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At least 271 records · Page 15
All sourcesNASA NTRSDOE OSTI
NASA NTRS1998

Protein Crystal Movements and Fluid Flows During Microgravity Growth

The growth of protein crystals suitable for x-ray crystal structure analysis is an important topic. The quality (perfection) of protein crystals is now being evaluated by mosaicity analysis (rocking curves) and x-ray topographic images as well as the diffraction resolution limit and overall data quality. In yet another study, use of hanging drop vapour diffusion geometry on the IML-2 shuttle mission showed, again via CCD video monitoring, growing apocrustacyanin C(sub 1) protein crystal executing near cyclic movement, reminiscent of Marangoni convection flow of fluid, the crystals serving as "markers" of the fluid flow. A review is given here of existing results and experience over several microgravity missions. Some comment is given on gel protein crystal growth in attempts to 'mimic' the benefits of microgravity on Earth. Finally, the recent new results from our experiments on the shuttle mission LMS are described. These results include CCD video as well as interferometry during the mission, followed, on return to Earth, by reciprocal space mapping at the NSLS, Brookhaven, and full X-ray data collection on LMS and Earth control lysozyme crystals. Diffraction data recorded from LMS and ground control apocrustacyanin C(sub 1) crystals are also described.

Boggon, Titus J.↗
NASA NTRS1998

Protein Crystal Growth Under Forced Solution Flow: Experimental Setup and General Response of Lysozyme

We have experimentally studied the effects of solution flow on the growth kinetics of the protein lysozyme. To this end, we have expanded our interferometry setup by a novel crystallization cell and solution recirculation system. This combination permits monitoring of interface morphology and kinetics with a depth resolution of 200 A at bulk flow rates of up to 2000 micron/s. Particular attention was paid to the prevention of protein denaturation that is often associated with the pumping of protein solutions. We found that at bulk flow rates it less than 250 microns/s the average growth rate and step velocity, R(sub avg) and upsilon(sub avg) increase with increasing it. This can be quantitatively understood in terms of the enhanced, convective solute supply to the interface. With high-purity solutions, it u greater than 250 microns/s lead to growth deceleration, and, at low supersaturations sigma, to growth cessation. When solutions containing approx. 1% of other protein impurities were used, growth deceleration occurred at any u greater than 0 and cessation in the low sigma experiments was reached at about half the it causing cessation with pure solution. The flow-induced changes in R(sub avg) and upsilon(sub avg) including growth cessation, were reversible and reproducible, independent of the direction of the u-changes and solution purity. Hence, we attribute the deceleration to the convection-enhanced supply of impurities to the interface, which at higher flow rates overpowers the effects of enhanced interfacial solute concentration. Most importantly, we found that convective transport leads to a significant reduction in kinetics fluctuations, in agreement with our earlier expectations for the lysozyme system. This supports our hypothesis that these long-term fluctuations represent an intrinsic response feature of the coupled bulk transport-interfacial kinetics system in the mixed growth control regime.

Vekilov, P. G.↗
NASA NTRS1998

Effects of Convective Solute and Impurity Transport in Protein Crystal Growth

High-resolution optical interferometry was used to investigate the effects of forced solution convection on the crystal growth kinetics of the model protein lysozyme. Most experiments were conducted with 99.99% pure protein solutions. To study impurity effects, approx. 1% of lysozyme dimer (covalently bound) was added in some cases. We show that the unsteady kinetics, corresponding to bunching of growth steps, can be characterized by the Fourier components of time traces of the growth rate. Specific Fourier spectra are uniquely determined by the solution conditions (composition, temperature, and flow rate) and the growth layer source activity. We found that the average step velocity and growth rate increase by approx. I0% with increasing flow rate, as a result of the enhanced solute supply to the interface. More importantly, faster convective transport results in lower fluctuation amplitudes. This observation supports our rationale for system-dependent effects of transport on the structural perfection of protein crystals. We also found that solution flow rates greater than 500 microns/s result in stronger fluctuations while the average growth rate is decreased. This can lead to growth cessation at low supersaturations. With the intentionally contaminated solutions, these undesirable phenomena occurred at about half the flow rates required in pure solutions. Thus, we conclude that they are due to enhanced convective supply of impurities that are incorporated into the crystal during growth. Furthermore, we found that the impurity effects are reduced at higher crystal growth rates. Since the exposure time of terraces is inversely proportional to the growth rate, this observation suggests that the increased kinetics instability results from impurity adsorption on the interface. Finally, we provide evidence relating earlier observations of "slow protein crystal growth kinetics" to step bunch formation in response to nonsteady step generation.

Vekilov, Peter G.↗
NASA NTRS1997

Protein Crystal Movements and Fluid Flows During Microgravity Growth

The growth of protein crystals suitable for X-ray crystal structure analysis is an important topic. The methods of protein crystal growth are under increasing study whereby different methods are being compared via diagnostic monitoring including Charge Coupled Device (CCD) video and interferometry. The quality (perfection) of protein crystals is now being evaluated by mosaicity analysis (rocking curves) and X-ray topographic images as well as the diffraction resolution limit and overall data quality. Choice of a liquid-liquid linear crystal growth geometry and microgravity can yield a spatial stability of growing crystals and fluid, as seen in protein crystallization experiments on the unmanned platform EURICA. A review is given here of existing results and experience over several microgravity missions. The results include CCD video as well as interferometry during the mission, followed, on return to earth, by rocking curve experiments and full X-ray data collection on LMS and earth control lysozyme crystals. Diffraction data recorded from LMS and ground control apocrustacyanin C(sub 1) crystals are also described.

Boggon, Titus J.↗
NASA NTRS1999

DNA Repair-Protein Relocalization After Heavy Ion Exposure

Ionizing radiation is good at making DNA double strand breaks, and high linear energy transfer (LET) radiations such as heavy ion particles are particularly efficient. For this reason, the proteins belonging to repair systems that deal with double strand breaks are of particular interest. One such protein is Ku, a component in the non-homologous recombination repair system. The Ku protein is an abundant, heterodimeric DNA end-binding complex, composed of one 70 and one 86 kDa subunit. Ku protein binds to DNA ends, nicks, gaps, and regions of transition between single and double-stranded structure. These binding properties suggest an important role in DNA repair. The Ku antigen is important in this study because it is present in relatively large copy numbers and it is part of a double-strand-break repair system. More importantly, we consistently measure an apparent upregulation in situ that is not verified by whole-cell-lysate immunoblot measurements. This apparent upregulation is triggered by very low doses of radiation, thus showing a potentially useful high sensitivity. However, elucidation of the mechanism underlying this phenomenon is still to be done.

Metting, N. F.↗
NASA NTRS2000

Protein Crystal Growth Dynamics and Impurity Incorporation

The general concepts and theories of crystal growth are proven to work for biomolecular crystallization. This allowed us to extract basic parameters controlling growth kinetics - free surface energy, alpha, and kinetic coefficient, beta, for steps. Surface energy per molecular site in thermal units, alpha(omega)(sup 2/3)/kT approx. = 1, is close to the one for inorganic crystals in solution (omega is the specific molecular volume, T is the temperature). Entropic restrictions on incorporation of biomolecules into the lattice reduce the incorporation rate, beta, by a factor of 10(exp 2) - 10(exp 3) relative to inorganic crystals. A dehydration barrier of approx. 18kcal/mol may explain approx. 10(exp -6) times difference between frequencies of adding a molecule to the lattice and Brownian attempts to do so. The latter was obtained from AFM measurements of step and kink growth rates on orthorhombic lysozyme. Protein and many inorganic crystals typically do not belong to the Kossel type, thus requiring a theory to account for inequivalent molecular positions within its unit cell. Orthorhombic lysozyme will serve as an example of how to develop such a theory. Factors deteriorating crystal quality - stress and strain, mosaicity, molecular disorder - will be reviewed with emphasis on impurities. Dimers in ferritin and lysozyme and acetylated lysozyme, are microheterogeneous i.e. nearly isomorphic impurities that are shown to be preferentially trapped by tetragonal lysozyme and ferritin crystals, respectively. The distribution coefficient, K defined as a ratio of the (impurity/protein) ratios in crystal and in solution is a measure of trapping. For acetylated lysoyzme, K = 2.15 or, 3.42 for differently acetylated forms, is independent of both the impurity and the crystallizing protein concentration. The reason is that impurity flux to the surface is constant while the growth rate rises with supersaturation. About 3 times lower dimer concentration in space grown ferritin and lysozyme crystals might be examples explaining higher quality of the space grown protein crystal. Depletion of solution with respect to isomorphic impurities around a growing crystal may be K times deeper than with respect to the crystallizing protein.

Chernov, Alex A.↗
NASA NTRS2001

First Protein Crystallization Experiments on The International Space Station: Sweet Success in Space With Thaumatin

We determined the room temperature 1.2 A structure of thaumatin using a crystal grown in the first protein crystallization experiment conducted aboard the International Space Station (ISS). The crystals were grown in the Enhanced Gaseous Nitrogen Dewar (EGN) developed by Alexander McPherson and co-workers. EGN transports frozen solutions contained in tygon tubing in a liquid nitrogen Dewar to ISS where the tubes then thaw. Batch, free interface diffusion (FID), or vapor diffusion crystallization occurs after thawing. EGN was flown to the ISS on STS-106 on September 8, 2000. This was a "risk mitigation" flight that tested EGN performance and the process of conducting experiments on ISS. We focused on how to map a hanging drop crystallization recipe to the EGN FID method. Thaumatin was chosen as the test system. Three series of crystallization recipes were set-up. Each series tested different volume ratios of protein-rich solution to precipitant-rich solution. The series differed from each other by fixing either the protein concentration or the amount of protein in the solutions. Upon return of the samples to Earth on October 24 by STS-92, bubbles that spanned the diameter of the tubing were observed in all tubes. Such bubbles interrupt liquid-liquid diffusion and force vapor diffusion equilibration to occur instead. Nonetheless, crystals grew in 9 of 30 tubes. Many large crystals were grown, the largest being 2.0 x 1.1 x 1.0 cubic mm. The largest crystal was used to collect data at room temperature on beamline 7-1 of the Stanford Synchrotron Radiation Source to a maximum resolution of 1.2 A. The structure was refined anisotropically using SHELX with a data to parameter ratio of 4.5 to give an R(sub factor) of 15.8% (R(sub free) = 18.2%) for ail reflections without generated hydrogens. This refinement is proceeding. Comparisons of this 1.2 A microgravity structure to previous reports of the thaumatin structure at 1.75 A and to ground control crystals will be presented.

Kundrot, Craig E.↗
NASA NTRS2002

Equilibrium Kinetics Studies and Crystallization Aboard the International Space Station (ISS) Using the Protein Crystallization Apparatus for Microgravity (PCAM)

Protein Crystallization Apparatus in Microgravity (PCAM) trays have been used in Shuttle missions to crystallize proteins in a microgravity environment. The crystallization experiments are 'sitting drops' similar to that in Cryschem trays, but the reservoir solution is soaked in a wick. From early 2001, crystallization experiments are conducted on the International Space Station using mission durations of months rather than two weeks on previous shuttle missions. Experiments were set up in April 2001 on Flight 6A to characterize the time crystallization experiments will take to reach equilibrium in a microgravity environment using salts, polyethylene glycols and an organic solvent as precipitants. The experiments were set up to gather data for a series of days of activation with different droplet volumes and precipitants. The experimental set up on ISS and results of this study will be presented. These results will help future users of PCAM to choose precipitants to optimize crystallization conditions for their target macromolecules for a particular mission with known mission duration. Changes in crystal morphology and size between the ground and space grown crystals of a protein and a protein -DNA complex flown on the same mission will also be presented.

Achari, Aniruddha↗
NASA NTRS2002

The Kinetics of Crystallization of Colloids and Proteins: A Light Scattering Study

Hard-sphere colloidal systems serve as model systems for aggregation, nucleation, crystallization and gelation as well as interesting systems in their own right. Similar colloidal systems have important technological uses such as paints and pharmaceuticals. There is a strong current interest in using colloidal systems to form photonic crystals. A number of colloidal experiments have flown on the space shuttle and a suite of experiments is currently active on the ISS A major scientific thrust of NASA's microgravity research is the crystallization of proteins for structural determination. The crystallization of proteins is a complicated process that requires a great deal of trial and error experimentation. In spite of a great deal of work, "better" protein crystals cannot always be grown in microgravity and conditions for crystallization are not well understood. Crystallization of colloidal systems interacting as hard spheres and with an attractive potential induced by entropic forces have been studied in a series of static light scattering experiments. Additionally, aggregation of a protein as a function of pH has been studied using dynamic light scattering.

Jim McClymer↗
NASA NTRS2003

Protein-Precipitant-Specific Criteria for the Impact of Reduced Gravity on Crystal Perfection

The objective of this research is to provide quantitative criteria for the impact of reduced or enhanced convective transport on protein crystal perfection. Our earlier work strongly suggests that the magnitude of (lattice defect-inducing) fluctuations in the crystallization rate of proteins arise from the coupling of bulk transport and nonlinear interface kinetics. Hence, we surmised that, depending on the relative weight of bulk transport and interface kinetics in the control of the crystallization process on Earth, these fluctuations can either increase or decrease under reduced gravity conditions. The sign and magnitude of these changes depend on the specific protein-precipitant system. As a consequence, space environments can be either beneficial or detrimental for achieving structural perfection in protein crystals. The task objectives consist in systematic investigations of this hypothesis.

Vekilov, Peter G.↗
NASA NTRS2003

A Gravity-Responsive Time-Keeping Protein of the Plant and Animal Cell Surface

The hypothesis under investigation was that a ubiquinol (NADH) oxidase protein of the cell surface with protein disulfide-thiol interchange activity (= NOX protein) is a plant and animal time-keeping ultradian (period of less than 24 h) driver of both cell enlargement and the biological clock that responds to gravity. Despite considerable work in a large number of laboratories spanning several decades, this is, to my knowledge, our work is the first demonstration of a time-keeping biochemical reaction that is both gravity-responsive and growth-related and that has been shown to determine circadian periodicity. As such, the NOX protein may represent both the long-sought biological gravity receptor and the core oscillator of the cellular biological clock. Completed studies have resulted in 12 publications and two issued NASA-owned patents of the clock activity. The gravity response and autoentrainment were characterized in cultured mammalian cells and in two plant systems together with entrainment by light and small molecules (melatonin). The molecular basis of the oscillatory behavior was investigated using spectroscopic methods (Fourier transform infrared and circular dichroism) and high resolution electron microscopy. We have also applied these findings to an understanding of the response to hypergravity. Statistical methods for analysis of time series phenomena were developed (Foster et al., 2003).

Morre, D. James↗
NASA NTRS2003

Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

Source record↗
NASA NTRS2003

Biotechnology Protein Expression and Purification Facility

The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

Source record↗
NASA NTRS2004

Solutal Convection Around Growing Protein Crystal and Diffusional Purification in Space

At least some protein crystals were found to preferentially trap microheterogeneous impurities. The latter are, for example, dimmer molecules of the crystallizing proteines (e.g. ferritin, lysozyme), or the regular molecules on which surfaces small molecules or ions are adsorbed (e.g. acetilated lysozyme) and modi@ molecular charge. Impurities may induce lattice defects and deteriorate structural resolution. Distribution of impurities between mother solution and gorwing crystal is defined by two interrelated distribution coefficients: kappa = rho(sup c, sub 2) and K = (rho(sup c, sub 2)/rho(sup c, sub 1)/rho(sub 2)/rho(sub 1). Here, rho(sub 2), rho(sub 1) and rho(sup c, sub 2) are densities of impurity (2) and regular protein (1) in solution at the growing interface and within the crystal ("c"). For the microheterogeneous impurities studied, K approx. = 2 - 4, so that kappa approx. - 10(exp 2) - 10(exp 3), since K = kappa (rho(sub 1)/rho(sup c, sub 1) and protein solubility ratio rho(sub 1)/rho(sub=p c, sub 2) much less than 1. Therefore, a crystal growing in absence of convection purifies mother solution around itself, grows cleaner and, probably, more perfect. If convection is present, the solution flow permanently brings new impurities to the crystal. This work theoretically addressed two subjects: 1) onset of convection, 2) distribution of impurities.

Lee, Chun P.↗
NASA NTRS2003

Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress stimulates an inflammatory response

Atherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NFkappaB-dependent mechanism. We suggest that BMP4 is a mechanosensitive, inflammatory factor playing a critical role in early steps of atherogenesis in the lesion-prone areas.

NASA Program Fundamental Space Biology↗
NASA NTRS2003

Hypercortisolemia alters muscle protein anabolism following ingestion of essential amino acids

Debilitating injury is accompanied by hypercortisolemia, muscle wasting, and disruption of the normal anabolic response to food. We sought to determine whether acute hypercortisolemia alters muscle protein metabolism following ingestion of a potent anabolic stimulus: essential amino acids (EAA). A 27-h infusion (80 microg. kg(-1). h(-1)) of hydrocortisone sodium succinate mimicked cortisol (C) levels accompanying severe injury (>30 microg/dl), (C + AA; n = 6). The control group (AA) received intravenous saline (n = 6). Femoral arteriovenous blood samples and muscle biopsies were obtained during a primed (2.0 micromol/kg) constant infusion (0.05 micromol. kg(-1). min(-1)) of l-[ring-(2)H(5)]phenylalanine before and after ingestion of 15 g of EAA. Hypercortisolemia [36.5 +/- 2.1 (C + AA) vs. 9.0 +/- 1.0 microg/dl (AA)] increased postabsorptive arterial, venous, and muscle intracellular phenylalanine concentrations. Hypercortisolemia also increased postabsorptive and post-EAA insulin concentrations. Net protein balance was blunted (40% lower) following EAA ingestion but remained positive for a greater period of time (60 vs. 180 min) in the C + AA group. Thus, although differences in protein metabolism were evident, EAA ingestion improved muscle protein anabolism during acute hypercortisolemia and may help minimize muscle loss following debilitating injury.

Clinical Trial↗
NASA NTRS1997

Immunolocalization of integrin-like proteins in Arabidopsis and Chara

Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken beta1 integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the internodal cells and rhizoids of Chara. These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.

Non-NASA Center↗
NASA NTRS1986

In vitro and in vivo protein phosphorylation in Avena sativa L. coleoptiles: effects of Ca2+, calmodulin antagonists, and auxin

In vitro and in vivo protein phosphorylations in oat (Avena sativa L.) coleoptile segments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. In vitro phosphorylation of several polypeptides was distinctly promoted at 1 to 15 micromolar free Ca2+ concentrations. Ca2(+)-stimulated phosphorylation was markedly reduced by trifluoperazine, chlorpromazine, and naphthalene sulfonamide (W7). Two polypeptides were phosphorylated both under in vitro and in vivo conditions, but the patterns of phosphorylation of several other polypeptides were different under the two conditions indicating that the in vivo phosphorylation pattern of proteins is not truly reflected by in vitro phosphorylation studies. Trifluoperazine, W7, or ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) + calcium ionophore A23187 treatments resulted in reduced levels of in vivo protein phosphorylation of both control and auxin-treated coleoptile segments. Analysis by two-dimensional electrophoresis following in vivo phosphorylation revealed auxin-dependent changes of certain polypeptides. A general inhibition of phosphorylation by calmodulin antagonists suggested that both control and auxin-treated coleoptiles exhibited Ca2+, and calmodulin-dependent protein phosphorylation in vivo.

NASA Discipline Plant Biology↗