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At least 523 records · Page 29
All sourcesNASA NTRSDOE OSTI
NASA NTRS1998

The Effect of Temperature and Solution pH on Tetragonal Lysozyme Nucleation Kinetics

Part of the challenge of macromolecular crystal growth for structure determination is obtaining an appropriate number of crystals with a crystal 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 one week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions, Duplicate experiments indicate the reproducibility of the technique, Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable however, was pH, where crystal numbers changed by two orders of magnitude over the pH range 4.0 to 5.2. Crystal size varied also with solution conditions, with the largest crystals being obtained at pH 5.2. Having optimized the crystallization conditions, a batch of crystals were prepared under exactly the same conditions and fifty of these crystals were analyzed by x-ray techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.

Judge, Russell A.↗
NASA NTRS1999

IMF Length Scales and Predictability: The Two Length Scale Medium

We present preliminary results from a systematic study using simultaneous data from three spacecraft, Wind, IMP 8 (Interplanetary Monitoring Platform) and Geotail to examine interplanetary length scales and their implications on predictability for magnetic field parcels in the typical solar wind. Time periods were selected when the plane formed by the three spacecraft included the GSE (Ground Support Equipment) x-direction so that if the parcel fronts were strictly planar, the two adjacent spacecraft pairs would determine the same phase front angles. After correcting for the motion of the Earth relative to the interplanetary medium and deviations in the solar wind flow from radial, we used differences in the measured front angle between the two spacecraft pairs to determine structure radius of curvature. Results indicate that the typical radius of curvature for these IMF parcels is of the order of 100 R (Sub E). This implies that there are two important IMF (Interplanetary Magnetic Field) scale lengths relevant to predictability: (1) the well-established scale length over which correlations observed by two spacecraft decay along a given IMF parcel, of the order of a few tens of Earth radii and (2) the scale length over which two spacecraft are unlikely to even observe the same parcel because of its curvature, of the order of a hundred Earth radii.

Collier, Michael R.↗
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.↗
NASA NTRS1998

Effect of halogenation on the nonlinear optical properties of porthyrin and substituted porphyrins

The effect that fluorine and chlorine substitution has on the nonlinear optical properties of porphyrin, tetramethylporphyrin and tetraphenylporphyrin has been theoretically studied. The calculations of nonlinear optical properties have been obtained by performing finite-field calculations on structures determined by semiempirical methods. In addition, tetra(p-chlorophenyl)porphyrin and tetra(p-bromophenyl)porphyrin were synthesized by the condensation of pyrrol and the appropriate aldehyde. Thin films of polymethylmethacrylate were obtained containing these materials, by spin coating onto glass substrates. The films were characterized by third-harmonic generation. It was determined that the experimental conditions enhance the third-order polarizability of the tetraphenylporphyrins by a factor of about 1.6.

Cardelino, Beatriz H.↗
NASA NTRS2000

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

Hong, Y. S.↗
NASA NTRS1999

Cell Culture in Microgravity: Opening the Door to Space Cell Biology

Adaptational response of human cell populations to microgravity is investigated using simulation, short-term Shuttle experiments, and long-term microgravity. Simulation consists of a clinostatically-rotated cell culture system. The system is a horizontally-rotated cylinder completely filled with culture medium. Low speed rotation results in continuous-fall of the cells through the fluid medium. In this setting, cells: 1) aggregate, 2) propagate in three dimensions, 3) synthesize matrix, 4) differentiate, and 5) form sinusoids that facilitate mass transfer. Space cell culture is conducted in flight bioreactors and in static incubators. Cells grown in microgravity are: bovine cartilage, promyelocytic leukemia, kidney proximal tubule cells, adrenal medulla, breast and colon cancer, and endothelium. Cells were cultured in space to test specific hypotheses. Cartilage cells were used to determine structural differences in cartilage grown in space compared to ground-based bioreactors. Results from a 130-day experiment on Mir revealed that cartilage grown in space was substantially more compressible due to insufficient glycosaminoglycan in the matrix. Interestingly, earth-grown cartilage conformed better to the dimensions of the scaffolding material, while the Mir specimens were spherical. The other cell populations are currently being analyzed for cell surface properties, gene expression, and differentiation. Results suggest that some cells spontaneously differentiate in microgravity. Additionally, vast changes in gene expression may occur in response to microgravity. In conclusion, the transition to microgravity may constitute a physical perturbation in cells resulting in unique gene expressions, the consequences of which may be useful in tissue engineering, disease modeling, and space cell biology.

Pellis, Neal R.↗
NASA NTRS2001

A Miniaturized Seismometer for Subsurface Probing on Mars

Seismology is one of the most powerful tools for investigating the subsurface structure of a planet. The mechanical structure information derived from seismic measurements is complementary to other methods of probing the subsurface (such as gravity and electromagnetics), both in terms of spatial and depth resolution and the relevant types of material properties being sensed. In the near-surface, the propagation of seismic waves is especially sensitive to density and degree of compaction. In addition, interfaces between layer with contrasting properties can be relatively easily delineated. The subsurface of Mars provides an obvious target for seismic investigations. Searching for the presence of water is among the highest priority goals of the Mars Exploration Program, but there are many other important science questions that could be addressed by a seismic profile of the upper layers of the crust. We have developed an extremely small, lightweight, low-power seismometer for planetary applications which is ideally suited for use on Mars. This instrument has previously been proposed and selected for use on a comet (on the Rosetta Lander, subsequently deselected for programmatic reasons) and Mars (on the NetLander mission, with an emphasis on global structure determination).

Banerdt, W. B.↗
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 NTRS2002

Characterization of Truncated Tumor-Associated NADH Oxidase (ttNOX)

Bacterial, plant and animal cells possess novel surface proteins that exhibit both NADH oxidation (NOX) or hydroquinone and protein disulfide-thiol interchange. These enzymatic activities alternate to yield oscillating patterns wjth period lengths of approximately 24 minutes. The catalytic period of NOX proteins are temperature compensated and gravity responsive. We report the cloning, expression and characterization of truncated tumor-associated NADH oxidase (ttNOX), in which the membrane spanning region has been deleted. The cDNA (originated from HeLa cells) was cloned into pET-34b and pET-14b (Novagen) vectors for E. coli expression. Optimized expression and purification protocols yielded greater than 300mg per liter of culture with greater than 95% purity. Circular dichroism data was collected from a 2.7mg/ml solution in a 0.1mm cuvette with variable scanning using an Olis RSM CD spectrophotometer. The ellipticity values were scanned from 190 to 260nm. The spectra recorded have characteristics for alpha proteins with band maxima at 216nm and a possible shoulder at 212nm at 12OC and 250 C. Protein crystal screens are in progress and, to date, only small crystals have been observed. The regular periodic oscillatory change in the ttNOX protein is indicative of a possible time-keeping functional role. A single protein possessing alternating catalytic activities, with a potential biological clock function, is unprecedented and structural determination is paramount to understanding this role.

Karr, Laurel J.↗
NASA NTRS2002

System IDentification Programs for AirCraft (SIDPAC)

A collection of computer programs for aircraft system identification is described and demonstrated. The programs, collectively called System IDentification Programs for AirCraft, or SIDPAC, were developed in MATLAB as m-file functions. SIDPAC has been used successfully at NASA Langley Research Center with data from many different flight test programs and wind tunnel experiments. SIDPAC includes routines for experiment design, data conditioning, data compatibility analysis, model structure determination, equation-error and output-error parameter estimation in both the time and frequency domains, real-time and recursive parameter estimation, low order equivalent system identification, estimated parameter error calculation, linear and nonlinear simulation, plotting, and 3-D visualization. An overview of SIDPAC capabilities is provided, along with a demonstration of the use of SIDPAC with real flight test data from the NASA Glenn Twin Otter aircraft. The SIDPAC software is available without charge to U.S. citizens by request to the author, contingent on the requestor completing a NASA software usage agreement.

Morelli, Eugene 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

Synchrotron X-Ray Diffraction Analysis of Meteorites in Thin Section: Preliminary Results

X-ray diffraction is the pre-eminent technique for mineral identification and structure determination, but is difficult to apply to grains in thin section, the standard meteorite preparation. Bright focused X-ray beams from synchrotrons have been used extensively in mineralogy and have been applied to extraterrestrial particles. The intensity and small spot size achievable in synchrotron X-ray beams makes them useful for study of materials in thin sections. Here, we describe Synchrotron X-ray Diffraction (SXRD) in thin section as done at the National Synchrotron Light Source, and cite examples of its value for studies of meteorites in thin section.

Treiman, A. H.↗
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

Intricate Crystal Structure of Dihydrolipoamide Dehydrogenase (E3) with its Binding Protein: Multiple Copies, Dynamic and Static Disorders

Human E3 and binding protein E3BP are two components of the pyruvate dehydrogenase complex. Crystallization of E3 with 221-amino acid fragment of E3BP (E3BPdd) led to crystals that diffracted to a resolution of 2.6 Angstroms. Structure determination involved molecular replacement using a dimer of E3 homolog as a search model and de novo building of the E3BPdd peptide. Solution was achieved by inclusion of one E3 dimer at a time, followed by refinement until five E3 dimers were located. This complete content of E3 provided electron density maps suitable for tracing nine peptide chains of E3BPdd, eight of them being identified with partial occupancies. Final content of the asymmetric unit consists of five E3 dimers, each binding one E3BPdd molecule. In four of these molecular complexes, E3BPdd is in static disorder resulting in E3BPdd binding to either one or the other monomer of the E3 dimer. However, E3BPdd of the fifth E3 dimer forms specific contacts that lock it at one monomer. In addition to this static disorder, E3BPdd reveals high mobility in the limited space of the crystal lattice. Support from NIH and NASA.

Makal, A.↗
NASA NTRS2003

Fiber-Optic Bragg Gratings and Optical Holography Compared as Vibration Detectors

The NASA Glenn Research Center is interested in determining structural damage in engine components during flight to evaluate the health of aerospace propulsion systems. On the ground, we can use holography to detect structural damage by examining the characteristic mode shapes and frequencies of vibrating objects. We are studying the feasibility of using embedded fiber Bragg gratings (FBGs) to accomplish this goal in a flight-worthy system, by using the minimal intrusion and high sensitivity afforded by fiber optics. We have recently compared holographically imaged modes of vibrating plates with the corresponding dynamic strains detected by embedded FBGs. We constructed an experimental setup for studying the responses of FBGs to dynamic excitations. One of the plates was made of a polymer matrix composite (PMC) with an FBG embedded in it, and the other one was made of copper with surface-mounted FBGs. The instrumented plates were mounted and vibrated, and time-averaged holography was used to measure their surface displacements. Simultaneously, the signals from the FBGs were detected and sent via fiber-optic cable to a quiet location about 20 m away for interrogation. The the test configuration used for the PMC plate is shown. Experimental results are also shown. The FBG was embedded in the middle of the PMC plates, roughly within the center circular fringe in each of the interferograms shown. Two resonant excitation frequencies were used: 706 and 3062 Hz. The plot in this paper shows a larger FBG signal at the higher frequency; this is because the plate bends more at higher order resonant modes, causing higher strain. This contrasts to the smaller displacements characteristic of higher frequencies, which are measured by holographic techniques.

Adamovsky, Grigory↗
NASA NTRS2006

Estimation of Longitudinal Unsteady Aerodynamics of a Wing-Tail Combination From Wind Tunnel Data

This paper presents an initial step toward model identification from wind tunnel data for an airliner configuration. Two approaches to modeling a transport configuration are considered and applied to both steady and large-amplitude forced-oscillation wind tunnel data taken over a wide range of angles of attack. Only limited conclusions could be drawn from this initial data set. Although model estimated time histories of normal force and pitching moment agree reasonably well with the corresponding measured values, model damping parameters did not, for some cases, have values consistent with small amplitude oscillatory data. In addition, large parameter standard errors implied poor information content for model structure determination and parameter estimation. Further investigation of the modeling problem for more general aerodynamic models is recommended with close attention to experiment design for obtaining parameters with high accuracy.

Murphy, Patrick C.↗
NASA NTRS2011

Estimation of Unsteady Aerodynamic Models from Dynamic Wind Tunnel Data

Demanding aerodynamic modelling requirements for military and civilian aircraft have motivated researchers to improve computational and experimental techniques and to pursue closer collaboration in these areas. Model identification and validation techniques are key components for this research. This paper presents mathematical model structures and identification techniques that have been used successfully to model more general aerodynamic behaviours in single-degree-of-freedom dynamic testing. Model parameters, characterizing aerodynamic properties, are estimated using linear and nonlinear regression methods in both time and frequency domains. Steps in identification including model structure determination, parameter estimation, and model validation, are addressed in this paper with examples using data from one-degree-of-freedom dynamic wind tunnel and water tunnel experiments. These techniques offer a methodology for expanding the utility of computational methods in application to flight dynamics, stability, and control problems. Since flight test is not always an option for early model validation, time history comparisons are commonly made between computational and experimental results and model adequacy is inferred by corroborating results. An extension is offered to this conventional approach where more general model parameter estimates and their standard errors are compared.

Murphy, Patrick↗
NASA NTRS2013

Conductance of Ion Channels - Theory vs. Experiment

Transmembrane ion channels mediate a number of essential physiological processes in a cell ranging from regulating osmotic pressure to transmission of neural signals. Kinetics and selectivity of ion transport is of critical importance to a cell and, not surprisingly, it is a subject of numerous experimental and theoretical studies. In this presentation we will analyze in detail computer simulations of two simple channels from fungi - antiamoebin and trichotoxin. Each of these channels is made of an alpha-helical bundle of small, nongenomically synthesized peptides containing a number of rare amino acids and exhibits strong antimicrobial activity. We will focus on calculating ionic conductance defined as the ratio of ionic current through the channel to applied voltage. From molecular dynamics simulations, conductance can be calculated in at least two ways, each involving different approximations. Specifically, the current, given as the number of charges transferred through the channel per unit of time, can be obtained from the number of events in which ions cross the channel during the simulation. This method works well for large currents (high conductance values and/or applied voltages). If the number of crossing events is small, reliable estimates of current are difficult to achieve. Alternatively, conductance can be estimated assuming that ion transport can be well approximated as diffusion in the external potential given by the free energy profile. Then, the current can be calculated by solving the one-dimensional diffusion equation in this external potential and applied voltage (the generalized Nernst-Planck equation). To do so three ingredients are needed: the free energy profile, the position-dependent diffusion coefficient and the diffusive flux of ions into the channel. All these quantities can be obtained from molecular dynamics simulations. An important advantage of this method is that it can be used equally well to estimating large and small currents. In addition, once the free energy profile becomes available the full current-voltage dependence can be readily obtained. For both channels we carried out calculations using both approaches. We also tested the main assumptions underlying the diffusive model, such as uncorrelated nature of individual crossing events and Fickian diffusion. The accuracy and consistency of different methods will be discussed. Finally we will discuss how comparisons between calculated and measured ionic conductance and selectivity of transport can be used for determining structural models of the channels.

Pohorille, Andrew↗