Engineering topics

Judge, Russell A.

Publications and source records attributed to Judge, Russell A..

At least 19 records

NASA NTRS2006

A Quasi-Laue Neutron Crystallographic Study of D-Xylose Isomerase

Hydrogen atom location and hydrogen bonding interaction determination are often critical to explain enzymatic mechanism. Whilst it is difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (less than 1.0 Angstrom) resolution data available, neutron crystallography provides an experimental tool to directly localise hydrogeddeuteriwn atoms in biological macromolecules at resolution of 1.5-2.0 Angstroms. Linearisation and isomerisation of xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data were collected on Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism (Myles et al. 1998). The neutron structure unambiguously reveals the protonation state of His 53 in the active site, identifying the model for the enzymatic pathway.

Meilleur, Flora↗

NASA NTRS2005

Extracting trends from two decades of microgravity macromolecular crystallization history

Since the 1980s hundreds of macromolecular crystal growth experiments have been performed in the reduced acceleration environment of an orbiting spacecraft. Significant enhancements in structural knowledge have resulted from X-ray diffraction of the crystals grown. Similarly, many samples have shown no improvement or degradation in comparison to those grown on the ground. A complex series of interrelated factors affect these experiments and by building a comprehensive archive of the results it was aimed to identify factors that result in success and those that result in failure. Specifically, it was found that dedicated microgravity missions increase the chance of success when compared with those where crystallization took place as a parasitic aspect of the mission. It was also found that the chance of success could not be predicted based on any discernible property of the macromolecule available to us.

Review↗

NASA NTRS2003

Crystal Growth Rate Dispersion: A Predictor of Crystal Quality in Microgravity?

In theory macromolecular crystals grow through a process involving at least two transport phenomena of solute to the crystal surface: diffusion and convection. In absence of standard gravitational forces, the ratio of these two phenomena can change and explain why crystal growth in microgravity is different from that on Earth. Experimental evidence clearly shows, however, that crystal growth of various systems is not equally sensitive to reduction in gravitational forces, leading to quality improvement in microgravity for some crystals but not for others. We hypothesize that the differences in final crystal quality are related to crystal growth rate dispersion. If growth rate dispersion exists on Earth, decreases in microgravity, and coincides with crystal quality improvements then this dispersion is a predictor for crystal quality improvement. In order to test this hypothesis, we will measure growth rate dispersion both in microgravity and on Earth and will correlate the data with previously established data on crystal quality differences for the two environments. We present here the first crystal growth rate measurement data for three proteins (lysozyme, xylose isomerase and human recombinant insulin), collected on Earth, using hardware identical to the hardware to be used in microgravity and show how these data correlate with crystal quality improvements established in microgravity.

Kephart, Richard D.↗

NASA NTRS2002

Decades of Data: Extracting Trends from Microgravity Crystallization History

The reduced acceleration environment of an orbiting spacecraft has been posited as an ideal environment for biological crystal growth since buoyancy driven convection and sedimentation are greatly reduced. Since the first sounding rocket flight in 1981 many crystallization experiments have flown with some showing improvement and others not. To further explore macromolecule crystal improvement in microgravity we have accumulated data from published reports and reports submitted by individual investigators to NASA, forming a database called BIOSEArCH (Biological Space Experiment Archive of Crystallization History). To date it contains information from 63 missions including, the Space Shuttle program, unmanned satellites, the Russian Space Station MIR and sounding rocket experiments, containing reports for more than 736 macromolecule experiments. While it is not at this point in time a comprehensive record of all flight crystallization experimental results, there is however sufficient information for emerging trends to be identified. These trends will be highlighted.

Judge, Russell A.↗

NASA NTRS2001

Delta L: An Apparatus for Measuring Macromolecule Crystal Growth Rates in Microgravity

Strongly diffracting high quality macromolecule crystals of suitable volume are keenly sought for X-ray diffraction analysis so that high-resolution molecular structure data can be obtained. Such data is of tremendous value to medical research, agriculture and commercial biotechnology. In previous studies by many investigators microgravity has been reported in some instances to improve biological macromolecule X-ray crystal quality while little or no improvement was observed in other cases. A better understanding of processes effecting crystal quality improvement in microgravity will therefore be of great benefit in optimizing crystallization success in microgravity. In ground based research with the protein lysozyme we have previously shown that a population of crystals grown under the same solution conditions, exhibit a variation in X-ray diffraction properties (Judge et al., 1999). We have also observed that under the same solution conditions, individual crystals will grow at slightly different growth rates. This phenomenon is called growth rate dispersion. For small molecule materials growth rate dispersion has been directly related to crystal quality (Cunningham et al., 1991; Ristic et al., 1991). We therefore postulate that microgravity may act to improve crystal quality by reducing growth rate dispersion. If this is the case then as different, Materials exhibit different degrees of growth rate dispersion on the ground then growth rate dispersion could be used to screen which materials may benefit the most from microgravity crystallization. In order to assess this theory the Delta L hardware is being developed so that macromolecule crystal growth rates can be measured in microgravity. Crystal growth rate is defined as the change or delta in crystal size (defined as a characteristic length, L) over time; hence the name of the hardware. Delta L will consist of an optics, a fluids, and a data acquisition sub-assemblies. The optics assembly will consist of a video microscope camera mounted on three axis computer controlled translation stages. The fluids assembly consists of macromolecule and precipitant reservoirs, a temperature controlled growth cell and waste container, The data acquisition is achieved by using a frame-gabber, with images being stored on a hard drive. In operation, macromolecule and precipitant solution will be injected into the temperature controlled growth cell. As macromolecule crystals grow, the video microscope camera controlled by the translation stages, will be used to locate and record images of individual crystals, returning to the same crystals at specific time intervals. The images will be stored on the hard drive and used to calculate the crystal growth rate. To prevent vibrations interfering in the crystal growth rate measurements (Snell et al., 1997) Delta L will be used in connection with the Glovebox Integrated Microgravity Isolation Technology (g-LIMIT) inside the Microgravity Science Glovebox (MSG), onboard the International Space Station (ISS).

Judge, Russell A.↗

NASA NTRS2001

Investigating the Effect of Impurities on Macromolecule Crystal Growth in Microgravity

Chicken egg-white lysozyme (CEWL) crystals were grown in microgravity and on the ground in the presence of various amounts of a naturally occurring lysozyme dimer impurity. No significant favorable differences in impurity incorporation between microgravity and ground crystal samples were observed. At low impurity concentration the microgravity crystals preferentially incorporated the dimer. The presence of the dimer in the crystallization solutions in microgravity reduced crystal size, increased mosaicity and reduced the signal to noise ratio of the X-ray data. Microgravity samples proved more sensitive to impurity. Accurate indexing of the reflections proved critical to the X-ray analysis. The largest crystals with the best X-ray diffraction properties were grown from pure solution in microgravity.

Snell, Edward H.↗

NASA NTRS2001

Quantifying Main Trends in Lysozyme Nucleation: The Effects of Precipitant Concentration, Supersaturation and Impurities

Full factorial experimental design incorporating multi-linear regression analysis of the experimental data allows quick identification of main trends and effects using a limited number of experiments. In this study these techniques were employed to identify the effect of precipitant concentration, supersaturation, and the presence of an impurity, the physiological lysozyme dimer, on the nucleation rate and crystal dimensions of the tetragonal forin of chicken egg white lysozyme. Decreasing precipitant concentration, increasing supers aturation, and increasing impurity, were found to increase crystal numbers. The crystal axial ratio decreased with increasing precipitant concentration, independent of impurity.

Burke, Michael W.↗

NASA NTRS2001

The Effect of Solution Thermal History on Chicken Egg White Lysozyme Nucleation

Proteins are highly flexible molecules and often exhibit defined conformational changes in response to changes in the ambient temperature. Chicken egg white lysozyme has been previously shown to undergo an apparent structural change when warmed above the tetragonal/orthorhombic phase transition temperature. This is reflected by a change in the habit of the tetragonal and orthorhombic crystals so formed. In this study, we show that possible conformational changes induced by heating are stable and apparently non-reversible by simple cooling. Exposure of protein solutions to temperatures above the phase change transition temperature, before combining with precipitant solution to begin crystallization, reduces final crystal numbers. Protein that is briefly warmed to 37 C, then cooled shows no sign of reversal to the unheated nucleation behavior even after storage for four weeks at 4 C. The change in nucleation behavior of tetragonal lysozyme crystals, attributed to a structural shift, occurs faster the greater the exposure to temperature above the equi-solubility point for the two phases. Heating for 2 hours at 48 C reduces crystal numbers by 20 fold in comparison to the same solution heated for the same time at 30 C. Thermal treatment of solutions is therefore a possible tool to reduce crystal numbers and increase crystal size. The effects of a protein's previous thermal history are now shown to be a potentially critical factor in subsequent macromolecule crystal nucleation and growth studies.

Burke, Michael W.↗

NASA NTRS2001

Free-falling Crystals: Biological Macromolecular Crystal Growth Studies in Low Earth Orbit

Spacecraft orbiting the earth experience a reduced acceleration environment due to being in a state of continuous free-fall. This state colloquially termed microgravity, has produced improved X-ray diffraction quality crystals of biological macromolecules. Improvements in X-ray diffraction resolution (detail) or signal to noise, provide greater detail in the three-dimensional molecular structure providing information about the molecule, how it works, how to improve its function or how to impede it. Greater molecular detail obtained by crystallization in microgravity, has important implications for structural biology. In this article we examine the theories behind macromolecule crystal quality improvement in microgravity using results obtained from studies with the model protein, chicken egg white lysozyme.

Judge, Russell A.↗

NASA NTRS2001

Delta L: An Apparatus for Measuring Macromolecular Crystal Growth Rates in Microgravity

In order to determine how macromolecule crystal quality improvement in microgravity is related to crystal growth characteristics, is was necessary to develop new hardware that could measure the crystal growth rates of a population of crystals growing under the same solution conditions. As crystal growth rate is defined as the change or delta in a defined dimension or length (L) of a crystal over time, the hardware was named Delta L. Delta L consists of fluids, optics, and data acquisition, sub-assemblies. Temperature control is provided for the crystal growth chamber. Delta L will be used in connection with the Glovebox Integrated Microgravity Isolation Technology (g-LIMIT) inside the Microgravity Science Glovebox (MSG), onboard the International Space Station (ISS). Delta L prototype hardware has been assembled. This paper will describe an overview of the design of Delta L and present preliminary crystal growth rate data.

Judge, Russell A.↗

NASA NTRS2000

Fluid Physics and Macromolecular Crystal Growth in Microgravity

The first protein crystallization experiment in microgravity was launched in April, 1981 and used Germany's Technologische Experimente unter Schwerelosigkeit (TEXUS 3) sounding rocket. The protein P-galactosidase (molecular weight 465Kda) was chosen as the sample with a liquid-liquid diffusion growth method. A sliding device brought the protein, buffer and salt solution into contact when microgravity was reached. The sounding rocket gave six minutes of microgravity time with a cine camera and schlieren optics used to monitor the experiment, a single growth cell. In microgravity a strictly laminar diffusion process was observed in contrast to the turbulent convection seen on the ground. Several single crystals, approx 100micron in length, were formed in the flight which were of inferior but of comparable visual quality to those grown on the ground over several days. A second experiment using the same protocol but with solutions cooled to -8C (kept liquid with glycerol antifreeze) again showed laminar diffusion. The science of macromolecular structural crystallography involves crystallization of the macromolecule followed by use of the crystal for X-ray diffraction experiments to determine the three dimensional structure of the macromolecule. Neutron protein crystallography is employed for elucidation of H/D exchange and for improved definition of the bound solvent (D20). The structural information enables an understanding of how the molecule functions with important potential for rational drug design, improved efficiency of industrial enzymes and agricultural chemical development. The removal of turbulent convection and sedimentation in microgravity, and the assumption that higher quality crystals will be produced, has given rise to the growing number of crystallization experiments now flown. Many experiments can be flown in a small volume with simple, largely automated, equipment - an ideal combination for a microgravity experiment. The term "protein crystal growth" is often historically used to describe these microgravity experiments. This is somewhat inaccurate as the field involves the study of many varied biological molecules including viruses, proteins, DNA, RNA and complexes of those structures. For this reason we use the term macromolecular crystal growth. In this chapter we review a series of diagnostic microgravity crystal growth experiments carried out principally using the European Space Agency (ESA) Advanced Protein Crystallization Facility (APCF). We also review related research, both experimental and theoretical, on the aspects of microgravity fluid physics that affect microgravity protein crystal growth. Our experiments have revealed some surprises that were not initially expected. We discuss them here in the context of practical lessons learnt and how to maximize the limited microgravity opportunities available.

Helliwell, John R.↗

NASA NTRS2000

Macromolecule Crystal Quality Improvement in Microgravity: The Role of Impurities

While macromolecule impurities may affect crystal size and morphology the over-riding question is; "How do macromolecule impurities effect crystal X-ray quality and diffraction resolution?" In the case of chicken egg white lysozyme, crystals can be grown in the presence of a number of impurities without affecting diffraction resolution. One impurity however, the lysozyme dimer, does negatively impact the X-ray crystal properties. Crystal quality improvement as a result of better partitioning of this impurity during crystallization in microgravity has been reported'. In our recent experimental work dimer partitioning was found to be not significantly different between the two environments. Mosaicity analysis of pure crystals showed a reduced mosaicity and increased signal to noise for the microgravity grown crystals. Dimer incorporation however, did greatly reduce the resolution limit in both ground and microgravity grown crystals. These results indicate that impurity effects in microgravity are complex and may rely on the conditions or techniques employed.

Judge, Russell A.↗

NASA NTRS2000

Synchrotron X-Ray Reciprocal Space Mapping, Topography and Diffraction Resolution Studies of Macromolecular Crystal Quality

A comprehensive study of microgravity and ground grown chicken egg white lysozyme crystals is presented using synchrotron X-ray reciprocal space mapping, topography techniques and diffraction resolution. Microgravity crystals displayed, on average, reduced intrinsic mosaicities but no differences in terms of stress over their earth grown counterparts. Topographic analysis revealed that in the microgravity case the majority of the crystal was contributing to the peak of the reflection at the appropriate Bragg angle. In the earth case at the diffraction peak only a small volume of the crystal contributed to the intensity. The techniques prove to be highly complementary with the reciprocal space mapping providing a quantitative measure of the crystal mosaicity and stress (or variation in lattice spacing) and topography providing a qualitative overall assessment of the crystal in terms of its X-ray diffraction properties. Structural data collection was also carried out both at the synchrotron and in the laboratory.

Boggon, T. J.↗

NASA NTRS2000

The Question of Impurities in Macromolecule Crystal Quality Improvement in Microgravity

While macromolecule impurities may affect crystal size and morphology the over-riding question is how do macromolecule impurities effect crystal X-ray quality and diffraction resolution. In the case of chicken egg white lysozyme previous researchers have reported that crystals grown in the presence of ovalbumin, ovotransferrin, and turkey egg white lysozyme show no difference in diffraction resolution compared to those grown in pure solutions. One impurity however, a naturally occurring lysozyme dimer, does negatively impact the X-ray crystal properties. For this impurity it has been reported that crystal quality improvement in microgravity may be due to improved impurity partitioning during crystallization. In this study we have examined the incorporation of the dimer into lysozyme crystals, both on the ground and in microgravity experiments, and have performed detailed X-ray analysis of the crystals using a new technique for finely probing the mosaicity of the crystal at the Stanford Synchrotron Radiation Laboratory. Dimer partitioning was not significantly different in microgravity compared to the ground based experiments, although it is significantly better than that previously reported in microgravity. Mosaicity analysis of pure crystals, 1422 indexed reflections (microgravity) and 752 indexed reflections (ground), gave average results of 0.0066 and 0.0092 degrees (FWHM) respectively. The microgravity crystals also provided an increased signal to noise. Dimer incorporation increased the average mosaicity in microgravity but not on the ground. However, dimer incorporation did greatly reduce the resolution limit in both ground and microgravity grown crystals. The data is being treated anisotropically to explore these effects. These results indicate that impurity effects in microgravity are complex and that the conditions or techniques employed may greatly affect the role of impurities.

Judge, Russell A.↗

NASA NTRS2000

The Effect of Solution Thermal History on Chicken Egg White Lysozyme Nucleation

Proteins are highly flexible molecules and often exhibit defined conformational changes in response to changes in the ambient temperature. Chicken egg white lysozyme has been previously shown to undergo an apparent structural change when warmed above the tetragonal/orthorhombic phase transition temperature. This is reflected by a change in the habit of the tetragonal and orthorhombic crystals so formed. In this study we show that possible conformational changes induced by heating are stable and apparently non- reversible by simple cooling. Exposure of protein solutions to temperatures above the phase change transition temperature, before combining with precipitant solution to begin crystallization, reduces final crystal numbers. Protein that is briefly warmed to 37 C, then cooled shows no sign of reversal to the unheated nucleation behavior even after storage for 4 weeks at 4 C. The change in nucleation behavior of tetragonal lysozyme crystals, attributed to a structural shift, occurs faster the greater the exposure to temperature above the equi-solubility point for the two phases. Heating for 2 h at 48 C reduces crystal numbers by 20 fold in comparison to the same solution heated for the same time at 30 C. Thermal treatment of solutions is therefore a possible tool to reduce crystal numbers and increase crystal size. The effects of a protein's previous thermal history are now shown to be a potentially critical factor in subsequent macromolecule crystal nucleation and growth studies.

Burke, Michael W.↗

NASA NTRS2000

Quantifying Main Trends in Lysozyme Nucleation: The Effect of Precipitant Concentration and Impurities

Full factorial experiment design incorporating multi-linear regression analysis of the experimental data allows the main trends and effects to be quickly identified while using only a limited number of experiments. These techniques were used to identify the effect of precipitant concentration and the presence of an impurity, the physiological lysozyme dimer, on the nucleation rate and crystal dimensions of the tetragonal form of chicken egg white lysozyme. Increasing precipitant concentration was found to decrease crystal numbers, the magnitude of this effect also depending on the supersaturation. The presence of the dimer generally increased nucleation. The crystal axial ratio decreased with increasing precipitant concentration independent of impurity.

Burke, Michael W.↗

NASA NTRS2000

Microgravity and Macromolecular Crystallography

Macromolecular crystal growth has been seen as an ideal experiment to make use of the reduced acceleration environment provided by an orbiting spacecraft. The experiments are small, simply operated and have a high potential scientific and economic impact. In this review we examine the theoretical reasons why microgravity should be a beneficial environment for crystal growth and survey the history of experiments on the Space Shuttle Orbiter, on unmanned spacecraft, and on the Mir space station. Finally we outline the direction for optimizing the future use of orbiting platforms.

Kundrot, Craig E.↗

NASA NTRS1999

The Effect of Temperature and Solution pH on the Nucleation of Tetragonal Lysozyme Crystals

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.

Judge, Russell A.↗