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At least 1,243 records · Page 69
All sourcesNASA NTRSDOE OSTI
NASA NTRS1991

Containerless protein crystal growth method

A method of growing protein crystals from levitated drops is introduced and unique features of containerless approach in 1-g and micro-G laboratories are discussed. Electrostatic multidrop levitation system which is capable of simultaneous four drop levitation is described. A method of controlling protein saturation level in a programmed way is introduced and discussed. Finally, some of the unique features of containerless approach of protein crystal growth in space are discussed and summarized.

Rhim, Won-Kyu↗
NASA NTRS1991

Protein crystal growth in space

Studies of protein crystal growth in the microgravity environment in space are described with special attention given to the crystal growth facilities and the techniques used in Space Shuttle experiments. The properties of large space-grown crystals of gamma interferon, elastase, lathyros ochrus lectin I, and few other proteins grown on various STS flights are described. A comparison of the microgravity-grown crystals with the bast earth-grown crystals demonstrated that the space-grown crystals are more highly ordered at the molecular level than their earth-grown counterparts. When crystallization conditions were optimized, the microgravity-grown protein crystals were larger, displayed more uniform morphologies, and yielded diffraction data to significantly higher resolution than their earth-grown counterparts.

Delucas, Lawrence J.↗
NASA NTRS1992

Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

Mednieks, M. I.↗
NASA NTRS1993

Device For Controlling Crystallization Of Protein

Variable sandwich spacer enables optimization of evaporative driving force that governs crystallization of protein from solution. Mechanically more rigid than hanging-drop and sitting-drop devices. Large oscillations and dislodgment of drop of solution in response to vibrations suppressed by glass plates. Other advantages include: suitable for automated delivery, stable handling, and programmable evaporation of protein solution; controlled configuration enables simple and accurate determination of volume of solution without disrupting crystallization; pH and concentration of precipitant controlled dynamically because pH and concentration coupled to rate of evaporation, controllable via adjustment of gap between plates; and enables variation of ratio between surface area and volume of protein solution. Alternative version, plates oriented vertically instead of horizontally.

Noever, David A.↗
NASA NTRS1992

Laser scattering in a hanging drop vapor diffusion apparatus for protein crystal growth in a microgravity environment

One type of hardware used to grow protein crystals in the microgravity environment aboard the U.S. Space Shuttle is a hanging drop vapor diffusion apparatus (HDVDA). In order to optimize crystal growth conditions, dynamic control of the HDVDA is desirable. A critical component in the dynamically controlled system is a detector for protein nucleation. We have constructed a laser scattering detector for the HDVDA capable of detecting the nucleation stage. The detector was successfully tested for several scatterers differing in size using dynamic light scattering techniques. In addition, the ability to detect protein nucleation using the HDVDA was demonstrated for lysozyme.

Casay, G. A.↗
NASA NTRS1988

Intracellular proteins produced by mammalian cells in response to environmental stress

The nature of the response of mammalian cells to environmental stress is examined by reviewing results of studies where cultured mouse L cells and baby hamster kidney cells were exposed to heat shock and the synthesis of heat-shock proteins and stress-response proteins (including HSP70, HSC70, HSP90, ubiquitin, and GRP70) in stressed and unstressed cells was evaluated using 2D-PAGE. The intracellular roles of the individual stress response proteins are discussed together with the regulation of the stress response system.

Goochee, Charles F.↗
NASA NTRS1992

Advanced protein crystal growth programmatic sensitivity study

The purpose of this study is to define the costs of various APCG (Advanced Protein Crystal Growth) program options and to determine the parameters which, if changed, impact the costs and goals of the programs and to what extent. This was accomplished by developing and evaluating several alternate programmatic scenarios for the microgravity Advanced Protein Crystal Growth program transitioning from the present shuttle activity to the man tended Space Station to the permanently manned Space Station. These scenarios include selected variations in such sensitivity parameters as development and operational costs, schedules, technology issues, and crystal growth methods. This final report provides information that will aid in planning the Advanced Protein Crystal Growth Program.

Source record↗
NASA NTRS1993

Method for controlling protein crystallization

A method and apparatus for controlling the crystallization of protein by solvent evaporation including placing a drop of protein solution between and in contact with a pair of parallel plates and driving one of the plates toward and away from the other plate in a controlled manner to adjust the spacing between the plates is presented. The drop of solution forms a liquid cylinder having a height dependent upon the plate spacing thereby effecting the surface area available for solvent evaporation. When the spacing is close, evaporation is slow. Evaporation is increased by increasing the spacing between the plates until the breaking point of the liquid cylinder. One plate is mounted upon a fixed post while the other plate is carried by a receptacle movable relative to the post and driven by a belt driven screw drive. The temperature and humidity of the drop of protein solution are controlled by sealing the drop within the receptacle and mounting a heater and dessicant within the receptacle.

Noever, David A.↗
NASA NTRS1994

Convective flow effects on protein crystal growth

The long-term stability of the interferometric setup for the monitoring of protein morphologies has been improved. Growth or dissolution of a crystal on a 100 A scale can now be clearly distinguished from dimensional changes occurring within the optical path of the interferometer. This capability of simultaneously monitoring the local interfacial displacement at several widely-spaced positions on the crystal surface with high local depth resolution, has already yielded novel results. We found with lysozyme that (1) the normal growth rate is oscillatory, and (2) the mean growth step density is greater at the periphery of a facet than in its center. The repartitioning of Na(+) and Cl(-) 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 a unified way. The results strongly suggests that (1) the ion to lysozyme ratio in the crystal depends mostly on kinetic rather than crystallographic parameters, and (2) lysozyme crystals possess a salt-rich core with a diameter on the order of 10 microns. The computational model for diffusive-convective transport in protein crystallization (see the First Report) has been applied to a realistic growth cell geometry, taking into account the findings of the above repartitioning studies. These results show that some elements of a moving boundary problem must be incorporated into the model in order to obtain a more realistic description. Our experimental setup for light scattering investigations of aggregation and nucleation in protein solutions has been extensively tested. Scattering intensity measurements with a true Rayleigh scatterer produced systematically increased forward scattering, indicating problems with glare. These have been resolved. Preliminary measurements with supersaturated lysozyme solutions revealed that the scatterers grow with time. Work has begun on a computer program for the unified evaluation of simultaneously obtained, multi-angle static and dynamic light scattering data.

Rosenberger, Franz↗
NASA NTRS1994

Protein crystal growth results from the United States Microgravity Laboratory-1 mission

Protein crystal growth experiments have been performed by this laboratory on 18 Space Shuttle missions since April, 1985. In addition, a number of microgravity experiments also have been performed and reported by other investigators. These Space Shuttle missions have been used to grow crystals of a variety of proteins using vapor diffusion, liquid diffusion, and temperature-induced crystallization techniques. The United States Microgravity Laboratory - 1 mission (USML-1, June 25 - July 9, 1992) was a Spacelab mission dedicated to experiments involved in materials processing. New protein crystal growth hardware was developed to allow in orbit examination of initial crystal growth results, the knowledge from which was used on subsequent days to prepare new crystal growth experiments. In addition, new seeding hardware and techniques were tested as well as techniques that would prepare crystals for analysis by x-ray diffraction, a capability projected for the planned Space Station. Hardware that was specifically developed for the USML-1 mission will be discussed along with the experimental results from this mission.

Delucas, Lawrence J.↗
NASA NTRS1995

Convective flow effects on protein crystal growth

During the fourth semi-annual period under this grant we have pursued the following activities: (1) crystal growth morphology and kinetics studies with tetragonal lysozyme. These clearly revealed the influence of higher molecular weight protein impurities on interface shape; (2) characterization of the purity and further purification of lysozyme solutions. These efforts have, for the first time, resulted in lysozyme free of higher molecular weight components; (3) continuation of the salt repartitioning studies with Seikagaku lysozyme, which has a lower protein impurity content that Sigma stock. These efforts confirmed our earlier findings of higher salt contents in smaller crystals. However, less salt is in corporated into the crystals grown from Seikagaku stock. This strongly suggests a dependence of salt repartitioning on the concentration of protein impurities in lysozyme. To test this hypothesis, repartitioning studies with the high purity lysozyme prepared in-house will be begun shortly; (4) numerical modelling of the interaction between bulk transport and interface kinetics. These simulations have produced interface shapes which are in good agreement with out experimental observations; and (5) light scattering studies on under- and supersaturated lysozyme solutions. A consistent interpretation of the static and dynamic data leaves little doubt that pre-nucleation clusters, claimed to exist even in undersaturated solutions, are not present. The article: 'Growth morphology response to nutrient and impurity nonuniformities' is attached.

Rosenberger, Franz↗
NASA NTRS1994

Immunolocalization of an annexin-like protein in corn

Although calcium has been proposed to be an important regulatory element in plant gravitropic growth, as yet no specific function of Ca2(+) in growth regulation has been discovered. Our recent studies on a Ca2(+)-binding protein in pea seedlings called p35 indicate that it is a member of the annexin family of proteins and may play a key role in growth regulation through its function in delivering polysaccharides needed for wall construction. We previously reported the isolation of p35 from pea plumules and the production of polyclonal antibodies to it. Immunolocalization analyses of p35 in pea tissues revealed high levels of staining in secretory cell types such as developing vascular cells and outer root cap cells. To test how general was the occurrence and distribution of this annexin-like protein in plant cells we initiated an analysis of annexins in the monocot corn using immunological techniques. Our results indicate the immunochemical properties and localization of corn annexins are very similar to those reported for pea. They are consistent with the postulate that annexins may play a general role in the regulation of the secretion of wall polysaccharides needed for growth, and thus could be an important target of calcium action during gravitropic growth.

Clark, G. B.↗
NASA NTRS1994

Protein crystal growth in microgravity: Temperature induced large scale crystallization of insulin

One of the major stumbling blocks that prevents rapid structure determination using x-ray crystallography is macro-molecular crystal growth. There are many examples where crystallization takes longer than structure determination. In some cases, it is impossible to grow useful crystals on earth. Recent experiments conducted in conjuction with NASA on various Space Shuttle missions have demonstrated that protein crystals often grow larger and display better internal molecular order than their earth-grown counterparts. This paper reports results from three Shuttle flights using the Protein Crystallization Facility (PCF). The PCF hardware produced large, high-quality insulin crystals by using a temperature change as the sole means to affect protein solubility and thus, crystallization. The facility consists of cylinders/containers with volumes of 500, 200, 100, and 50 ml. Data from the three Shuttle flights demonstrated that larger, higher resolution crystals (as evidenced by x-ray diffraction data) were obtained from the microgravity experiments when compared to earth-grown crystals.

Long, Marianna M.↗
NASA NTRS1996

Protein crystallization studies

The Structural Biology laboratory at NASA Marshall Spaceflight Center uses x-ray crystallographic techniques to conduct research into the three-dimensional structure of a wide variety of proteins. A major effort in the laboratory involves an ongoing study of human serum albumin (the principal protein in human plasma) and its interaction with various endogenous substances and pharmaceutical agents. Another focus is on antigenic and functional proteins from several pathogenic organisms including the human immunodeficiency virus (HIV) and the widespread parasitic genus, Schistosoma. My efforts this summer have been twofold: first, to identify clinically significant drug interactions involving albumin binding displacement and to initiate studies of the three-dimensional structure of albumin complexed with these agents, and secondly, to establish collaborative efforts to extend the lab's work on human pathogens.

Lyne, James Evans↗
NASA NTRS1997

Nucleation and Convection Effects in Protein Crystal Growth

Work during the second year under this grant (NAG8-1161) resulted in several major achievements. We have characterized protein impurities as well as microheterogeneities in the proteins hen egg white lysozyme and horse spleen apoferritin, and demonstrated the effects of these impurities on nucleation and crystallization. In particular, the purification of apoferritin resulted in crystals with an X-ray diffraction resolution of better than 1.8 A, i.e. a 1 A improvement over earlier work on the cubic form. Furthermore, we have shown, in association with studies of liquid-liquid phase separation, that depending on the growth conditions, lysozyme can produce all growth morphologies that have been observed with other proteins. Finally, in connection with our experimental and simulation work on growth step bunching, we have developed a system-dependent criterion for advantages and disadvantages of crystallization from solution under reduced gravity. In the following, these efforts are described in some detail.

Rosenberger, Franz↗
NASA NTRS1987

Early Fluid and Protein Shifts in Men During Water Immersion

High precision blood and plasma densitometry was used to measure transvascular fluid shifts during water immersion to the neck. Six men (28-49 years) undertook 30 min of standing immersion in water at 35.0 +/- 0.2 C; immersion was preceded by 30 min control standing in air at 28 +/- 1 C. Blood was sampled from an antecubital catheter for determination of Blood Density (BD), Plasma Density (PD), Haematocrit (Ht), total Plasma Protein Concentration (PPC), and Plasma Albumin Concentration (PAC). Compared to control, significant decreases (p less than 0.01) in all these measures were observed after 20 min immersion. At 30 min, plasma volume had increased by 11.0 +/- 2.8%; the average density of the fluid shifted from extravascular fluid into the vascular compartment was 1006.3 g/l; albumin moved with the fluid and its albumin concentration was about one-third of the plasma protein concentration during early immersion. These calculations are based on the assumption that the F-cell ratio remained unchanged. No changes in erythrocyte water content during immersion were found. Thus, immersion-induced haemodilution is probably accompanied by protein (mainly albumin) augmentation which accompanies the intra-vascular fluid shift.

Hinghofer-Szalkay, H.↗
NASA NTRS1998

Advanced Protein Crystallization Facility (APCF)

This section of the Life and Microgravity Spacelab (LMS) publication contains articles entitled: (1) Crystallization of EGFR-EGF; (2) Crystallization of Apocrustacyanin C1; (3) Crystallization and X-ray Analysis of 5S rRNA and the 5S rRNA Domain A; (4) Growth of Lysozyme Crystals at Low Nucleation Density; (5) Comparative Analysis of Aspartyl tRNA-synthetase and Thaumatin Crystals Grown on Earth and In Microgravity; (6) Lysosome Crystal Growth in the Advanced Protein Crystallization Facility Monitored via Mach-Zehnder Interferometry and CCD Video; (7) Analysis of Thaumatin Crystals Grown on Earth and in Microgravity; (8) Crystallization of the Nucleosome Core Particle; (9) Crystallization of Photosystem I; (10) Mechanism of Membrane Protein Crystal Growth: Bacteriorhodopsin-mixed Micelle Packing at the Consolution Boundary, Stabilized in Microgravity; (11) Crystallization in a Microgravity Environment of CcdB, a Protein Involved in the Control of Cell Death; and (12) Crystallization of Sulfolobus Solfataricus

Source record↗
NASA NTRS1998

Determining the Molecular Packing Arrangements on Protein Crystal Faces by Atomic Force Microscopy

Periodic Bond Chain (PBC) analysis of the packing of tetragonal lysozyme crystals have revealed that there are two possible molecular packing arrangements for the crystal faces. The analysis also predicted that only one of these, involving the formation of helices about the 4(sub 3) axes, would prevail during crystal growth. In this study high resolution atomic force microscopy (AFM) was employed to verify these predictions for the (110) crystal face. A computer program was developed which constructs the expected AFM image for a given tip shape for each possible molecular packing arrangement. By comparing the actual AFM image with the predicted images the correct packing arrangement was determined. The prediction of an arrangement involving 4(sub 3) helices was confirmed in this manner,"while the alternate arrangement was not observed. The investigation also showed the protein molecules were packed slightly closer about the 4(sub 3) axes than in the crystallographic arrangement of the crystal interior. This study demonstrates a new approach for determining the molecular packing arrangements on protein crystal faces. It also shows the power of combining a theoretical PBC analysis with experimental high resolution AFM techniques in probing protein crystal growth processes at the molecular level.

Li, Huayu↗