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At least 199 records · Page 11
All sourcesNASA NTRSDOE OSTI
NASA NTRS2001

Lab-on-a-Chip Based Protein Crystallization

We are developing a novel technique with which we will grow protein crystals in very small volumes, utilizing chip-based, microfluidic ("LabChip") technology. This development, which is a collaborative effort between NASA's Marshall Space Flight Center and Caliper Technologies Corporation, promises a breakthrough in the field of protein crystal growth. Our initial results obtained from two model proteins, Lysozyme and Thaumatin, show that it is feasible to dispense and adequately mix protein and precipitant solutions on a nano-liter scale. The mixtures have shown crystal growth in volumes in the range of 10 nanoliters to 5 microliters. In addition, large diffraction quality crystals were obtained by this method. X-ray data from these crystals were shown to be of excellent quality. Our future efforts will include the further development of protein crystal growth with LabChip(trademark) technology for more complex systems. We will initially address the batch growth method, followed by the vapor diffusion method and the liquid-liquid diffusion method. The culmination of these chip developments is to lead to an on orbit protein crystallization facility on the International Space Station. Structural biologists will be invited to utilize the on orbit Iterative Biological Crystallization facility to grow high quality macromolecular crystals in microgravity.

vanderWoerd, Mark J.↗
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

A Model for Macromolecular Crystallization

Macromolecular crystallization is a complex process. involving a system which typically has 5 or more components (macromolecule, water, buffer + counter ion, and precipitant). Whereas small molecules have only several well defined contacts in the crystal lattice, macromolecules generally have 10's or even 100's of contacts between molecules. These can range from hydrogen bonds (direct or water-mediated), through van der Waals, hydrophobic, salt bridges, and ion-mediated contacts. The latter interactions are stronger and require some specificity in the molecular alignment, while the others are weaker, more prevalent, and more promiscuous, i.e., can often be readily broken and reformed between other sites. Formation of a consistent, ordered, 3D structure may be impossible in the absence of any or presence of too many strong interactions. Further complicating the process is the inherent structural asymmetry of monomeric single chain macromolecules. The process of crystal nucleation and growth involves the ordered assembly of growth units into a defined 3D lattice. We suggest that for many macromolecules, particularly those that are monomeric, this involves a preliminary solution-phase assembly process into a growth unit having some symmetry prior to addition to the lattice, recapitulating the initial stages of the nucleation process. If this model is correct then fluids and crystal growth models assuming a strictly monodisperse nutrient solution need to be revised. Experimental evidence, based upon face growth rate, AFM, and fluorescence energy transfer data, for a postulated model of the nucleation of tetragonal lysozyme crystals and how it transitions into crystal growth will be presented.

Pusey, Marc L.↗
NASA NTRS2002

Morphology and the Strength of Intermolecular Contact in Protein Crystals

The strengths of intermolecular contacts (macrobonds) in four lysozyme crystals were estimated based on the strengths of individual intermolecular interatomic interaction pairs. The periodic bond chain of these macrobonds accounts for the morphology of protein crystals as shown previously. Further in this paper, the surface area of contact, polar coordinate representation of contact site, Coulombic contribution on the macrobond strength, and the surface energy of the crystal have been evaluated. Comparing location of intermolecular contacts in different polymorphic crystal modifications, we show that these contacts can form a wide variety of patches on the molecular surface. The patches are located practically everywhere on this surface except for the concave active site. The contacts frequently include water molecules, with specific intermolecular hydrogen-bonds on the background of non-specific attractive interactions. The strengths of macrobonds are also compared to those of other protein complex systems. Making use of the contact strengths and taking into account bond hydration we also estimated crystal-water interfacial energies for different crystal faces.

Matsuura, Yoshiki↗
NASA NTRS2003

Hot Views on Cold Crystals: The Application of Thermal Imaging in Cryocrystallography

We have used thermal imaging techniques to visualize the cryocooling processes of macromolecular crystals. Cryocooling is a common technique used for structural data collection to reduce radiation damage in intense X-ray beams and decrease the thermal motion of the atoms. From the thermal images it was clear that during cryocooling a cold wave progresses through a crystal starting at the face closest to the origin of the cold stream and ending at the point furthest away. As an extension to this work, we used thermal imaging to study small crystals, held in a cryo-loop, in the presence of vitrified mother liquor. The different infrared transmission and reflectance properties of the crystal in comparison to the mother liquor surrounding it are thought to be the parameter that produces the contrast that makes the crystal visible. An application of this technology may be the determination of the exact location of small crystals in a cryo-loop for automated structural genomics studies. Data from initial tests in support of application development was recorded for lysozyme crystals and for bFGF/dna complex crystals, which were cryocooled and imaged in large loops, both with visible light and with infrared radiation. The crystals were clearly distinguished from the vitrified solution in the infrared spectrum, while in the case of the bFGF/dna complex the illumination had to be carefully manipulated to make the crystal visible in the visible spectrum. These results suggest that the thermal imaging may be more sensitive than visual imaging for automated location of small crystals. However, further work on small crystals robotically mounted at SSRL did not clearly visualize those crystals. The depth of field of the camera proved to be limiting and a different cooling geometry was used, compared to the previous, successful experiments. Analysis to exploit multiple images to improve depth of field and experimental work to understand cooling geometry effects is ongoing. These results will be presented along with advantages and disadvantages of the technique and a discussion of how it might be applied.

Snell, Eddie↗
NASA NTRS2003

Hot Views on Cold Crystals: The Application of Thermal Imaging in Cryocrystallography

In the past we have used thermal imaging techniques to visualize the cryocooling processes of macromolecular crystals. From these images it was clear that a cold wave progresses through a crystal starting at the face closest to the origin of the cold stream and ending at the point furthest away. During these studies we used large volume crystals, which were clearly distinguished from the loop holding them. These large crystals, originally grown for neutron diffraction studies, were chosen deliberately to enhance the imaging. As an extension to this work, we used thermal imaging to study small crystals, held in a cryo- loop, in the presence of vitrified mother liquor. The different infrared transmission and reflectance properties of the crystal in comparison to the mother liquor surrounding it are thought to be the parameter that produces the contrast that makes the crystal visible. An application of this technology may be the determination of the exact location of small crystals in a cryo-loop. Data from initial tests in support of application development was recorded for lysozyme crystals and for bFGF/dna complex crystals, which were cryo-cooled and imaged in large loops, both with visible light and with infrared radiation. The crystals were clearly distinguished from the vitrified solution in the infrared spectrum, while in the case of the bFGF/dna complex the illumination had to be carefully manipulated to make the crystal visible in the visible spectrum. These results suggest that the thermal imaging may be more sensitive than visual imaging for automated location of small crystals. However, further work on small crystals robotically mounted at SSRL did not clearly visualize those crystals. The depth of field of the camera proved to be limiting and a different cooling geometry was used, compared to the previous, successful experiments. Analysis to exploit multiple images to improve depth of field and experimental work to understand cooling geometry effects is ongoing. These results will be presented along with advantages and disadvantages of the technique and a discussion of how it might be applied .

Snell, Eddie H.↗
NASA NTRS2003

Finding the Cold Needle in a Warm Haystack: Infrared Imaging Applied to Locating Cryo-cooled Crystals in Loops

Thermally imaging the cryocooling processes of crystals has been demonstrated showing the progression of a cold wave through a crystal from the face closest to the origin of the coldstream ending at the point furthest away. During these studies large volume crystals were clearly distinguished from the loop holding them. Large volume crystals, used for neutron studies, were chosen deliberately to enhance the imaging. The different infrared transmission and reflectance properties of the crystal in comparison to the cryo-protectant are thought to be the parameter that produces the contrast making the crystal visible. As an application of the technology to locating crystals, more small crystals of lysozyme and a bFGF/dna complex were cryo-protected and imaged in large loops. The crystals were clearly distinguished from the vitrified solution. In the case of the bFGF/dna complex the illumination had to be carefully manipulated to enable the crystal to be seen in the visible spectrum. These preliminary results will be presented along with advantages and disadvantages of the technique and a discussion of how it might be applied.

Snell, Edward↗
NASA NTRS2002

Changes in the Molar Ellipticities of HEWL Observed by Circular Dichroism and Quantitated by Time Resolved Fluorescence Anisotropy Under Crystallizing Conditions

Fluid models for simple colloids predict that as the protein concentration is increased, crystallization should occur at some sufficiently high concentration regardless of the strength of attraction. However, empirical measurements do not fully support this assertion. Measurements of the second virial coefficient (B22) indicate that protein crystallization occurs only over a discrete range of solution parameters. Furthermore, observations of a strong correlation between protein solubility and B22, has led to an ongoing debate regarding the relationship between the two. Experimental work in our lab, using Hen Egg White Lysozyme (HEWL), previously revealed that the rotational anisotropy of the protein under crystallizing conditions changes systematically with pH, ionic strength and temperature. These observations are now supported by recent work revealing that small changes in the molar ellipticity also occur systematically with changes in ionic strength and temperature. This work demonstrates that under crystallization conditions, the protein native state is characterized by a conformational heterogeneity that may prove fundamental to the relationship between protein crystallization and protein solubility.

Sumida, John↗
NASA NTRS2003

Hot Views on Cold Crystals: The Application of Thermal Imaging in Cryo-crystallography

In the past we have used thermal imaging techniques to visualize the cryocooling processes of macromolecular crystals. From these images it was clear that a cold wave progresses through a crystal starting at the face closest to the origin of the cold stream and ending at the point furthest away. During these studies we used large volume crystals, which were clearly distinguished from the loop holding them. These large crystals, originally grown for neutron diffraction studies, were chosen deliberately to enhance the imaging. As an extension to this work, we present used thermal imaging to study small crystals, held in a cryo-loop, in the presence of vitrified mother liquor. The different infrared transmission and reflectance properties of the crystal in comparison to the mother liquor surrounding it are thought to be the parameter that produces the contrast that makes the crystal visible. An application of this technology may be the determination of the exact location of small crystals in a cryo-loop. Data from initial tests in support of application development was recorded for lysozyme crystals and for bFGF/dna complex crystals, which were cryo-cooled and imaged in large loops, both with visible light and with infrared radiation. The crystals were clearly distinguished from the vitrified solution in the infrared spectrum, while in the case of the bFGF/dna complex the illumination had to be carefully manipulated to make the crystal visible in the visible spectrum. These results suggest that the thermal imaging may be more sensitive than visual imaging for automated location of small crystals. However, further work on small crystals robotically mounted at SSRL did not clearly visualize those crystals. The depth of field of the camera proved to be limiting and a different cooling geometry was used, compared to the previous, successful experiments. Analysis to exploit multiple images to improve depth of field and experimental work to understand cooling geometry effects is ongoing. These results will be presented along with advantages and disadvantages of the technique and a discussion of how it might be applied.

Snell, E. H.↗
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 NTRS2003

Gibbs-Thomson Law for Singular Step Segments: Thermodynamics Versus Kinetics

Classical Burton-Cabrera-Frank theory presumes that thermal fluctuations are so fast that at any time density of kinks on a step is comparable with the reciprocal intermolecular distance, so that the step rate is about isotropic within the crystal plane. Such azimuthal isotropy is, however, often not the case: Kink density may be much lower. In particular, it was recently found on the (010) face of orthorhombic lysozyme that interkink distance may exceed 500-600 intermolecular distances. Under such conditions, Gibbs-Thomson law (GTL) may not be applicable: On a straight step segment between two corners, communication between the comers occurs exclusively by kink exchange. Annihilation between kinks of opposite sign generated at the comers results in the grain in step energy entering GTL. If the step segment length l much greater than D/v, where D and v are the kink diffusivity and propagation rate, respectively, the opposite kinks have practically no chance to annihilate and GTL is not applicable. The opposite condition of the GTL applicability, l much less than D/v, is equivalent to the requirement that relative supersaturation Delta(sub mu)/kT much less than alpha/l, where alpha is molecular size. Thus, GTL may be applied to a segment of 10(exp 3)alpha approx. 3 x 10(exp -5)cm approx 0.3 micron only if supersaturation is less than 0.1%, while practically used driving forces for crystallization are much larger. Relationships alternative to the GTL for different, but low, kink density have been discussed. They confirm experimental evidences that the Burton-Cabrera-Frank theory of spiral growth is growth rates twice as low as compared to the observed figures. Also, application of GTL results in unrealistic step energy while suggested kinetic law give reasonable figures.

Chernov, A. A.↗
NASA NTRS2003

Phase Sensitive X-Ray Diffraction Imaging Study of Protein Crystals

The study of defects and growth of protein crystals is of importance in providing a fundamental understanding of this important category of systems and the rationale for crystallization of better ordered crystals for structural determination and drug design. Yet, as a result of the extremely weak scattering power of x-rays in protein and other biological macromolecular crystals, the extinction lengths for those crystals are extremely large and, roughly speaking, of the order of millimeters on average compared to the scale of micrometers for most small molecular crystals. This has significant implication for x-ray diffraction and imaging study of protein crystals, and presents an interesting challenge to currently available x-ray analytical techniques. We proposed that coherence-based phase sensitive x-ray diffraction imaging could provide a way to augment defect contrast in x-ray diffraction images of weakly diffracting biological macromolecular crystals. I shall examine the principles and ideas behind this approach and compare it to other available x-ray topography and diffraction methods. I shall then present some recent experimental results in two model protein systems-cubic apofemtin and tetragonal lysozyme crystals to demonstrate the capability of the coherence-based imaging method in mapping point defects, dislocations, and the degree of perfection of biological macromolecular crystals with extreme sensitivity. While further work is under way, it is intended to show that the observed new features have yielded important information on protein crystal perfection and nucleation and growth mechanism otherwise unobtainable.

Hu, Z. W.↗
NASA NTRS2004

Hot Stuff? Thermal Imaging Applied to Cryocrystallography

In the past we have used thermal imaging techniques to visualize the cryocooling processes of macromolecular crystals. From these images it was clear that a cold wave progresses through a crystal starting at the face closest to the origin of the cold stream and ending at the point furthest away. During these studies we used large volume crystals, which were clearly distinguished fiom the loop holding them. These large crystals, originally grown for neutron diffiaction studies, were chosen deliberately to enhance the imaging. As an extension to this work, we present used thermal imaging to study small crystals, held in a cryo-loop, in the presence of vitrified mother liquor. The different d a r e d transmission and reflectance properties of the crystal in comparison to the mother liquor surrounding it are thought to be the parameter that produces the contrast that makes the crystal visible. An application of this technology may be the determination of the exact location of small crystals in a cryo-loop. Data fkom initial tests in support of application development was recorded for lysozyme crystals and for bFGF/dna complex crystals, which were cryocooled and imaged in large loops, both with visible light mad with h i k e d rdi&tion. The crystals were clearly distinguished from the vitrified solution in the infiared spectrum, while in the case of the bFGF/dna complex the illumination had to be carefully manipulated to make the crystal visible in the visible spectrum. These results suggest that the thermal imaging may be more sensitive than visual imaging for automated location of small crystals. However, further work on small crystals robotically mounted at SSRL did not clearly visualize those crystals. The depth of field of the camera proved to be limiting and a different cooling geometry was used, compared to the previous, successful experiments. Analysis to exploit multiple images to improve depth of field and experimental work to understand cooling geometry effects is ongoing. These results will be presented along with advantages and disadvantages of the technique and a discussion of how it might be applied.

Snell, E. H.↗
NASA NTRS2004

Solution-Phase Processes of Macromolecular Crystallization

We have proposed, for the tetragonal form of chicken egg lysozyme, that solution phase assembly processes are needed to form the growth units for crystal nucleation and growth. The starting point for the self-association process is the monomeric protein, and the final crystallographic symmetry is defined by the initial dimerization interactions of the monomers and subsequent n-mers formed, which in turn are a function of the crystallization conditions. It has been suggested that multimeric proteins generally incorporate the underlying multimers symmetry into the final crystallographic symmetry. We posed the question of what happens to a protein that is known to grow as an n-mer when it is placed in solution conditions where it is monomeric. The trypsin-treated, or cut, form of the protein canavalin (CCAN) has been shown to nucleate and grow crystals as a trimer from neutral to slightly acidic solutions. Under these conditions the solution is composed almost wholly of trimers. The insoluble protein can be readily dissolved by weakly basic solution, which results in a solution that is monomeric. There are three possible outcomes to an attempt at crystallization of the protein under monomeric (high pH) conditions: 1) we will obtain the same crystals as under trimer conditions, but at different protein concentrations governed by the self association equilibria; 2) we will obtain crystals having a different symmetry, based upon a monomeric growth unit; 3) we will not obtain crystals. Obtaining the first result would be indicative that the solution-phase self-association process is critical to the crystal nucleation and growth process. The second result would be less clear, as it may also reflect a pH-dependent shift in the trimer-trimer molecular interactions. The third result, particularly for experiments in the transition pH's between trimeric and monomeric CCAN, would indicate that the monomer does not crystallize, and that solution phase self association is not part of the crystal nucleation and growth path. Results are presented for crystallization experiments of CCAN over the pH 6.8 to 9.6 range.

Pusey, Marc L.↗
NASA NTRS2004

Diffraction and Imaging Study of Imperfections of Protein Crystals with Coherent X-rays

High angular-resolution x-ray diffraction and phase contrast x-ray imaging were combined to study defects and perfection of protein crystals. Imperfections including line defects, inclusions and other microdefects were observed in the diffraction images of a uniformly grown lysozyme crystal. The observed line defects carry distinct dislocation features running approximately along the <110> growth front and have been found to originate mostly in a central growth area and occasionally in outer growth regions. Slow dehydration led to the broadening of a fairly symmetric 4 4 0 rocking curve by a factor of approximately 2.6, which was primarily attributed to the dehydration-induced microscopic effects that are clearly shown in diffraction images. X-ray imaging and diffraction characterization of the quality of apoferritin crystals will also be discussed in the presentation.

Hu, Z. W.↗
NASA NTRS2001

Phase sensitive x-ray diffraction imaging of defects in biological macromolecular crystals

Conventional x-ray diffraction topography is currently used to map defects in the bulk of protein crystals, but the lack of sufficient contrast is frequently a limiting factor. We experimentally demonstrate that this barrier can be circumvented using a method that combines phase sensitive and diffraction imaging principles. Details of defects revealed in tetragonal lysozyme and cubic ferritin crystals are presented and discussed. The approach enabling the detection of the phase changes of diffracted x rays should prove to be useful in the study of defect structures in a broad range of biological macromolecular crystals.

Muramidase/chemistry↗
NASA NTRS2004

Steps in Solution Growth: Revised Gibbs-Thomson Law, Turbulence and Morphological Stability

Two groups of new phenomena revealed by AFM and high resolution optical interferometry on crystal faces growing from solutions will be discussed. 1. Spacing between strongly polygonized spiral steps with low less than 10(exp -2) kink density on lysozyme and K- biphtalate do not follow the Burton-cabrera-Frank theory. The critical length of the yet immobile first Short step segment adjacent to a pinning defect (dislocation, stacking fault) is many times longer than that following from the step free energy. The low-kink density steps are typical of many growth conditions and materials, including low temperature gas phase epitaxy and MBE. 2. The step bunching pattern on the approx. 1 cm long { 110) KDP face growing from the turbulent solution flow (Re (triple bonds) 10(exp 4), solution flow rate approx. 1 m/s) suggests that the step bunch height does not increase infinitely as the bunch path on the crystal face rises, as is usually observed on large KDP crystals. The mechanism controlling the maximal bunch width and height is based on the drag of the solution depleted by the step bunch down thc solution stream. It includes splitting, coagulation and interlacing of bunches

Chernov, A. A.↗
NASA NTRS1999

Antiorthostatic suspension stimulates profiles of macrophage activation in mice

The antiorthostatic suspension model simulates certain physiological effects of spaceflight. We have previously reported BDF1 mice suspended by the tail in the antiorthostatic orientation for 4 days express high levels of resistance to virulent Listeria monocytogenesinfection. In the present study, we examined whether the increased resistance to this organism correlates with profiles of macrophage activation, given the role of the macrophage in killing this pathogen in vivo. We infected BDF1 mice with a lethal dose of virulent L. monocytogenes on day 4 of antiorthostatic suspension and 24 h later constructed profiles of macrophage activation. Viable listeria could not be detected in mice suspended in the antiorthostatic orientation 24 h after infection. Flow cytometric analysis revealed the numbers of granulocytes and mononuclear phagocytes in the spleen of infected mice were not significantly altered as a result of antiorthostatic suspension. Splenocytes from antiorthostatically suspended infected mice produced increased titers of IL-1. Serum levels of neopterin, a nucleotide metabolite secreted by activated macrophages, were enhanced in mice infected during antiorthostatic suspension, but not in antiorthostatically suspended naive mice. Splenic macrophages from mice infected on day 4 of suspension produced enhanced levels of lysozyme. In contrast to the results from antiorthostatically suspended infected mice, macrophages from antiorthostatically suspended uninfected mice did not express enhanced bactericidal activities. The collective results indicate that antiorthostatic suspension can stimulate profiles of macrophage activation which correlate with increased resistance to infection by certain classes of pathogenic bacteria.

Non-NASA Center↗