space travel has the potential of accelerating the aging process (ref. 1). A patented compact device (ref. 2) based on the technique of dynamic light scattering (DLS) was designed for monitoring an astronaut's ocular health during long-duration space travel. This capability of early diagnosis, unmatched by any other clinical technique in use today, may enable prompt initiation of preventive/curative therapy. An Internet web-based system integrating photon correlation data and controlling the hardware to monitor cataract development in vivo at a remote site in real time (teleophthalmology) is currently being developed. The new technology detects cataracts very early (at the molecular level). Cataract studies onboard the International Space Station will be helpful in quantifying any adverse effect of radiation to ocular health. The normal lens in a human eye, situated behind the cornea, is a transparent tissue. It contains 35 wt % protein and 65 wt % water. Aging, disease (e.g., diabetes), smoking, dehydration, malnutrition, and exposure to ultraviolet light and ionizing radiation can cause agglomeration of the lens proteins. Protein aggregation can take place anywhere in the lens, causing lens opacity. The aggregation and opacification could produce nuclear (central portion of the lens) or cortical (peripheral) cataracts. Nuclear and posterior subcapsular (the membrane's capsule surrounds the whole lens) cataracts, being on the visual optical axis of the eye, cause visual impairment that can finally lead to blindness. The lens proteins, in their native state, are small in size. As a cataract develops, this size grows from a few nanometers (transparent) to several micrometers (cloudy). Ansari and Datiles have shown that DLS can detect cataracts at least two to three orders of magnitude earlier noninvasively and quantitatively than the best imaging (Scheimpflug) techniques in clinical use today (ref. 3).

Ansari, Rafat R.↗
NASA NTRS1998

Modeling the Growth Rates of Tetragonal Lysozyme Crystal Faces

The measured macroscopic growth rates of the (110) and (101) faces of tetragonal lysozyme show an unexpectedly complex dependence on the supersaturation. The growth rates decay asymptotically to zero when the supersaturation is lowered to zero and increase rapidly when the supersaturation is increased. When supersaturations are increased still further the growth rates attain a maximum before starting to decrease. However, growth of these crystals is known to proceed by the classical dislocation and 2D nucleation growth mechanisms. This anomaly can be explained if growth is assumed to occur not by monomer units but by lysozyme aggregates. Analysis of the molecular packing of these crystals revealed that they were constructed of strongly bonded 4(sub 3) helices, while weaker bonds were responsible for binding the helices to each other. It follows that during crystal growth the stronger bonds are formed before the weaker ones. Thus, the growth of these crystals could be viewed as a two step process: aggregate growth units corresponding to the 4(sub 3) helix are first formed in the bulk solution by stronger intermolecular bonds and then attached to the crystal face by weaker bonds on dislocation hillocks or 2D islands. This will lead to a distribution of aggregates in the solution with monomers and lower order aggregates being predominant at low supersaturations and higher order aggregates being predominant at high supersaturations. If the crystal grows mostly by higher order aggregates, such as tetramers and octamers, it would explain the anomalous dependence of the growth rates on the supersaturation. Besides the analysis of molecular packing, a comprehensive analysis of the measured (110) and (101) growth rates was also undertaken in this study. The distribution of aggregates in lysozyme nutrient solutions at various solution conditions were determined from reversible aggregation reactions at equilibrium. The supersaturation was defined for each aggregate species with respect to its concentration at saturation in order to apply growth rate models to this process. The measured growth rates were then compared with the predicted ones from several dislocation and 2D nucleation growth models, employing tetramer and octamer growth units in polydisperse solutions and monomer units in monodisperse solutions. For the (110) face, the calculations consistently showed that the measured growth rates followed the expected model relations with octamer growth units. For the (101) face, it is not possible to obtain a clear agreement between the predicted and measured growth rates for a single growth unit as done for the (110) face. However, the calculations do indicate that the average size of the growth unit is between a tetramer and an octamer. This suggests that tetramers, octamers and other intermediate size growth units all participate in the growth process for this face. These calculations show that it is possible to model the macroscopic protein crystal growth rates if the molecular level processes can be account for, particularly protein aggregation processes in the bulk solution. Our recent investigations of tetragonal lysozyme crystals employing high resolution atomic force microscopy scans have further confirmed the growth of these crystals by aggregate growth units corresponding to 4(sub 3) helices.

Li, Meirong↗
NASA NTRS1993

Characterization of auxin-binding proteins from zucchini plasma membrane

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

NASA Program Space Biology↗
NASA NTRS2006

Stress, and pathogen response gene expression in modeled microgravity

Purpose: Immune suppression in microgravity has been well documented. With the advent of human exploration and long-term space travel, the immune system of the astronaut must be optimally maintained. It is important to investigate the expression patterns of cytokine genes, because they are directly related to immune response. Heat shock proteins (HSPs), also called stress proteins, are a group of proteins that are present in the cells of every life form. These proteins are induced when a cell responds to stressors such as heat, cold and oxygen deprivation. Microgravity is another stressor that may regulate HSPs. Heat shock proteins trigger immune response through activities that occur both inside the cell (intracellular) and outside the cell (extracellular). Knowledge about these two gene groups could lead to establishment of a blueprint of the immune response and adaptation-related genes in the microgravity environment. Methods: Human peripheral blood cells were cultured in 1g (T flask) and modeled microgravity (MMG, rotating-wall vessel) for 24 and 72 hours. Cell samples were collected and subjected to gene array analysis using the Affymetrix HG_U95 array. Data was collected and subjected to a two-way analysis of variance. The genes related to immune and stress responses were analyzed. Results and Conclusions: HSP70 was up-regulated by more than two fold in microgravity culture, while HSP90 was significantly down-regulated. HSP70 is not typically expressed in all kinds of cells, but it is expressed at high levels in stress conditions. HSP70 participates in translation, protein translocation, proteolysis and protein folding, suppressing aggregation and reactivating denatured proteins. Increased serum HSP70 levels correlate with a better outcome for heat-stroke or severe trauma patients. At the same time, elevated serum levels of HSP70 have been detected in patients with peripheral or renal vascular disease. HSP90 has been identified in the cytosol, nucleus and endoplasmic reticulum, and exists in many tissue types. HSP90 associates with actin filaments in certain conditions and aids cell motility. The down-regulation of HSP90 could lead to deleterious effects in the lymphocytes, thereby contributing to suppressed immune function in microgravity. Interleukins such as IL 1 alpha, IL11 receptor chain alpha, IL7R, and IL4R were significantly down regulated in modeled microgravity. Further analysis of the genes involved in immune response at the protein level may provide a basis for prophylactic and countermeasure strategies to augment the human immune system for space exploration.

Sundaresan, Alamelu↗
NASA NTRS2002

Peptides at Membrane Surfaces and their Role in Prebiotic Evolution

Protocells had to transport ions and organic matter across membranes separating the interior of the cell from the environment, capture and utilize energy and transduce environmental signals. In a series of detailed, molecular-level computer simulations we show how these peptides in contact with membranes can acquire ordered structures and functions. We have investigated the stability of a simple alpha-helical peptide containing Leucine (L) and Serine (S) of the form (LSLLLSL)3 in a model membrane system. The parallel in-plane state is the most stable configuration. The transmembrane state is metastable, and about 15 kcal/mol is required to insert the peptide into the membrane. We investigated dimes of both (LSLLLSL)3 and glycophorin A, and show how the free energy of helix association can, at least partially, offset the free energy of insertion. We have also investigated the transmembrane pore of the influenza M2 protein. This aggregate of four identical alpha-helices, each built of 25 amino acids, forms an efficient and selective voltage-gated proton channel. Our simulations explain the gating mechanism, which can involve strands of hydrogen-bonded water through the pore or proton transfer through tautomerization of protein residues. The channel can be re-engineered to act as a simple proton pump.

Pohorille, Andrew↗
NASA NTRS1981

Origins of the protein synthesis cycle

Largely derived from experiments in molecular evolution, a theory of protein synthesis cycles has been constructed. The sequence begins with ordered thermal proteins resulting from the self-sequencing of mixed amino acids. Ordered thermal proteins then aggregate to cell-like structures. When they contained proteinoids sufficiently rich in lysine, the structures were able to synthesize offspring peptides. Since lysine-rich proteinoid (LRP) also catalyzes the polymerization of nucleoside triphosphate to polynucleotides, the same microspheres containing LRP could have synthesized both original cellular proteins and cellular nucleic acids. The LRP within protocells would have provided proximity advantageous for the origin and evolution of the genetic code.

Fox, S. W.↗
NASA NTRS1980

Physical chemistry and evolution of salt tolerance in halobacteria

The cellular constituents of extremely halophilic bacteria not only tolerate high salt concentration, but in many cases require it for optical functioning. The characteristics affected by salt include enzyme activity, stability, allosteric regulation, conformation and subunit association. The salt effects are of two major kinds: electrostatic shielding of negative charges by cations at low salt concentration, and hydrophobic stabilization by salting-out type salts at high salt concentration. The composition of halobacterial proteins shows an excess of acidic amino acids and a deficiency of nonpolar amino acids, which accounts for these effects. Since the cohesive forces are weaker and the repulsing forces are stronger in these proteins, preventing aggregation in salt, these structures are no longer suited for functioning in the absence of high salt concentrations. Unlike these nonspecific effects, ribosomes in halobacteria show marked preference for potassium over sodium ions. To ensure the proper intracellular ionic composition, powerful ion transport systems have evolved in the halobacteria, resulting in the extrusion of sodium ions and their replacement by potassium. It is likely that such membrane transport system for ionic movements is a necessary requisite for salt tolerance.

Lanyi, J. K.↗
NASA NTRS2002

Peptides at Membrane Surfaces and their Role in the Origin of Life

All ancestors of contemporary cells (protocells) had to transport ions and organic matter across membranous walls, capture and utilize energy and transduce environmental signals. In modern organisms, all these functions are preformed by membrane proteins. We make the parsimonious assumption that in the protobiological milieu the same functions were carried out by their simple analogs - peptides. This, however, required that simple peptides could self-organize into ordered, functional structures. In a series of detailed, molecular-level computer simulations we demonstrated how this is possible. One example is the peptide (LSLLLSL)3 which forms a trameric bundle capable of transporting protons across membranes. Another example is the transmembrane pore of the influenza M2 protein. This aggregate of four identical alpha-helices, each built of 25 amino acids, forms an efficient and selective voltage-gated proton channel. Our simulations explain the gating mechanism in this channel. The channel can be re-engineered into a simple proton pump.

Pohorille, Andrew↗
NASA NTRSDate not supplied

A Brief Review of Chaotropic vs. Kosmotropic Salts on the Degree of Saturation of Membrane Fatty Acids: Implications for Preservation of Environmental Biomarkers

Water activity is well known to have a significant impact on habitability, due to the presence of salts. However, salt composition is also important for habitability, as it can impact microbial activity and biomolecular interactions since certain salts are known to disrupt hydrogen bonding. Chaotropic agents disrupt hydrogen bonding in aqueous solutions leading to increased entropy (chaos) whereas kosmotropic agents are compounds that promote hydrogen bonding (order). The Hofmeister series (Fig. 1) shows the ordering of ions in terms of their ability to stabilize and induce precipitation or aggregation of proteins.

M B Wilhelm↗
NASA NTRS2005

Chemical colloids versus biological colloids: a comparative study for the elucidation of the mechanism of protein fiber formation

Fiber formation from murine serum amyloid A1 (SAA) was compared to the linear aggregation and fiber formation of colloidal gold particles. Here we report the similarities of these processes. Upon incubation with acetic acid, SAA misfolds and adopts a new conformation, which we termed saa. saa apparently is less soluble than SAA in aqueous solution; it aggregates and forms nucleation units and then fibers. The fibers appear as a string of the nucleation units. Additionally, an external electric field promotes saa fiber formation. These properties of saa are reminiscent of colloidal gold formation from gold ions and one-dimensional aggregation of the gold colloids. Colloidal gold particles were also found to be capable of aggregating one-dimensionally under an electric field or in the presence of polylysine. These gold fibers resembled in structure that of saa fibers. In summary, protein aggregation and formation of fibers appear to follow the generalized principles derived in colloidal science for the aggregation of atoms and molecules, including polymers such as polypeptides. The analysis of colloidal gold formation and of one-dimensional aggregation provides a simple model system for the elucidation of some aspects of protein fiber formation.

Colloids↗