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At least 235 records · Page 13
All sourcesNASA NTRSDOE OSTI
NASA NTRS1996

Thermal Optimization of Growth and Quality in Protein Crystals

Experimental evidence suggests that larger and higher quality crystals can be attained in the microgravity of space; however, the effect of growth rate on protein crystal quality is not well documented. This research is the first step towards providing strategies to grow crystals under constant rates of growth. Controlling growth rates at a constant value allows for direct one-to-one comparison of results obtained in microgravity and on earth. The overall goal of the project was to control supersaturation at a constant value during protein crystal growth by varying temperature in a predetermined manner. Applying appropriate theory requires knowledge of specific physicochemical properties of the protein solution including the effect of supersaturation on growth rates and the effect of temperature on protein solubility. Such measurements typically require gram quantities of protein and many months of data acquisition. A second goal of the project applied microcalorimetry for the rapid determination of these physicochemical properties using a minimum amount of protein. These two goals were successfully implemented on hen egg-white lysozyme. Results of these studies are described in the attached reprints.

Wiencek, John M.↗
NASA NTRS1997

Effects of Silver and Other Metals on the Cytoskeleton

Directly or indirectly, trace concentrations of silver ion (Ag(+)) stabilize microtubules (Conrad, A.H., et al. Cell Motil. & Cytoskel. 27:117-132), as does taxol (Conrad, A.H., et al. J. Exp. Zool. 262:154-165), an effect with major consequences for cellular shape changes and development. Polymerization of microtubules is gravity-sensitive (Tabony and Job, Proc. Natl. Acad. Sci. USA 89:6948-6952), so trace amounts of Ag(+) may alter cellular ability to respond to gravity. If Ag electrolysis is used to purify water on NASA space vehicles, plants and animals/astronauts will be exposed continuously to Ag(+), a regimen with unknown cellular and developmental consequences. Fertilized eggs of the marine mudsnail, Ilyanassa obsoleta, are the cells in which the effects of A(+) on microtubules were discovered. They distribute visible cytoplasmic contents according to gravity and contain cytoplasmic morphogenetic determinants for heart development. The objectives are to determine if the effects of Ag(+), AU(3+), (of biosensor relevance), or Gd(3+) (inhibitor of some stretch-activated ion channels) on the cytoskeleton (in the presence and absence of mechanical loading) will affect cellular responses to gravity.

Conrad, Gary W.↗
NASA NTRS1997

The polarimetric capabilities of NICMOS

The polarimetric capabilities of Near-Infrared Camera and Multi-Object Spectrometer (NICMOS) are demonstrated from data obtained during the Early Release Observations of IRC+10216 and CRL 2688 (the Egg Nebula). Preflight Thermal Vacuum tests revealed that each polarizer has a unique polarizing efficiency, and that the position angle offsets differ from the nominal positions of O deg, 120 deg and 240 deg. Therefore an algorithm different from that of an ideal polarizer is required for proper reduction of astronomical polarimetry data. We discuss this new algorithm and the results of its application to NICMOS data. We also present preliminary estimates of the Instrumental Polarization, the sensitivity of the grisms to polarized light, and the accuracy of NICMOS imaging polarimetry for faint and low polarization objects. Finally, we suggest strategies for maximizing the success of NICMOS polarimetry observations.

Hines, D. C.↗
NASA NTRS1996

Relationship Between Equilibrium Forms of Lysozyme Crystals and Precipitant Anions

Molecular forces, such as electrostatic, hydrophobic, van der Waals and steric forces, are known to be important in determining protein interactions. These forces are affected by the solution conditions and changing the pH, temperature or the ionic strength of the solution can sharply affect protein interactions. Several investigations of protein crystallization have shown that this process is also strongly dependent on solution conditions. As the ionic strength of the solution is increased, the initially soluble protein may either crystallize or form an amorphous precipitate at high ionic strengths. Studies done on the model protein hen egg white lysozyme have shown that different crystal forms can be easily and reproducibly obtained, depending primarily on the anion used to desolubilize the protein. In this study we employ pyranine to probe the effect of various anions on the water structure. Additionally, lysozyme crystallization was carried out at these conditions and the crystal form was determined by X-ray crystallography. The goal of the study was to understand the physico-chemical basis for the effect of changing the anion concentration on the equilibrium form of lysozyme crystals. It will also verify the hypothesis that the anions, by altering the bulk water structure in the crystallizing solutions, alter the surface energy of the between the crystal faces and the solution and, consequently, the equilibrium form of the crystals.

Nadarajah, Arunan↗
NASA NTRS1997

Measurement of Optical Properties of Small Particles

We have measured the optical constants of montmorillonite and the separated coats and cores of B. subtilis spores over the wavelength interval from 200 nm to 2500 nm. The optical constants of kaolin were obtained over the wavelength interval from 130 nm to 2500 nm. Our results are applicable to the development of systems for detection of airborne biological contaminants. Future work will include measurement of the optical constants of B. cereus spores, B. sub tilts vegetative cells, egg albumin, illite, and a mixture (by weight) of one third kaolin, one third montmorillonite, and one third illite.

Arakawa, E. T.↗
NASA NTRS1998

The Effects of Impurities on Protein Crystal Growth and Nucleation: A Preliminary Study

Kubota and Mullin (1995) devised a simple model to account for the effects of impurities on crystal growth of small inorganic and organic molecules in aqueous solutions. Experimentally, the relative step velocity and crystal growth of these molecules asymptotically approach zero or non-zero values with increasing concentrations of impurities. Alternatively, the step velocity and crystal growth can linearly approach zero as the impurity concentration increases. The Kubota-Mullin model assumes that the impurity exhibits Langmuirian adsorption onto the crystal surface. Decreases in step velocities and subsequent growth rates are related to the fractional coverage (theta) of the crystal surface by adsorbed impurities; theta = Kx / (I +Kx), x = mole fraction of impurity in solution. In the presence of impurities, the relative step velocity, V/Vo, and the relative growth rate of a crystal face, G/Go, are proposed to conform to the following equations: V/Vo approx. = G/Go = 1 - (alpha)(theta). The adsorption of impurity is assumed to be rapid and in quasi-equilibrium with the crystal surface sites available. When the value of alpha, an effectiveness factor, is one the growth will asymptotically approach zero with increasing concentrations of impurity. At values less than one, growth approaches a non-zero value asymptotically. When alpha is much greater than one, there will be a linear relationship between impurity concentration and growth rates. Kubota and Mullin expect alpha to decrease with increasing supersaturation and shrinking size of a two dimensional nucleus. It is expected that impurity effects on protein crystal growth will exhibit behavior similar to that of impurities in small molecule growth. A number of proteins were added to purified chicken egg white lysozyme, the effect on crystal nucleation and growth assessed.

Schall, Constance A.↗
NASA NTRS1998

Preparation and Characterization of Fluorescent Derivatives of Lysozyme

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. However, its use in macromolecular crystal growth studies is hampered by the necessity of preparing fluorescent derivatives where the probe does not markedly affect the crystal packing. Alternatively, one can prepare derivatives of limited utility if it is known that they will not affect the specific goals of a given study. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme, covalently attaching fluorescent probes to two different sites on the protein molecule. The first site is the side chain carboxyl group of ASP 101. Amine containing probes such as lucifer yellow, cascade blue, and 5- (2-aminoethyl) aminonapthalene-l-sulfonic acid (EDANS) have been attached using a carbodiimide coupling procedure. ASP 101 lies within the active site cleft, and it is believed that the probes are "buried" within that cleft. This is supported by the fact that all such derivatives have been found to crystallize, with the crystals being fluorescent. Tetragonal crystals of the lucifer yellow derivative have been found to diffract to at least 1.9 A resolution. X-ray diffraction data has been acquired and we are now working on the structure of this derivative. The second group of derivatives is to the N-terminal amine group. The derivatization reaction is performed by using a succinimidyl ester of the probe to be attached. Fluorescent probes such as pyrene acetic acid, 5-carboxyfluorescein, and Oregon green have been attached to this site. We have had little success in crystallizing these derivatives, probably because this site is part of the contact region between the 43 helix chains. However, these sites do not interfere with formation of the 43 helices and the derivatives are suitable for study of their formation in solution. The derivatives are being characterized by steady state and lifetime fluorescence methods, and the presentation will discuss these results.

Smith, Lori↗
NASA NTRS1998

The Effect of Solution Conditions on the Nucleation Kinetics of Tetragonal Lysozyme Crystals

An understanding of protein crystal nucleation rates and the effect of solution conditions upon them, is fundamental to the preparation of protein crystals of the desired size and shape for X-ray diffraction analysis. The ability to predict the effect of supersaturation, temperature, pH and precipitant concentration on the number and size of crystals formed is of great benefit in the pursuit of protein structure analysis. In this study we experimentally examine the effect of supersaturation, temperature, pH and sodium chloride concentration on the nucleation rate of tetragonal chicken egg white lysozyme crystals. In order to do this batch crystallization plates were prepared at given solution concentrations and incubated at three different temperatures over the period of one week. The number of crystals per well with their size and dimensions were recorded and correlated against solution conditions. Duplicate experiments indicate the reproducibility of the technique. Although it is well known that crystal numbers increase with increasing supersaturation, large changes in crystal number were also correlated against solution conditions of temperature, pH and salt concentration over the same supersaturation ranges. Analysis of these results enhance our understanding of the effect of solution conditions such as the dramatic effect that small changes in charge and ionic strength can have on the number of tetragonal lysozyme crystals that form and grow in solution.

Judge, Russell A.↗
NASA NTRS1998

The Feasibility of Bulk Crystallization as an Industrial Purification and Production Technique for Proteins

Bulk crystallization in stirred vessels is used industrially for the recovery and purification of many inorganic and organic materials. Although much has been written on the crystallization of proteins for X-ray diffraction analysis, very little has been reported on the application of bulk crystallization in stirred vessels. In this study, a 1-liter, seeded, stirred, batch crystallizer was used with ovalbumin as a model protein to test the feasibility of this crystallization method as a recovery and purification process for proteins. Results were obtained for ovalbumin solubility, nucleation thresholds, crystal breakage and crystal growth kinetics in bulk solution under a range of operating conditions of pH and ammonium sulphate concentration (Judge et al., 1996). Experiments were also performed to determine the degree of purification that can be achieved by the crystallization of ovalbumin from a mixture of proteins. The effect of the presence of these proteins upon the ovalbumin crystal growth kinetics was also investigated (Judge et al., 1995). All of these aspects are essential for the design of bulk crystallization processes which have not previously been reported for proteins. Results from a second study that investigated the effect of structurally different proteins on the solubility, crystal growth rates and crystal purity of chicken egg white lysozyme are also presented (Judge et al., 1997). In this case face growth rates were measured using lysozyme purified by liquid chromatography and the effect of the addition of specific protein impurities were observed on the (110) and (101) crystal faces. In these two studies the results are presented to show the feasibility and purifying ability of crystallization as a production process for proteins.

Judge, Russell A.↗
NASA NTRS1998

Ecological Support of Larval Fish During Multigenerational Studies on Space Station

Live, microscopic food is required by larval Zebrafish, Danio rerio, which are candidates for the Aquatic Habitat of the Space Station Biological Research Project (SSBRP). Zebrafish have proven to be convenient research animals, and their embryology and genetics are extensively documented. Their ability to mature at 3 months of age, and the transparent eggs which hatches in 2 days, are attractive attributes for space research. Among the goals of the SSBRP Aquatic Habitat is the ability to study three generations, with the objective of maintaining adults, their offspring, and the maintaining of these offspring through maturity and spawning. For Zebrafish, it is anticipated that sexually mature fish (PI) would be delivered to Space Station and spawned in space. The challenge would be it to provide appropriate microscopic foods for the offspring (FI), and 3 months later for the next generation (F2); if these were raised to maturity and bred, live foods would be required at approximately 6 months. In laboratories where Zebrafish are traditionally reared, the larval foods are the protozoan Parameciwn micromultinucleatwn and later brine shrimp Artemia nauplii. Under normal laboratory conditions, the rearing of these foods are relatively easy, although time consuming because of the food organisms must be separated from their rearing medium which is discarded. A freshwater food chain that would ensur-e healthy on- orbit research animals is needed. ne food chain should (a) be reared in conditions that are compatible with the larval fish (water chemistry, pH, temperature and light), (b) assist in maintaining water quality (by removing ammonia, nitrate, phosphate, carbon dioxide, and bacteria) and (c) be convenient for the space crew (minimize handling and waste production).

Taub, Frieda B.↗
NASA NTRS1999

Application of CFD to Explain Anomalous Stall Behavior of the SSME Flowmeter

The Space Shuttle Main Engine (SSME) Fuel Flowmeter is located in the duct between the low and high pressure fuel turbopumps. In the flowmeter the rotation rate of a 4-blade rotor positioned downstream of two flow straighteners is employed to measure the engine fuel flow rate and thereby control the engine mixture ratio via the engine controller. Hence, inaccurate operation of the flowmeter could have serious consequences for SSME engine operation and performance, forcing, for example, tanking of extra fuel to allow for inaccurate flowmeter measurement. Since the current flight flowmeter configuration was incorporated into the SSME in the early eighties, some anomalies in flowmeter behavior have been observed. The initial flowmeter incorporated an "egg crate" design for the two flow straighteners which turn the duct flow to make it more uniform and parallel after it has come out of the 90 degree bend just upstream of the flowmeter.

Ascoli, E.↗
NASA NTRS1997

Liquid-Liquid Phase Separation in Supersaturated Lysozyme Solutions and Associated Precipitate Formation/Crystallization

Using cloud point determinations, the phase boundaries (binodals) for metastable liquid-liquid (L-L) separation in supersaturated hen egg white lysozyme solutions with 3%, 5%, and 7% (wlv) NaCl at pH= 4.5 and protein concentrations c between 40 and 400 mg/ml were determined. The critical temperature for the binodal increased approximately linearly with salt concentration. The coexisting liquid phases both remained supersaturated but differed widely in protein concentration. No salt repartitioning was observed between the initial and the two separated liquid phases. After the L-L separation, due to the presence of the high protein concentration phase, crystallization occurred much more rapidly than in the initial solution. At high initial protein concentrations, a metastable gel phase formed at temperatures above the liquid binodal. Both crystal nucleation and gel formation were accelerated in samples that had been cycled through the binodal. Solutions in the gel and L-L regions yielded various types of precipitates. Based on theoretical considerations, previous observations with other proteins, and our experimental results with lysozyme, a generic phase diagram for globular proteins is put forth. A limited region in the (T,c) plane favorable for the growth of protein single crystals is delineated.

Muschol, Martin↗
NASA NTRS1999

Microgravity Effecs During Fertilization, Cell Division, Development, and Calcium Metabolism in Sea Urchins

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. For long-term exposure to space it is crucial to understand the underlying mechanisms for altered physiological functions. Fundamental occurrences in cell biology which are likely to depend on gravity include cytoskeletal dynamics, chromatin and centrosome cycling, and ion immobilization. These events can be studied during fertilization and embryogenesis within invertebrate systems. We have chosen the sea urchin system to study the effects of microgravity on cytoskeletal processes and calcium metabolism during fertilization, cell division, development, and embryogenesis. Experiments during an aircraft parabolic flight (KC-135) demonstrated: (1) the viability of sea urchin eggs prior to fertilization, (2) the suitability of our specimen containment system, (3) the feasibility of fertilization in a reduced gravity environment (which was achieved during 25 seconds of reduced gravity under parabolic flight conditions). Two newly developed pieces of spaceflight hardware made further investigations possible on a spaceflight (STS-77); (1) the Aquatic Research Facility (ARF), and (2) the Fertilization Syringe Unit (FSU). The Canadian Space Agency developed ARF to conduct aquatic spaceflight experiments requiring controlled conditions of temperature, humidity, illumination, and fixation at predetermined time points. It contained a control centrifuge which simulated the 1 g environment of earth during spaceflight. The FSU was developed at the Kennedy Space Center (KSC) by the Bionetics Corporation specifically to enable the crew to perform sea urchin fertilization operations in space.

Schatten, Heide↗
NASA NTRS1996

Nucleation and Crystallization of Globular Proteins: What we Know and What is Missing

Recently. much progress has been made in understanding the nucleation and crystallization of globular proteins, including the formation of compositional and structural crystal defects, Insight into the interactions of (screened) protein macro-ions in solution, obtained from light scattering, small angle X-ray scattering and osmotic pressure studies. can guide the search for crystallization conditions. These studies show that the nucleation of globular proteins is governed by the same principles as that of small molecules. However, failure to account for direct and indirect (hydrodynamic) protein interactions in the solutions results in unrealistic aggregation scenarios. Microscopic studies of numerous proteins reveal that crystals grow by the attachment of growth units through the same layer-spreading mechanisms as inorganic crystals. Investigations of the growth kinetics of hen-egg-white lysozyme (HEWL) reveal non-steady behavior under steady external conditions. Long-term variations in growth rates are due to changes in step-originating dislocation groups. Fluctuations on a shorter timescale reflect the non-linear dynamics of layer growth that results from the interplay between interfacial kinetics and bulk transport. Systematic gel electrophoretic analyses suggest that most HEWL crystallization studies have been performed with material containing other proteins at percent levels. Yet, sub-percent levels of protein impurities impede growth step propagation and play a role in the formation of structural/compositional inhomogeneities. In crystal growth from highly purified HEWL solutions, however, such inhomogeneities are much weaker and form only in response to unusually large changes in growth conditions. Equally important for connecting growth conditions to crystal perfection and diffraction resolution are recent advances in structural characterization through high-resolution Bragg reflection profiling and X-ray topography.

Rosenberger, F.↗
NASA NTRS1999

Application of CFD to Explain Anomalous Stall Behavior of the SSME Flowmeter

Anomalous behavior manifested in the apparent SSME fuel flowmeter constant which relates the rotor speed to the engine flowrate has been shown to be the result of wakes of the upstream hexagonal web flow straightener periodically stalling the rotor blades, thereby changing the lift on the blades and the rotation speed of the rotor. Moreover, an unsteady, two-dimensional computational fluid dynamics model of the flowmeter has shown this wake-induced stall disappearing as the straightener-rotor distance is doubled, in accord with the existing SSME flowmeter database for the previous "egg crate" flowmeter. These observations have led to a new flowmeter design which has been shown in three-dimensional CFD computations (consistent with both the previous two-dimensional analyses and with existing correlations for airfoil stall) to be much less susceptible to stalling instabilities.

Ascoli, E.↗
NASA NTRS1998

Locations of Bromide Ions in Tetragonal Lysozyme Crystals

Anions have been shown to play a dominant role in the crystallization of chicken egg-white lysozyme from salt solutions. Previous studies employing X-ray crystallography have found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystals grown in bromide and chloride solutions. Five possible anion-binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four further sites were found which corresponded to the four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion-binding sites in lysozyme remain unchanged even when different anions and different crystal forms of lysozyme are employed.

Lim, Kap↗
NASA NTRS1998

Location of Bromide Ions in Tetragonal Lysozyme Crystals

Anions have been shown to play a dominant role in the crystallization of chicken egg white lysozyme from salt solutions. Previous studies employing X-ray crystallography had found one chloride ion binding site in the tetragonal crystal form of the protein and four nitrate ion binding sites in the monoclinic form. In this study the anion positions in the tetragonal form were determined from the difference Fourier map obtained from lysozyme crystal grown in bromide and chloride solutions. Five possible anion binding sites were found in this manner. Some of these sites were in pockets containing basic residues while others were near neutral, but polar, residues. The sole chloride ion binding site found in previous studies was confirmed, while four of these sites corresponded to four binding sites found for nitrate ions in monoclinic crystals. The study suggests that most of the anion binding sites in lysozyme remain unchanged, even when different anions and different crystal forms of lysozyme are employed.

Lim, Kap↗
NASA NTRS1999

Fluorescence Studies of Protein Crystallization Interactions

We are investigating protein-protein interactions in under- and over-saturated crystallization solution conditions using fluorescence methods. The use of fluorescence requires fluorescent derivatives where the probe does not markedly affect the crystal packing. A number of chicken egg white lysozyme (CEWL) derivatives have been prepared, with the probes covalently attached to one of two different sites on the protein molecule; the side chain carboxyl of ASP 101, within the active site cleft, and the N-terminal amine. The ASP 101 derivatives crystallize while the N-terminal amine derivatives do not. However, the N-terminal amine is part of the contact region between adjacent 43 helix chains, and blocking this site does would not interfere with formation of these structures in solution. Preliminary FRET data have been obtained at pH 4.6, 0.1M NaAc buffer, at 5 and 7% NaCl, 4 C, using the N-terminal bound pyrene acetic acid (PAA, Ex 340 nm, Em 376 nm) and ASP 101 bound Lucifer Yellow (LY, Ex 425 nm, Em 525 nm) probe combination. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10 (exp 5) M respectively), and all experiments were carried out at approximately Csat or lower total protein concentration. The data at both salt concentrations show a consistent trend of decreasing fluorescence yield of the donor species (PAA) with increasing total protein concentration. This decrease is apparently more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations (reflected in the lower solubility). The estimated average distance between protein molecules at 5 x 10 (exp 6) M is approximately 70 nm, well beyond the range where any FRET can be expected. The calculated RO, where 50% of the donor energy is transferred to the acceptor, for the PAA-CEWL * LY-CEWL system is 3.28 nm, based upon a PAA-CEWL quantum efficiency of 0.41.

Pusey, Marc L.↗