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Sample records for advanced protein crystal

  1. Advanced Protein Crystallization Facility (APCF)

    NASA Technical Reports Server (NTRS)

    1998-01-01

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

  2. Advanced protein crystal growth programmatic sensitivity study

    NASA Technical Reports Server (NTRS)

    1992-01-01

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

  3. FNAS/advanced protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz

    1992-01-01

    A scintillation method is presented for determination of the temperature dependence of the solubility, S(T), of proteins in 50-100 micro-l volumes of solution. S(T) data for lysozyme and horse serum albumin were obtained for various combinations of pH and precipitant concentrations. The resulting kinetics and equilibrium information was used for dynamic control, that is the separation of nucleation and growth stages in protein crystallization. Individual lysozyme and horse serum albumin crystals were grown in 15-20 micro-l solution volumes contained in x-ray capillaries.

  4. Definition study for temperature control in advanced protein crystal growth

    NASA Technical Reports Server (NTRS)

    Nyce, Thomas A.; Rosenberger, Franz; Sowers, Jennifer W.; Monaco, Lisa A.

    1990-01-01

    Some of the technical requirements for an expedient application of temperature control to advanced protein crystal growth activities are defined. Lysozome was used to study the effects of temperature ramping and temperature gradients for nucleation/dissolution and consecutive growth of sizable crystals and, to determine a prototype temperature program. The solubility study was conducted using equine serum albumin (ESA) which is an extremely stable, clinically important protein due to its capability to bind and transport many different small ions and molecules.

  5. Advanced protein crystal growth flight hardware for the Space Station

    NASA Technical Reports Server (NTRS)

    Herrmann, Frederick T.

    1988-01-01

    The operational environment of the Space Station will differ considerably from the previous short term missions such as the Spacelabs. Limited crew availability combined with the near continuous operation of Space Station facilities will require a high degree of facility automation. This paper will discuss current efforts to develop automated flight hardware for advanced protein crystal growth on the Space Station. Particular areas discussed will be the automated monitoring of key growth parameters for vapor diffusion growth and proposed mechanisms for control of these parameters. A history of protein crystal growth efforts will be presented in addition to the rationale and need for improved protein crystals for X-ray diffraction. The facility will be capable of simultaneously processing several hundred protein samples at various temperatures, pH's, concentrations etc., and provide allowances for real time variance of growth parameters.

  6. Breadboard activities for advanced protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Banish, Michael

    1993-01-01

    The proposed work entails the design, assembly, testing, and delivery of a turn-key system for the semi-automated determination of protein solubilities as a function of temperature. The system will utilize optical scintillation as a means of detecting and monitoring nucleation and crystallite growth during temperature lowering (or raising, with retrograde solubility systems). The deliverables of this contract are: (1) turn-key scintillation system for the semi-automatic determination of protein solubilities as a function of temperature, (2) instructions and software package for the operation of the scintillation system, and (3) one semi-annual and one final report including the test results obtained for ovostatin with the above scintillation system.

  7. Physical aspects of protein crystal growth investigated with the Advanced Protein Crystallization Facility in reduced-gravity environments.

    PubMed

    Vergara, Alessandro; Lorber, Bernard; Zagari, Adriana; Giegé, Richard

    2003-01-01

    The physicochemical aspects of protein crystallization in reduced-gravity environments ( micro g) have been investigated with the Advanced Protein Crystallization Facility during six space missions. This review summarizes the results, dealing with the mechanisms of nucleation and crystal growth and with the quality of the crystals that were obtained under reduced gravity as well as under normal gravity on earth. Statistical analyses of the experimental data strongly support the fact that micro g has a positive effect on crystallization and on crystal quality. A comparison of experiments and theories of protein crystallization in reduced-gravity environments is presented. Recommendations for improving the performance of protein crystallization experiments in micro g and on earth are discussed.

  8. In situ observation of elementary growth processes of protein crystals by advanced optical microscopy.

    PubMed

    Sazaki, Gen; Van Driessche, Alexander E S; Dai, Guoliang; Okada, Masashi; Matsui, Takuro; Otálora, Fermin; Tsukamoto, Katsuo; Nakajima, Kazuo

    2012-07-01

    To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.

  9. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  10. [Advances in effects of insecticidal crystal proteins released from transgenic Bt crops on soil ecology].

    PubMed

    Zhou, Xue-Yong; Liu, Ning; Zhao, Man; Li, He; Zhou, Lang; Tang, Zong-Wen; Cao, Fei; Li, Wei

    2011-05-01

    With the large scale cultivation of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystal proteins in the world, the problem of environmental safety caused by these Bt crops has received extensive attention. These insecticidal crystal proteins can be released into the soil continuously in the growing period of Bt plants. If their accumulation of the insecticidal crystal proteins exceeds consumption by insect larvae and degradation by the environmental factors, these insecticidal crystal proteins could constitute a hazard to non-target insects and soil microbiota. There are three main ways to release insecticidal crystal proteins into soil for Bt plants: root exudates, pollen falling, and crop reside returning. The Bt insecticidal crystal proteins released into soil can be adsorbed rapidly by active soil particles and the absorption equilibrium attained within 1-3 h. The adsorption protects Bt insecticidal crystal proteins against soil microbial degradation or enzyme degradation, which leads to remarkable prolong of the persistence of insecticidal activity. The change of soil microorganism species is an important index for evaluating the effect of Bt plants on soil ecology. The research showed that these insecticidal crystal proteins released by the Bt plant root exudates or Bt organism had no toxicity to the soil earthworms, nematodes, protozoa, bacteria and fungi; however, it could reduce the mycelium length of the arbuscular mycorrhizal fungi (AMF) and restrain AMF to form invasion unit. The influencing degree of Bt protein on soil enzyme activity varied with the releasing modes or growth period of Bt crops. Bt Cry1Ab protein can be taken up from soil by parts of following crops; however, different results were obtained with different commercial kits. To better understand the soil ecological evaluation about the insecticidal crystal proteins released from transgenic Bt crops, this review provides a comprehensive overview about the release

  11. Protein crystal growth in microgravity

    NASA Technical Reports Server (NTRS)

    Rosenblum, William M.; Delucas, Lawrence J.; Wilson, William W.

    1989-01-01

    Major advances have been made in several of the experimental aspects of protein crystallography, leaving protein crystallization as one of the few remaining bottlenecks. As a result, it has become important that the science of protein crystal growth is better understood and that improved methods for protein crystallization are developed. Preliminary experiments with both small molecules and proteins indicate that microgravity may beneficially affect crystal growth. For this reason, a series of protein crystal growth experiments using the Space Shuttle was initiated. The preliminary space experiments were used to evolve prototype hardware that will form the basis for a more advanced system that can be used to evaluate effects of gravity on protein crystal growth. Various optical techniques are being utilized to monitor the crystal growth process from the incipient or nucleation stage and throughout the growth phase. The eventual goal of these studies is to develop a system which utilizes optical monitoring for dynamic control of the crystallization process.

  12. Lessons from crystals grown in the Advanced Protein Crystallisation Facility for conventional crystallisation applied to structural biology.

    PubMed

    Vergara, Alessandro; Lorber, Bernard; Sauter, Claude; Giegé, Richard; Zagari, Adriana

    2005-12-01

    The crystallographic quality of protein crystals that were grown in microgravity has been compared to that of crystals that were grown in parallel on earth gravity under otherwise identical conditions. A goal of this comparison was to assess if a more accurate 3D-structure can be derived from crystallographic analysis of the former crystals. Therefore, the properties of crystals prepared with the Advanced Protein Crystallisation Facility (APCF) on earth and in orbit during the last decade were evaluated. A statistical analysis reveals that about half of the crystals produced under microgravity had a superior X-ray diffraction limit with respect of terrestrial controls. Eleven protein structures could be determined at previously unachieved resolutions using crystals obtained in the APCF. Microgravity induced features of the most relevant structures are reported. A second goal of this study was to identify the cause of the crystal quality enhancement useful for structure determination. No correlations between the effect of microgravity and other system-dependent parameters, such as isoelectric point or crystal solvent content, were found except the reduced convection during the crystallisation process. Thus, crystal growth under diffusive regime appears to be the key parameter explaining the beneficial effect of microgravity on crystal quality. The mimicry of these effects on earth in gels or in capillary tubes is discussed and the practical consequences for structural biology highlighted.

  13. Protein Crystal Based Nanomaterials

    NASA Technical Reports Server (NTRS)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  14. Pressure cryocooling protein crystals

    DOEpatents

    Kim, Chae Un; Gruner, Sol M.

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  15. Protein crystallization with paper

    NASA Astrophysics Data System (ADS)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  16. Approaches to automated protein crystal harvesting

    SciTech Connect

    Deller, Marc C. Rupp, Bernhard

    2014-01-28

    Approaches to automated and robot-assisted harvesting of protein crystals are critically reviewed. While no true turn-key solutions for automation of protein crystal harvesting are currently available, systems incorporating advanced robotics and micro-electromechanical systems represent exciting developments with the potential to revolutionize the way in which protein crystals are harvested.

  17. Protein crystallization in microgravity.

    PubMed

    Aibara, S; Shibata, K; Morita, Y

    1997-12-01

    A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992. The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization. Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126, were obtained as single crystals of good diffraction quality. Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR. Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.

  18. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1993-01-01

    Proteins account for 50% or more of the dry weight of most living systems and play a crucial role in virtually all biological processes. Since the specific functions of essentially all biological molecules are determined by their three-dimensional structures, it is obvious that a detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. At the present time, protein crystallography has no substitute, it is the only technique available for elucidating the atomic arrangements within complicated biological molecules. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting and promising projects have terminated at the crystal growth stage. There is a pressing need to better understand protein crystal growth, and to develop new techniques that can be used to enhance the size and quality of protein crystals. There are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor that might be expected to alter crystal growth processes in space is the elimination of density-driven convective flow. Another factor that can be readily controlled in the absence of gravity is the sedimentation of growing crystal in a gravitational field. Another potential advantage of microgravity for protein crystal growth is the option of doing containerless crystal growth. One can readily understand why the microgravity environment established by Earth-orbiting vehicles is perceived to offer unique opportunities for the protein crystallographer. The near term objectives of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

  19. Biomolecular membrane protein crystallization

    NASA Astrophysics Data System (ADS)

    Reddy Bolla, Jani; Su, Chih-Chia; Yu, Edward W.

    2012-07-01

    Integral membrane proteins comprise approximately 30% of the sequenced genomes, and there is an immediate need for their high-resolution structural information. Currently, the most reliable approach to obtain these structures is X-ray crystallography. However, obtaining crystals of membrane proteins that diffract to high resolution appears to be quite challenging, and remains a major obstacle in structural determination. This brief review summarizes a variety of methodologies for use in crystallizing these membrane proteins. Hopefully, by introducing the available methods, techniques, and providing a general understanding of membrane proteins, a rational decision can be made about now to crystallize these complex materials.

  20. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Atomic force microscopy uses laser technology to reveal a defect, a double-screw dislocation, on the surface of this crystal of canavalin, a major source of dietary protein for humans and domestic animals. When a crystal grows, attachment kinetics and transport kinetics are competing for control of the molecules. As a molecule gets close to the crystal surface, it has to attach properly for the crystal to be usable. NASA has funded investigators to look at those attachment kinetics from a theoretical standpoint and an experimental standpoint. Dr. Alex McPherson of the University of California, Irvine, is one of those investigators. He uses X-ray diffraction and atomic force microscopy in his laboratory to answer some of the many questions about how protein crystals grow. Atomic force microscopy provides a means of looking at how individual molecules are added to the surface of growing protein crystals. This helps McPherson understand the kinetics of protein crystal growth. McPherson asks, How fast do crystals grow? What are the forces involved? Investigators funded by NASA have clearly shown that such factors as the level of supersaturation and the rate of growth all affect the habit [characteristic arrangement of facets] of the crystal and the defects that occur in the crystal.

  1. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell, Post-Doctoral Fellow the National Research Council (NRC) uses a reciprocal space mapping diffractometer for macromolecular crystal quality studies. The diffractometer is used in mapping the structure of macromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystallized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  2. Protein crystal growth in a microgravity environment

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1988-01-01

    Protein crystal growth is a major experimental problem and is the bottleneck in widespread applications of protein crystallography. Research efforts now being pursued and sponsored by NASA are making fundamental contributions to the understanding of the science of protein crystal growth. Microgravity environments offer the possibility of performing new types of experiments that may produce a better understanding of protein crystal growth processes and may permit growth environments that are more favorable for obtaining high quality protein crystals. A series of protein crystal growth experiments using the space shuttle was initiated. The first phase of these experiments was focused on the development of micro-methods for protein crystal growth by vapor diffusion techniques, using a space version of the hanging drop method. The preliminary space experiments were used to evolve prototype hardware that will form the basis for a more advanced system that can be used to evaluate effects of gravity on protein crystal growth.

  3. Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    2003-01-01

    In order to rapidly and efficiently grow crystals, tools were needed to automatically identify and analyze the growing process of protein crystals. To meet this need, Diversified Scientific, Inc. (DSI), with the support of a Small Business Innovation Research (SBIR) contract from NASA s Marshall Space Flight Center, developed CrystalScore(trademark), the first automated image acquisition, analysis, and archiving system designed specifically for the macromolecular crystal growing community. It offers automated hardware control, image and data archiving, image processing, a searchable database, and surface plotting of experimental data. CrystalScore is currently being used by numerous pharmaceutical companies and academic and nonprofit research centers. DSI, located in Birmingham, Alabama, was awarded the patent Method for acquiring, storing, and analyzing crystal images on March 4, 2003. Another DSI product made possible by Marshall SBIR funding is VaporPro(trademark), a unique, comprehensive system that allows for the automated control of vapor diffusion for crystallization experiments.

  4. Using Inorganic Crystals To Grow Protein Crystals

    NASA Technical Reports Server (NTRS)

    Shlichta, Paul J.; Mcpherson, Alexander A.

    1989-01-01

    Solid materials serve as nucleating agents. Protein crystals induced by heterogeneous nucleation and in some cases by epitaxy to grow at lower supersaturations than needed for spontaneous nucleation. Heterogeneous nucleation makes possible to grow large, defect-free single crystals of protein more readily. Such protein crystals benefits research in biochemistry and pharmacology.

  5. Which Strategy for a Protein Crystallization Project?

    NASA Technical Reports Server (NTRS)

    Kundrot, Craig E.

    2003-01-01

    The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryo-crystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

  6. Which strategy for a protein crystallization project?

    NASA Technical Reports Server (NTRS)

    Kundrot, C. E.

    2004-01-01

    The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryocrystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

  7. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  8. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell (standing), Post-Doctoral Fellow the National Research Council (NRC),and Marc Pusey of Marshall Space Flight Center (MSFC) use a reciprocal space mapping diffractometer for marcromolecular crystal quality studies. The diffractometer is used in mapping the structure of marcromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystalized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  9. High-throughput protein crystallization.

    PubMed

    Stevens, R C

    2000-10-01

    The combinatorial chemistry industry has made major advances in the handling and mixing of small volumes, and in the development of robust liquid-handling systems. In addition, developments have been made in the area of material handling for the high-throughput drug screening and combinatorial chemistry fields. Lastly, improvements in beamline optics at synchrotron sources have enabled the use of flash-frozen micron-sized (10-50 microm) crystals. The combination of these and other recent advances will make high-throughput protein crystallography possible. Further advances in high-throughput methods of protein crystallography will require application of the above developments and the accumulation of success/failure data in a more systematic manner. Major changes in crystallography technology will emerge based on the data collected by first-generation high-throughput systems.

  10. Protein Crystal Bovine Insulin

    NASA Technical Reports Server (NTRS)

    1991-01-01

    The comparison of protein crystal, Bovine Insulin space-grown (left) and earth-grown (right). Facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  11. Protein crystal growth in microgravity

    NASA Technical Reports Server (NTRS)

    Delucas, Lawrence J.; Smith, Craig D.; Smith, H. Wilson; Vijay-Kumar, Senadhi; Senadhi, Shobha E.; Ealick, Steven E.; Carter, Daniel C.; Snyder, Robert S.

    1989-01-01

    The crystals of most proteins or other biological macromolecules are poorly ordered and diffract to lower resolutions than those observed for most crystals of simple organic and inorganic compounds. Crystallization in the microgravity environment of space may improve crystal quality by eliminating convection effects near growing crystal surfaces. A series of 11 different protein crystal growth experiments was performed on U.S. Space Shuttle flight STS-26 in September 1988. The microgravity-grown crystals of gamma-interferon D1, porcine elastase, and isocitrate lyase are larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth.

  12. Introduction to protein crystallization.

    PubMed

    McPherson, Alexander; Gavira, Jose A

    2014-01-01

    Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

  13. Introduction to protein crystallization

    PubMed Central

    McPherson, Alexander; Gavira, Jose A.

    2014-01-01

    Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid–liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies. PMID:24419610

  14. Characterizing protein crystal nucleation

    NASA Astrophysics Data System (ADS)

    Akella, Sathish V.

    We developed an experimental microfluidic based technique to measure the nucleation rates and successfully applied the technique to measure nucleation rates of lysozyme crystals. The technique involves counting the number of samples which do not have crystals as a function of time. Under the assumption that nucleation is a Poisson process, the fraction of samples with no crystals decays exponentially with the decay constant proportional to nucleation rate and volume of the sample. Since nucleation is a random and rare event, one needs to perform measurements on large number of samples to obtain good statistics. Microfluidics offers the solution of producing large number of samples at minimal material consumption. Hence, we developed a microfluidic method and measured nucleation rates of lysozyme crystals in supersaturated protein drops, each with volume of ˜ 1 nL. Classical Nucleation Theory (CNT) describes the kinetics of nucleation and predicts the functional form of nucleation rate in terms of the thermodynamic quantities involved, such as supersaturation, temperature, etc. We analyzed the measured nucleation rates in the context of CNT and obtained the activation energy and the kinetic pre-factor characterizing the nucleation process. One conclusion is that heterogeneous nucleation dominates crystallization. We report preliminary studies on selective enhancement of nucleation in one of the crystal polymorprhs of lysozyme (spherulite) using amorphous mesoporous bioactive gel-glass te{naomi06, naomi08}, CaO.P 2O5.SiO2 (known as bio-glass) with 2-10 nm pore-size diameter distribution. The pores act as heterogeneous nucleation centers and claimed to enhance the nucleation rates by molecular confinement. The measured kinetic profiles of crystal fraction of spherulites indicate that the crystallization of spherulites may be proceeding via secondary nucleation pathways.

  15. Protein crystal growth in space

    NASA Technical Reports Server (NTRS)

    Bugg, C. E.; Clifford, D. W.

    1987-01-01

    The advantages of protein crystallization in space, and the applications of protein crystallography to drug design, protein engineering, and the design of synthetic vaccines are examined. The steps involved in using protein crystallography to determine the three-dimensional structure of a protein are discussed. The growth chamber design and the hand-held apparatus developed for protein crystal growth by vapor diffusion techniques (hanging-drop method) are described; the experimental data from the four Shuttle missions are utilized to develop hardware for protein crystal growth in space and to evaluate the effects of gravity on protein crystal growth.

  16. Bacterial Ice Crystal Controlling Proteins

    PubMed Central

    Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  17. Bacterial ice crystal controlling proteins.

    PubMed

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  18. Path to protein crystallization

    SciTech Connect

    2010-01-01

    Growth of two-dimensional S-layer crystals on supported lipid bilayers observed in solution using in situ atomic force microscopy. This movie shows proteins sticking onto the supported lipid bilayer, forming a mobile phase that condenses into amorphous clusters, and undergoing a phase transition to crystalline clusters composed of 2 to 15 tetramers. These initial clusters then enter a growth phase in which new tetramers form exclusively at unoccupied lattice sites along the cluster edges.

  19. Protein Crystal Isocitrate Lyase

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The comparison of protein crystal, Isocitrate Lyase earth-grown (left) and space-grown (right). This is a target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast; it regulates the flow of metabolic intermediates required for cell growth. Principal Investigator is Larry DeLucas.

  20. Protein Crystals of Raf Kinase

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This image shows crystals of the protein raf kinase grown on Earth (photo a) and on USML-2 (photo b). The space-grown crystals are an order of magnitude larger. Principal Investigator: Dan Carter of New Century Pharmaceuticals

  1. Protein Crystals Grown in Space

    NASA Technical Reports Server (NTRS)

    2000-01-01

    A collage of protein and virus crystals, many of which were grown on the U.S. Space Shuttle or Russian Space Station, Mir. The crystals include the proteins canavalin; mouse monoclonal antibody; a sweet protein, thaumatin; and a fungal protease. Viruses are represented here by crystals of turnip yellow mosaic virus and satellite tobacco mosaic virus. The crystals are photographed under polarized light (thus causing the colors) and range in size from a few hundred microns in edge length up to more than a millimeter. All the crystals are grown from aqueous solutions and are useful for X-ray diffraction analysis. Credit: Dr. Alex McPherson, University of California, Irvine.

  2. Fusion-protein-assisted protein crystallization.

    PubMed

    Kobe, Bostjan; Ve, Thomas; Williams, Simon J

    2015-07-01

    Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a flexible linker is required that allows the native interaction between the fused proteins. The two approaches can also be combined. The recent applications of fusion-protein technology in protein crystallization from the work of our own and other laboratories are briefly reviewed.

  3. Protein crystal growth in space

    NASA Technical Reports Server (NTRS)

    Delucas, Lawrence J.; Bugg, Charles E.

    1991-01-01

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

  4. Protein Crystals and their Growth

    NASA Technical Reports Server (NTRS)

    Chernov, A. A.

    2004-01-01

    Recent results on binding between protein molecules in crystal lattice, crystal-solution surface energy, elastic properties and strength and spontaneous crystal cracking are reviewed and discussed in the first half of this paper (Sea 2-4). In the second par&, some basic approaches to solubility of proteins are followed by overview on crystal nucleation and growth (Sec 5). It is argued that variability of mixing in batch crystallization may be a source for scattering of crystal number ultimately appearing in the batch. Frequency at which new molecules join crystal lattice is measured by kinetic coefficient and related to the observable crystal growth rate. Numerical criteria to discriminate diffusion and kinetic limited growth are discussed on this basis in Sec 7. In Sec 8, creation of defects is discussed with the emphasis on the role of impurities and convection on macromolecular crystal I;erfection.

  5. (PCG) Protein Crystal Growth Canavalin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Canavalin. The major storage protein of leguminous plants and a major source of dietary protein for humans and domestic animals. It is studied in efforts to enhance nutritional value of proteins through protein engineerings. It is isolated from Jack Bean because of it's potential as a nutritional substance. Principal Investigator on STS-26 was Alex McPherson.

  6. High density protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rouleau, Robyn (Inventor); Delucas, Lawrence (Inventor); Hedden, Douglas Keith (Inventor)

    2004-01-01

    A protein crystal growth assembly including a crystal growth cell and further including a cell body having a top side and a bottom side and a first aperture defined therethrough, the cell body having opposing first and second sides and a second aperture defined therethrough. A cell barrel is disposed within the cell body, the cell barrel defining a cavity alignable with the first aperture of the cell body, the cell barrel being rotatable within the second aperture. A reservoir is coupled to the bottom side of the cell body and a cap having a top side is disposed on the top side of the cell body. The protein crystal growth assembly may be employed in methods including vapor diffusion crystallization, liquid to liquid crystallization, batch crystallization, and temperature induction batch mode crystallization.

  7. Lasing from fluorescent protein crystals.

    PubMed

    Oh, Heon Jeong; Gather, Malte C; Song, Ji-Joon; Yun, Seok Hyun

    2014-12-15

    We investigated fluorescent protein crystals for potential photonic applications, for the first time to our knowledge. Rod-shaped crystals of enhanced green fluorescent protein (EGFP) were synthesized, with diameters of 0.5-2 μm and lengths of 100-200 μm. The crystals exhibit minimal light scattering due to their ordered structure and generate substantially higher fluorescence intensity than EGFP or dye molecules in solutions. The magnitude of concentration quenching in EGFP crystals was measured to be about 7-10 dB. Upon optical pumping at 485 nm, individual EGFP crystals located between dichroic mirrors generated laser emission with a single-mode spectral line at 513 nm. Our results demonstrate the potential of protein crystals as novel optical elements for self-assembled, micro- or nano-lasers and amplifiers in aqueous environment.

  8. Scientist prepare Lysozyme Protein Crystal

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Dan Carter and Charles Sisk center a Lysozyme Protein crystal grown aboard the USML-2 shuttle mission. Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity crystal growth experiments. The goal is to compare kinetic data from microgravity experiments with data from laboratory experiments to study the equilibrium.

  9. Surface Relaxation in Protein Crystals

    NASA Technical Reports Server (NTRS)

    Boutet, S.; Robinson, I. K.; Hu, Z. W.; Thomas, B. R.; Chernov, A. A.

    2002-01-01

    Surface X-ray diffraction measurements were performed on (111) growth faces of crystals of the Cellular iron-storage protein horse spleen ferritin. Crystal Trunkation Rods (CTR) were measured. A fit of the measured profile of the CTR revealed a surface roughness of 48 +/- 4.5 A and a top layer spacing contraction of 3.9 +/- 1.5%. In addition to the peak from the CTR, the rocking curves of the crystals displayed unexpected extra peaks. Multiple-scattering is demonstrated to account for them. Future applications of the method could allow the exploration of hydration effects on the growth of protein crystals.

  10. Thermal crystallization mechanism of silk fibroin protein

    NASA Astrophysics Data System (ADS)

    Hu, Xiao

    stage before crystallization. (3) The beta-sheet crystallization kinetics in silk fibroin protein were measured using X-ray, FTIR and heat flow, and the structure reveals the formation mechanism of the silk crystal network. Avrami kinetics theories, which were established for studies of synthetic polymer crystal growth, were for the first time extended to investigate protein self-assembly in multiblock silk fibroin samples. The Avrami exponent, n, was close to two for all methods, indicating formation of beta sheet crystals in silk proteins is different from the 3-D spherulitic crystal growth found in most synthetic homopolymers. A microphase separation pattern after chymotrypsin enzyme biodegradation was shown in the protein structures using scanning electron microscopy. A model was then used to explain the crystallization of silk fibroin protein by analogy to block copolymers. (4) The effects of metal ions during the crystallization of silk fibroin was investigated using thermal analysis. Advanced thermal analysis methods were used to analyze the thermal protein-metallic ion interactions in silk fibroin proteins. Results show that K+ and Ca2+ metallic salts play different roles in silk fibroin proteins, which either reduce (K+) or increase (Ca2+ ) the glass transition (Tg) of pure silk protein and affect the thermal stability of this structure.

  11. Protein crystal growth in microgravity

    NASA Technical Reports Server (NTRS)

    Carter, Daniel

    1992-01-01

    The overall scientific goals and rationale for growing protein crystals in microgravity are discussed. Data on the growth of human serum albumin crystals which were produced during the First International Microgravity Laboratory (IML-1) are presented. Potential scientific advantages of the utilization of Space Station Freedom are discussed.

  12. The Physics of Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Vekilov, P. G.; Chernov, A. A.

    2002-01-01

    This paper covers review of recent research on protein crystal properties, nucleation, growth and perfection. Mechanical properties of crystals built of molecules strongly exceeding the range of molecular forces are very different from conventional ones. Similar scaling is responsible for specificity of phase equilibrium for macromolecular systems of which thermodynamics is discussed. Nucleation and growth peculiarity and similarity in protein solutions as compared to inorganic solutions is addressed. Hypotheses on why and when microgravity (lack of convection) conditions may result in more perfect crystals are discussed.

  13. The growth and characterization of membrane protein crystals

    NASA Astrophysics Data System (ADS)

    Garavito, R. Michael; Markovic-Housley, Zora; Jenkins, John A.

    1986-08-01

    A major advance in the study of integral membrane protein structure has been the development of methods for crystallizing these amphiphilic protein species. The crystals were obtained from isotropic solutions of protein and nonionic detergents and contain a substantial amount of detergent bound within the crystal lattice. Standard techniques for crystallizing water-soluble proteins can be applied to membrane proteins if the physical characteristics and behavior of the detergent system used for protein solubilization are adequately controlled. In this report we present the results of some crystallization experiments on porin, a protein forming transmembrane channels in the outer membrane of E. coli, and discuss the detergent-related phenomena which seem to affect the crystallization process.

  14. Protein Crystal Recombinant Human Insulin

    NASA Technical Reports Server (NTRS)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  15. Commercial Protein Crystal Growth: Protein Crystallization Facility (CPCG-H)

    NASA Astrophysics Data System (ADS)

    DeLucas, Lawrence J.

    2002-12-01

    Within the human body, there are thousands of different proteins that serve a variety of different functions, such as making it possible for red blood cells to carry oxygen in our bodies. Yet proteins can also be involved in diseases. Each protein has a particular chemical structure, which means it has a unique shape. It is this three-dimensional shape that allows each protein to do its job by interacting with chemicals or binding with other proteins. If researchers can determine the shape, or shapes, of a protein, they can learn how it works. This information can then be used by the pharmaceutical industry to develop new drugs or improve the way medications work. The NASA Commercial Space Center sponsoring this experiment - the Center for Biophysical Sciences and Engineering at the University of Alabama at Birmingham - has more than 60 industry and academic partners who grow protein crystals and use the information in drug design projects.

  16. Protein crystallization in hydrogel beads.

    PubMed

    Willaert, Ronnie; Zegers, Ingrid; Wyns, Lode; Sleutel, Mike

    2005-09-01

    The use of hydrogel beads for the crystallization of proteins is explored in this contribution. The dynamic behaviour of the internal precipitant, protein concentration and relative supersaturation in a gel bead upon submerging the bead in a precipitant solution is characterized theoretically using a transient diffusion model. Agarose and calcium alginate beads have been used for the crystallization of a low-molecular-weight (14.4 kDa, hen egg-white lysozyme) and a high-molecular-weight (636.0 kDa, alcohol oxidase) protein. Entrapment of the protein in the agarose-gel matrix was accomplished using two methods. In the first method, a protein solution is mixed with the agarose sol solution. Gel beads are produced by immersing drops of the protein-agarose sol mixture in a cold paraffin solution. In the second method (which was used to produce calcium alginate and agarose beads), empty gel beads are first produced and subsequently filled with protein by diffusion from a bulk solution into the bead. This latter method has the advantage that a supplementary purification step is introduced (for protein aggregates and large impurities) owing to the diffusion process in the gel matrix. Increasing the precipitant, gel concentration and protein loading resulted in a larger number of crystals of smaller size. Consequently, agarose as well as alginate gels act as nucleation promoters. The supersaturation in a gel bead can be dynamically controlled by changing the precipitant and/or the protein concentration in the bulk solution. Manipulation of the supersaturation allowed the nucleation rate to be varied and led to the production of large crystals which were homogeneously distributed in the gel bead.

  17. Compact Apparatus Grows Protein Crystals

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.; Delucas, Lawrence J.; Suddath, Fred L.; Snyder, Robert S.; Herren, Blair J.; Carter, Daniel C.; Yost, Vaughn H.

    1989-01-01

    Laboratory apparatus provides delicately balanced combination of materials and chemical conditions for growth of protein crystals. Apparatus and technique for growth based on hanging-drop method for crystallization of macromolecules. Includes pair of syringes with ganged plungers. One syringe contains protein solution; other contains precipitating-agent solution. Syringes intrude into cavity lined with porous reservoir material saturated with 1 mL or more of similar precipitating-agent solution. Prior to activation, ends of syringes plugged to prevent transport of water vapor among three solutions.

  18. Protein Crystal Serum Albumin

    NASA Technical Reports Server (NTRS)

    1998-01-01

    As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  19. Protein crystallization studies

    NASA Technical Reports Server (NTRS)

    Lyne, James Evans

    1996-01-01

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

  20. Overexpression, Isolation, and Crystallization of Proteins

    NASA Astrophysics Data System (ADS)

    Skelly, Jane V.; Madden, C. Bernadette

    Rapid developments in recombinant technology have made it possible to overproduce selected proteins of specific interest to the levels required for structural analysis by X-ray crystallography. High-level gene expression has facilitated the purification of many proteins that are normally only expressed at low concentrations, as well as those that have proven difficult to purify to homogeneity from natural sources. Furthermore, advances in oligonucleotide site-directed mutagenesis have enabled proteins to be engineered so as to possess certain features that may confer stability or assist in then isolation. There are several examples of proteins that, despite rigorous purification from their natural source, have defied crystallization attempts, e.g., human growth hormone, but have been successfully crystallized from recombinant sources (1). The lack of posttranslational processing in bacterial expressed proteins can often be an advantage to the crystallographer where microheterogeneity presents a problem. Indeed, certain features or residues of a protein that are believed to impede crystal formation by preventing a close-packing arrangement may be successfully deleted by genetic manipulation without destroying its essential functionality (2).

  1. Preliminary investigations of protein crystal growth using the Space Shuttle

    NASA Technical Reports Server (NTRS)

    Delucas, L. J.; Suddath, F. L.; Snyder, R.; Naumann, R.; Broom, M. B.; Pusey, M.; Yost, V.; Herren, B .; Carter, D.

    1986-01-01

    Four preliminary Shuttle experiments are described which have been used to develop prototype hardware for a more advanced system that will evaluate effects of gravity on protein crystal growth. The first phase of these experiments has centered on the development of micromethods for protein crystal growth by vapor-diffusion techniques (using a space version of the hanging-drop method) and on dialysis using microdialysis cells. Results suggest that the elimination of density-driven sedimentation can effect crystal morphology. In the dialysis experiment, space-grown crystals of concanavalin B were three times longer and 1/3 the thickness of earth-grown crystals.

  2. Protein and virus crystal growth on international microgravity laboratory-2.

    PubMed Central

    Koszelak, S; Day, J; Leja, C; Cudney, R; McPherson, A

    1995-01-01

    Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth. Images FIGURE 1 FIGURE 2 FIGURE 6 PMID:7669890

  3. Automated protein crystal growth facility

    NASA Technical Reports Server (NTRS)

    Donald, Stacey

    1994-01-01

    A customer for the protein crystal growth facility fills the specially designed chamber with the correct solutions, fills the syringes with their quenching solutions, and submits the data needed for the proper growth of their crystal. To make sure that the chambers and syringes are filled correctly, a NASA representative may assist the customer. The data needed is the approximate growth time, the growth temperature, and the desired crystal size, but this data can be changed anytime from the ground, if needed. The chambers are gathered and placed into numbered slots in special drawers. Then, data is entered into a computer for each of the chambers. Technicians map out when each chamber's growth should be activated so that all of the chambers have enough time to grow. All of this data is up-linked to the space station when the previous growth session is over. Anti-vibrational containers need to be constructed for the high forces encountered during the lift off and the landing of the space shuttle, and though our team has not designed these containers, we do not feel that there is any reason why a suitable one could not be made. When the shuttle reaches the space station, an astronaut removes a drawer of quenched chambers from the growth facility and inserts a drawer of new chambers. All twelve of the drawers can be replaced in this fashion. The optical disks can also be removed this way. The old drawers are stored for the trip back to earth. Once inside the growth facility, a chamber is removed by the robot and placed in one of 144 active sites at a time previously picked by a technician. Growth begins when the chamber is inserted into an active site. Then, the sensing system starts to determine the size of the protein crystal. All during the crystal's growth, the customer can view the crystal and read all of the crystal's data, such as growth rate and crystal size. When the sensing system determines that the crystal has reached the predetermined size, the robot is

  4. Process for Encapsulating Protein Crystals

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.; Mosier, Benjamin

    2003-01-01

    A process for growing protein crystals encapsulated within membranes has been invented. This process begins with the encapsulation of a nearly saturated aqueous protein solution inside semipermeable membranes to form microcapsules. The encapsulation is effected by use of special formulations of a dissolved protein and a surfactant in an aqueous first liquid phase, which is placed into contact with a second, immiscible liquid phase that contains one or more polymers that are insoluble in the first phase. The second phase becomes formed into the semipermeable membranes that surround microglobules of the first phase, thereby forming the microcapsules. Once formed, the microcapsules are then dehydrated osmotically by exposure to a concentrated salt or polymer solution. The dehydration forms supersaturated solutions inside the microcapsules, thereby enabling nucleation and growth of protein crystals inside the microcapsules. By suitable formulation of the polymer or salt solution and of other physical and chemical parameters, one can control the rate of transport of water out of the microcapsules through the membranes and thereby create physicochemical conditions that favor the growth, within each microcapsule, of one or a few large crystals suitable for analysis by x-ray diffraction. The membrane polymer can be formulated to consist of low-molecular-weight molecules that do not interfere with the x-ray diffraction analysis of the encapsulated crystals. During dehydration, an electrostatic field can be applied to exert additional control over the rate of dehydration. This protein-crystal-encapsulation process is expected to constitute the basis of protein-growth experiments to be performed on the space shuttle and the International Space Station. As envisioned, the experiments would involve the exposure of immiscible liquids to each other in sequences of steps under microgravitational conditions. The experiments are expected to contribute to knowledge of the precise

  5. Advancements of vertically aligned liquid crystal displays.

    PubMed

    Kumar, Pankaj; Jaggi, Chinky; Sharma, Vandna; Raina, Kuldeep Kumar

    2016-02-01

    This review describes the recent advancements in the field of the vertical aligned (VA) liquid crystal displays. The process and formation of different vertical alignment modes such as conventional VA, patterned VA, multi-domain VA, and polymer stabilised VA etc are widely discussed. Vertical alignment of liquid crystal due to nano particle dispersion in LC host, bifunctional PR-SAM formed by silane coupling reaction to oxide surfaces, azo dye etc., are also highlighted and discussed. Overall, the article highlights the advances in the research of vertical aligned liquid crystal in terms of their scientific and technological aspects.

  6. Protein Crystal Growth With the Aid of Microfluidics

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark

    2003-01-01

    Protein crystallography is one of three well-known methods to obtain the structure of proteins. A major rate limiting step in protein crystallography is protein crystal nucleation and growth, which is still largely a process conducted by trial-and-error methods. Many attempts have been made to improve protein crystal growth by performing growth in microgravity. Although the use of microgravity appears to improve crystal quality in some attempts, this method has been inefficient because several reasons: we lack a fundamental understanding of macromolecular crystal growth in general and of the influence of microgravity in particular, we have to start with crystal growth conditions in microgravity based on conditions on the ground and finally the hardware does not allow for experimental iteration without reloading samples on the ground. To partially accommodate the disadvantages of the current hardware, we have used microfluidic technology (Lab-on-a-Chip devices) to design the concept of a more efficient crystallization device, suitable for use on the International Space Station and in high-throughput applications on the ground. The concept and properties of microfluidics, the application design process, and the advances in protein crystal growth hardware will be discussed in this presentation. Some examples of proteins crystallized in the new hardware will be discussed, including the differences between conventional crystallization versus crystallization in microfluidics.

  7. Studying how protein crystals form

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Watching molecules of the iron-storing protein apoferritin come together to form a nucleus reveals some interesting behavior. In this series of images, researchers observed clusters of four molecules at the corners of a diamond shape (top). As more molecules attach to the cluster, they arrange themselves into rods (second from top), and a raft-like configuration of molecules forms the critical nucleus (third from top), suggesting that crystal growth is much slower than it could be were the molecules arranged in a more compact formation. In the final image, a crystallite consisting of three layers containing approximately 60 to 70 molecules each is formed. Atomic force microscopy made visualizing the process of nucleation possible for the first time. The principal investigator is Peter Vekilov, of the University of Alabama in Huntsville. Vekilov's team at UAH studies protein solutions as they change phases from liquids to crystalline solids. They want to know if the molecules in the solution interact with one another, and if so, how, from the perspectives of thermodynamics and kinetics. They want to understand which forces -- electrical, electrostatic, hydrodynamic, or other kinds of forces -- are responsible for the interactions. They also study nucleation, the begirning stage of crystallization. This process is important to understand because it sets the stage for crystal growth in all kinds of solutions and liquid melts that are important in such diverse fields as agriculture, medicine, and the fabrication of metal components. Nucleation can determine the rate of crystal growth, the number of crystals that will be formed, and the quality and size of the crystals.

  8. Can Solution Supersaturation Affect Protein Crystal Quality?

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar

    2013-01-01

    The formation of large protein crystals of "high quality" is considered a characteristic manifestation of microgravity. The physical processes that predict the formation of large, high quality protein crystals in the microgravity environment of space are considered rooted in the existence of a "depletion zone" in the vicinity of crystal. Namely, it is considered reasonable that crystal quality suffers in earth-grown crystals as a result of the incorporation of large aggregates, micro-crystals and/or large molecular weight "impurities", processes which are aided by density driven convective flow or mixing at the crystal-liquid interface. Sedimentation and density driven convection produce unfavorable solution conditions in the vicinity of the crystal surface, which promotes rapid crystal growth to the detriment of crystal size and quality. In this effort, we shall further present the hypothesis that the solution supersaturatoin at the crystal surface determines the growth mechanism, or mode, by which protein crystals grow. It is further hypothesized that protein crystal quality is affected by the mechanism or mode of crystal growth. Hence the formation of a depletion zone in microgravity environment is beneficial due to inhibition of impurity incorporatoin as well as preventing a kinetic roughening transition. It should be noted that for many proteins the magnitude of neither protein crystal growth rates nor solution supersaturation are predictors of a kinetic roughening transition. That is, the kinetic roughening transition supersaturation must be dtermined for each individual protein.

  9. (PCG) Protein Crystal Growth Horse Serum Albumin

    NASA Technical Reports Server (NTRS)

    1995-01-01

    Horse Serum Albumin crystals grown during the USML-1 (STS-50) mission's Protein Crystal Growth Glovebox Experiment. These crystals were grown using a vapor diffusion technique at 22 degrees C. The crystals were allowed to grow for nine days while in orbit. Crystals of 1.0 mm in length were produced. The most abundant blood serum protein, regulates blood pressure and transports ions, metabolites, and therapeutic drugs. Principal Investigator was Edward Meehan.

  10. Membrane protein structures without crystals, by single particle electron cryomicroscopy

    PubMed Central

    Vinothkumar, Kutti R

    2015-01-01

    It is an exciting period in membrane protein structural biology with a number of medically important protein structures determined at a rapid pace. However, two major hurdles still remain in the structural biology of membrane proteins. One is the inability to obtain large amounts of protein for crystallization and the other is the failure to get well-diffracting crystals. With single particle electron cryomicroscopy, both these problems can be overcome and high-resolution structures of membrane proteins and other labile protein complexes can be obtained with very little protein and without the need for crystals. In this review, I highlight recent advances in electron microscopy, detectors and software, which have allowed determination of medium to high-resolution structures of membrane proteins and complexes that have been difficult to study by other structural biological techniques. PMID:26435463

  11. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1991-01-01

    The objective of this research is to study the effect of low gravity on the growth of protein crystals and those parameters which will affect growth and crystal quality. The application of graphoepitaxy (artificial epitaxy) to proteins is detailed. The development of a method for the control of nucleation is discussed. The factor affecting the morphology of isocitrate lyase crystals is presented.

  12. Integrated Protein-Crystal-Growing Apparatus

    NASA Technical Reports Server (NTRS)

    Rhodes, Percy H.; Snyder, Robert S.; Pusey, Marc L.

    1991-01-01

    Proposed apparatus for research on growth of protein crystals dispenses drops of protein and precipitating solutions, provides controlled environment for crystalization, and stores crystals. Intended for use in microgravity of outer space, concept of apparatus also useful in design of self-contained terrestrial experiments for remote and/or automatic execution.

  13. Measurements of Protein Crystal Face Growth Rates

    NASA Technical Reports Server (NTRS)

    Gorti, S.

    2014-01-01

    Protein crystal growth rates will be determined for several hyperthermophile proteins.; The growth rates will be assessed using available theoretical models, including kinetic roughening.; If/when kinetic roughening supersaturations are established, determinations of protein crystal quality over a range of supersaturations will also be assessed.; The results of our ground based effort may well address the existence of a correlation between fundamental growth mechanisms and protein crystal quality.

  14. Protein single crystal growth under microgravity

    NASA Astrophysics Data System (ADS)

    Littke, Walter; John, Christina

    1986-08-01

    Crystal growth conditions for proteins under microgravity were investigated with two model compounds (β-galactosidase and lysozyme). The single crystals obtained have been found to be significantly larger than those prepared in the same environment on earth.

  15. Protein crystallization facilitated by molecularly imprinted polymers

    PubMed Central

    Saridakis, Emmanuel; Khurshid, Sahir; Govada, Lata; Phan, Quan; Hawkins, Daniel; Crichlow, Gregg V.; Lolis, Elias; Reddy, Subrayal M.; Chayen, Naomi E.

    2011-01-01

    We present a previously undescribed initiative and its application, namely the design of molecularly imprinted polymers (MIPs) for producing protein crystals that are essential for determining high-resolution 3D structures of proteins. MIPs, also referred to as “smart materials,” are made to contain cavities capable of rebinding protein; thus the fingerprint of the protein created on the polymer allows it to serve as an ideal template for crystal formation. We have shown that six different MIPs induced crystallization of nine proteins, yielding crystals in conditions that do not give crystals otherwise. The incorporation of MIPs in screening experiments gave rise to crystalline hits in 8–10% of the trials for three target proteins. These hits would have been missed using other known nucleants. MIPs also facilitated the formation of large single crystals at metastable conditions for seven proteins. Moreover, the presence of MIPs has led to faster formation of crystals in all cases where crystals would appear eventually and to major improvement in diffraction in some cases. The MIPs were effective for their cognate proteins and also for other proteins, with size compatibility being a likely criterion for efficacy. Atomic force microscopy (AFM) measurements demonstrated specific affinity between the MIP cavities and a protein-functionalized AFM tip, corroborating our hypothesis that due to the recognition of proteins by the cavities, MIPs can act as nucleation-inducing substrates (nucleants) by harnessing the proteins themselves as templates. PMID:21690356

  16. Advanced protein formulations.

    PubMed

    Wang, Wei

    2015-07-01

    It is well recognized that protein product development is far more challenging than that for small-molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long-term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site-specific mutagenesis, and preparation of nontraditional types of dosage forms-semiaqueous solutions, nonfreeze-dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution.

  17. Advanced protein formulations

    PubMed Central

    Wang, Wei

    2015-01-01

    It is well recognized that protein product development is far more challenging than that for small-molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long-term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site-specific mutagenesis, and preparation of nontraditional types of dosage forms—semiaqueous solutions, nonfreeze-dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution. PMID:25858529

  18. Crystal Dehydration in Membrane Protein Crystallography.

    PubMed

    Sanchez-Weatherby, Juan; Moraes, Isabel

    2016-01-01

    Crystal dehydration has been successfully implemented to facilitate the structural solution of a number of soluble and membrane protein structures over the years. This chapter will present the currently available tools to undertake controlled crystal dehydration, focusing on some successful membrane protein cases. Also discussed here will be some practical considerations regarding membrane protein crystals and the relationship between different techniques in order to help researchers to select the most suitable technique for their projects. PMID:27553236

  19. Protein-crystal growth experiment (planned)

    NASA Technical Reports Server (NTRS)

    Fujita, S.; Asano, K.; Hashitani, T.; Kitakohji, T.; Nemoto, H.; Kitamura, S.

    1988-01-01

    To evaluate the effectiveness of a microgravity environment on protein crystal growth, a system was developed using 5 cubic feet Get Away Special payload canister. In the experiment, protein (myoglobin) will be simultaneously crystallized from an aqueous solution in 16 crystallization units using three types of crystallization methods, i.e., batch, vapor diffusion, and free interface diffusion. Each unit has two compartments: one for the protein solution and the other for the ammonium sulfate solution. Compartments are separated by thick acrylic or thin stainless steel plates. Crystallization will be started by sliding out the plates, then will be periodically recorded up to 120 hours by a still camera. The temperature will be passively controlled by a phase transition thermal storage component and recorded in IC memory throughout the experiment. Microgravity environment can then be evaluated for protein crystal growth by comparing crystallization in space with that on Earth.

  20. Gold nanoparticle capture within protein crystal scaffolds.

    PubMed

    Kowalski, Ann E; Huber, Thaddaus R; Ni, Thomas W; Hartje, Luke F; Appel, Karina L; Yost, Jarad W; Ackerson, Christopher J; Snow, Christopher D

    2016-07-01

    DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)∼17 (nitrilotriacetic acid)∼1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was confirmed by single crystal X-ray crystallography. PMID:27264210

  1. Gold nanoparticle capture within protein crystal scaffolds

    NASA Astrophysics Data System (ADS)

    Kowalski, Ann E.; Huber, Thaddaus R.; Ni, Thomas W.; Hartje, Luke F.; Appel, Karina L.; Yost, Jarad W.; Ackerson, Christopher J.; Snow, Christopher D.

    2016-06-01

    DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)~17 (nitrilotriacetic acid)~1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was confirmed by single crystal X-ray crystallography.DNA assemblies have been used to organize inorganic nanoparticles into 3D arrays, with emergent properties arising as a result of nanoparticle spacing and geometry. We report here the use of engineered protein crystals as an alternative approach to biologically mediated assembly of inorganic nanoparticles. The protein crystal's 13 nm diameter pores result in an 80% solvent content and display hexahistidine sequences on their interior. The hexahistidine sequence captures Au25(glutathione)~17 (nitrilotriacetic acid)~1 nanoclusters throughout a chemically crosslinked crystal via the coordination of Ni(ii) to both the cluster and the protein. Nanoparticle loading was validated by confocal microscopy and elemental analysis. The nanoparticles may be released from the crystal by exposure to EDTA, which chelates the Ni(ii) and breaks the specific protein/nanoparticle interaction. The integrity of the protein crystals after crosslinking and nanoparticle capture was

  2. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1989-01-01

    The mechanisms involved in protein crystallization and those parameters which influence the growth process and crystalline perfection were studied. The analysis of the flows around growing crystals is detailed. The preliminary study of the growth of isocitrate lyase and the crystal morphologies found are discussed. Preliminary results of controlled nucleation studies are presented.

  3. Accelerated protein crystal growth by protein thin film template

    NASA Astrophysics Data System (ADS)

    Pechkova, Eugenia; Nicolini, Claudio

    2001-11-01

    A new method based on Langmuir-Blodgett (LB) technology is presented for the template stimulation of protein crystal growth. The new approach allows the acceleration of the hen egg white lysozyme (HEWL) crystal growth rate in comparison with such a classical vapour diffusion method as hanging drop. Protein thin films were coated on the cover slide of the common crystallization plates. Lysozyme crystal growth was observed on the LB thin films of HEWL.

  4. Growth of shaped single crystals of proteins

    NASA Astrophysics Data System (ADS)

    Moreno, Abel; Rondón, Deyanira; García-Ruiz, Juan Ma.

    1996-09-01

    We present a procedure for obtaining protein single crystals that fill the capillary tubes in which they grow. The implementation was typical of the gel acupuncture method and the four different proteins are used as examples: lysozyme (HEW), thaumatin I, ferritin and insulin. Rod- and prismatic-shaped protein single crystals of these four proteins were grown inside capillary tubes of 0.2, 0.3, 0.5 mm in diameter and, for the case of lysozyme, up to 1.2 mm in diameter. The maximum length measured along the long axes of the rod crystals was 1.6 mm again for lysozyme crystals. It was observed that, once the capillary tube was filled, the crystal continues to grow by diffusion of the precipitating agent throughout the porous network formed by the protein crystal structure. We also discuss the possibility of growing these cylinders of crystalline proteins by the addition of protein solution to the mother liquor through the upper end of the glass capillary while the precipitating agent diffuses through the protein crystal itself. X-ray diffraction patterns confirm the single crystal character of the protein rods.

  5. Advances in understanding damage by salt crystallization.

    PubMed

    Espinosa-Marzal, Rosa M; Scherer, George W

    2010-06-15

    The single most important cause of the deterioration of monuments in the Mediterranean basin, and elsewhere around the world, is the crystallization of salt within the pores of the stone. Considerable advances have been made in recent years in elucidating the fundamental mechanisms responsible for salt damage. As a result, new methods of treatment are being proposed that offer the possibility of attacking the cause of the problem, rather than simply treating the symptoms. In this Account, we review the thermodynamics and kinetics of crystallization, then examine how a range of technological innovations have been applied experimentally to further the current understanding of in-pore crystallization. We close with a discussion of how computer modeling now provides particularly valuable insight, including quantitative estimates of both the interaction forces between the mineral and the crystal and the stresses induced in the material. Analyzing the kinetics and thermodynamics of crystal growth within the pores of a stone requires sensitive tools used in combination. For example, calorimetry quantifies the amount of salt that precipitates in the pores of a stone during cooling, and dilatometric measurements on a companion sample reveal the stress exerted by the salt. Synchrotron X-rays can penetrate the stone and identify the metastable phases that often appear in the first stages of crystallization. Atomic force microscopy and environmental scanning electron microscopy permit study of the nanometric liquid film that typically lies between salt and stone; this film controls the magnitude of the pressure exerted and the kinetics of relaxation of the stress. These experimental advances provide validation for increasingly advanced simulations, using continuum models of reactive transport on a macroscopic scale and molecular dynamics on the atomic scale. Because of the fundamental understanding of the damage mechanisms that is beginning to emerge, it is possible to devise

  6. The MORPHEUS II protein crystallization screen.

    PubMed

    Gorrec, Fabrice

    2015-07-01

    High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions.

  7. Approaches to automated protein crystal harvesting

    PubMed Central

    Deller, Marc C.; Rupp, Bernhard

    2014-01-01

    The harvesting of protein crystals is almost always a necessary step in the determination of a protein structure using X-ray crystallographic techniques. However, protein crystals are usually fragile and susceptible to damage during the harvesting process. For this reason, protein crystal harvesting is the single step that remains entirely dependent on skilled human intervention. Automation has been implemented in the majority of other stages of the structure-determination pipeline, including cloning, expression, purification, crystallization and data collection. The gap in automation between crystallization and data collection results in a bottleneck in throughput and presents unfortunate opportunities for crystal damage. Several automated protein crystal harvesting systems have been developed, including systems utilizing microcapillaries, microtools, microgrippers, acoustic droplet ejection and optical traps. However, these systems have yet to be commonly deployed in the majority of crystallography laboratories owing to a variety of technical and cost-related issues. Automation of protein crystal harvesting remains essential for harnessing the full benefits of fourth-generation synchrotrons, free-electron lasers and microfocus beamlines. Furthermore, automation of protein crystal harvesting offers several benefits when compared with traditional manual approaches, including the ability to harvest microcrystals, improved flash-cooling procedures and increased throughput. PMID:24637746

  8. Approaches to automated protein crystal harvesting.

    PubMed

    Deller, Marc C; Rupp, Bernhard

    2014-02-01

    The harvesting of protein crystals is almost always a necessary step in the determination of a protein structure using X-ray crystallographic techniques. However, protein crystals are usually fragile and susceptible to damage during the harvesting process. For this reason, protein crystal harvesting is the single step that remains entirely dependent on skilled human intervention. Automation has been implemented in the majority of other stages of the structure-determination pipeline, including cloning, expression, purification, crystallization and data collection. The gap in automation between crystallization and data collection results in a bottleneck in throughput and presents unfortunate opportunities for crystal damage. Several automated protein crystal harvesting systems have been developed, including systems utilizing microcapillaries, microtools, microgrippers, acoustic droplet ejection and optical traps. However, these systems have yet to be commonly deployed in the majority of crystallography laboratories owing to a variety of technical and cost-related issues. Automation of protein crystal harvesting remains essential for harnessing the full benefits of fourth-generation synchrotrons, free-electron lasers and microfocus beamlines. Furthermore, automation of protein crystal harvesting offers several benefits when compared with traditional manual approaches, including the ability to harvest microcrystals, improved flash-cooling procedures and increased throughput. PMID:24637746

  9. Insecticidal crystal proteins of Bacillus thuringiensis.

    PubMed Central

    Höfte, H; Whiteley, H R

    1989-01-01

    A classification for crystal protein genes of Bacillus thuringiensis is presented. Criteria used are the insecticidal spectra and the amino acid sequences of the encoded proteins. Fourteen genes are distinguished, encoding proteins active against either Lepidoptera (cryI), Lepidoptera and Diptera (cryII), Coleoptera (cryIII), or Diptera (cryIV). One gene, cytA, encodes a general cytolytic protein and shows no structural similarities with the other genes. Toxicity studies with single purified proteins demonstrated that every described crystal protein is characterized by a highly specific, and sometimes very restricted, insect host spectrum. Comparison of the deduced amino acid sequences reveals sequence elements which are conserved for Cry proteins. The expression of crystal protein genes is affected by a number of factors. Recently, two distinct sigma subunits regulating transcription during different stages of sporulation have been identified, as well as a protein regulating the expression of a crystal protein at a posttranslational level. Studies on the biochemical mechanisms of toxicity suggest that B. thuringiensis crystal proteins induce the formation of pores in membranes of susceptible cells. In vitro binding studies with radiolabeled toxins demonstrated a strong correlation between the specificity of B. thuringiensis toxins and the interaction with specific binding sites on the insect midgut epithelium. The expression of B. thuringiensis crystal proteins in plant-associated microorganisms and in transgenic plants has been reported. These approaches are potentially powerful strategies for the protection of agriculturally important crops against insect damage. Images PMID:2666844

  10. Crystallization of viruses and virus proteins

    NASA Astrophysics Data System (ADS)

    Sehnke, Paul C.; Harrington, Melissa; Hosur, M. V.; Li, Yunge; Usha, R.; Craig Tucker, R.; Bomu, Wu; Stauffacher, Cynthia V.; Johnson, John E.

    1988-07-01

    Methods for crystallizing six isometric plant and insect viruses are presented. Procedures developed for modifying, purifying and crystallizing coat protein subunits isolated from a virus forming asymmetric, spheroidal particles, stabilized almost exclusively by protein-RNA interactions, are also discussed. The tertiary and quaternary structures of small RNA viruses are compared.

  11. Protein crystal growth (5-IML-1)

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1992-01-01

    Proteins (enzymes, hormones, immunoglobulins) account for 50 pct. or more of the dry weight of most living systems. A detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting projects have terminated at the crystal growth stage. In principle, there are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor is the elimination of density driven convective flow. Other factors that can be controlled in the absence of gravity is the sedimentation of growing crystals in a gravitational field, and the potential advantage of doing containerless crystal growth. As a result of these theories and facts, one can readily understand why the microgravity environment of an Earth orbiting vehicle seems to offer unique opportunities for the protein crystallographer. This perception has led to the establishment of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project. The results of experiments already performed during STS missions have in many cases resulted in large protein crystals which are structurally correct. Thus, the near term objective of the PCG/ME project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

  12. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1990-01-01

    The effect of low gravity on the growth of protein crystals and those parameters which will affect growth and crystal quality was studied. The proper design of the flight hardware and experimental protocols are highly dependent on understanding the factors which influence the nucleation and growth of crystals of biological macromolecules. Thus, those factors are investigated and the body of knowledge which has been built up for small molecule crystallization. These data also provide a basis of comparison for the results obtained from low-g experiments. The flows around growing crystals are detailed. The preliminary study of the growth of isocitrate lyase, the crystal morphologies found and the preliminary x ray results are discussed. The design of two apparatus for protein crystal growth by temperature control are presented along with preliminary results.

  13. The MORPHEUS II protein crystallization screen

    SciTech Connect

    Gorrec, Fabrice

    2015-06-27

    MORPHEUS II is a 96-condition initial crystallization screen formulated de novo. The screen incorporates reagents selected from the Protein Data Bank to yield crystals that are not observed in traditional conditions. In addition, the formulation facilitates the optimization and cryoprotection of crystals. High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions.

  14. The Protein Crystallization Facility STS-95

    NASA Technical Reports Server (NTRS)

    2004-01-01

    The Protein Crystallization Facility will be used to grow crystals of human insulin. Insulin is the primary treatment for diabetes, the fourth leading cause of death by disease. Research on STS-95 is aimed at producing crystals of even higher quality, which when combined with new analysis techniques will permit a better understanding of the interaction between insulin and its receptor. This has the potential to aid in the development of a new commercially available insulin product with unique time release properties that could reduce fluctuations in a patient's blood sugar level. The Protein Crystallization Facility supports large-scale commercial investigations.

  15. Electrostatic Stabilization Of Growing Protein Crystals

    NASA Technical Reports Server (NTRS)

    Shlichta, Paul J.

    1991-01-01

    Proposed technique produces large crystals in compact, economical apparatus. Report presents concept for supporting protein crystals during growth in microgravity. Yields crystals larger and more-nearly perfect than those grown on Earth. Combines best features of sandwich-drop and electrostatic-levitation methods of support. Drop of protein solution inserted between pair of glass or plastic plates, as in sandwich-drop-support method. Electrostatically charged ring confines drop laterally and shapes it, as in electrostatic technique. Apparatus also made to accommodate several drops simultaneously between same pair of supporting plates. Drops can be inserted and crystals removed through ducts in plates.

  16. Protein Crystallization Using Room Temperature Ionic Fluids

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Paley, Mark Steve; Turner, Megan B.; Rogers, Robin D.

    2006-01-01

    The ionic liquids (ILs) 1-butyl-3-methylimidizolium chloride (C4mim-C1), 1-butyl-3- methylimidizolium diethyleneglycol monomethylethersulfate ([C4mim]DEMGS), and 1-butyl-1 -methylpyrollidinium dihydrogenphosphate ([p1,4]dhp) were tested for their effects on the crystallization of the proteins canavalin, beta-lactoglobulin B, xylanase, and glucose isomerase, using a standard high throughput screen. The crystallization experiments were set up with the ILs added to the protein solutions at 0.2 and 0.4 M final concentrations. Crystallization droplets were set up at three proteixprecipitant ratios (1:1, 2:1, and 4:l), which served to progressively dilute the effects of the screen components while increasing the equilibrium protein and IL concentrations. Crystals were obtained for all four proteins at a number of conditions where they were not obtained from the IL-free control experiment. Over half of the protein-IL combinations tested had more successful outcomes than negative, where the IL-free crystallization was better than the corresponding IL-containing outcome, relative to the control. One of the most common causes of a negative outcome was solubilization of the protein by the IL, resulting in a clear drop. In one instance, we were able to use the IL-induced solubilizing to obtain beta-lactoglobulin B crystals from conditions that gave precipitated protein in the absence of IL. The results suggest that it may be feasible to develop ILs specifically for the task of macromolecule crystallization.

  17. Crystallization of Membrane Proteins by Vapor Diffusion

    PubMed Central

    Delmar, Jared A.; Bolla, Jani Reddy; Su, Chih-Chia; Yu, Edward W.

    2016-01-01

    X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to expression and diffraction, eventually producing high quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization. PMID:25950974

  18. (PCG) Protein Crystal Growth Porcine Elastase

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Porcine Elastase. This enzyme is associated with the degradation of lung tissue in people suffering from emphysema. It is useful in studying causes of this disease. Principal Investigator on STS-26 was Charles Bugg.

  19. The Nucleation and Growth of Protein Crystals

    NASA Technical Reports Server (NTRS)

    Pusey, Marc

    2004-01-01

    Obtaining crystals of suitable size and high quality continues to be a major bottleneck in macromolecular crystallography. Currently, structural genomics efforts are achieving on average about a 10% success rate in going from purified protein to a deposited crystal structure. Growth of crystals in microgravity was proposed as a means of overcoming size and quality problems, which subsequently led to a major NASA effort in microgravity crystal growth, with the agency also funding research into understanding the process. Studies of the macromolecule crystal nucleation and growth process were carried out in a number of labs in an effort to understand what affected the resultant crystal quality on Earth, and how microgravity improved the process. Based upon experimental evidence, as well as simple starting assumptions, we have proposed that crystal nucleation occurs by a series of discrete self assembly steps, which 'set' the underlying crystal symmetry. This talk will review the model developed, and its origins, in our laboratory for how crystals nucleate and grow, and will then present, along with preliminary data, how we propose to use this model to improve the success rate for obtaining crystals from a given protein.

  20. Protein crystallization on liquid surfaces: Forced versus natural crystallization

    NASA Astrophysics Data System (ADS)

    Hirsa, A.

    2005-11-01

    Two-dimensional crystallization of proteins has recently been reported where streptavidin protein dissolved in the bulk liquid anchors to binding sites on a biotinylated lipid monolayer initially spread on the liquid surface. Thermodynamic aspects investigated include the effects of subphase buffer and pH, dilution of bulk protein and monolayer. Here, we investigate three possible avenues where flow can influence protein crystallization: i) change the initial state of monolayer, ii) advect dissolved protein to the interface, iii) apply direct hydrodynamic force on the crystals at the interface. The flow system consists of a stationary open cylinder driven by constant rotation of the floor, in the axisymmetric flow regime with inertia. Direct imaging of the interface illuminated by forward scattering of a laser was utilized to avoid labeling proteins for conventional fluorescence microscopy. These images provide greater detail than Brewster angle microscopy. Scientific motivation is to use flow to probe protein structure, and the application is to make designer protein thin-films, e.g. for biosensors.

  1. Can Supersaturation Affect Protein Crystal Quality?

    NASA Technical Reports Server (NTRS)

    Gorti, Sridhar

    2013-01-01

    In quiescent environments (microgravity, capillary tubes, gels) formation of a depletion zone is to be expected, due either to limited sedimentation, density driven convection or a combination of both. The formation of a depletion zone can: Modify solution supersaturation near crystal; Give rise to impurity partitioning. It is conjectured that both supersaturation and impurity partitioning affect protein crystal quality and size. Further detailed investigations on various proteins are needed to assess above hypothesis.

  2. Two puzzling aspects of protein crystal growth

    NASA Technical Reports Server (NTRS)

    Grant, M. L.; Saville, D. A.

    1988-01-01

    A study is presented of several mechanisms which may reduce crystal growth rates and or terminate crystal growth. It is found that salt gradients which change the local chemical potential of the protein are insufficient to account for the slow crystal growth rates which have been reported. Contaminants which adsorb protein from solution may reduce the effective protein concentration, but the impurity's concentration and its affinity for protein are unknown. Association of protein molecules in bulk solution can reduce the monomer concentration significantly, but extant theory and experiment are not sensitive enough to determine the actual concentration of aggregates in solution. For systems of interest, shear-induced effects were found to be too weak to interfere with normal binding of incoming protein molecules. Although we found that most crystal growth occurs in a regime where both interfacial kinetics and diffusion influence crystal growth, the role of mass transfer rates on the terminal size of crystals is unknown, primarily because no data exist which cover the size range of interest (0.1 mm to 1 mm in length).

  3. Trace fluorescent labeling for protein crystallization

    PubMed Central

    Pusey, Marc; Barcena, Jorge; Morris, Michelle; Singhal, Anuj; Yuan, Qunying; Ng, Joseph

    2015-01-01

    Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent. PMID:26144224

  4. On the elementary processes of protein crystallization: Bond selection mechanism

    NASA Astrophysics Data System (ADS)

    Nanev, Christo N.

    2014-09-01

    The paper explores the application of bond selection mechanism (BSM) in protein crystal growth; previously, BSM was employed to explain the slow rate of protein crystal nucleation, equilibrium crystal shape and energy barrier in nucleus formation (C.N. Nanev, Prog. Cryst. Growth Charact. Mater. 59 (2013) 133-169). Now, the elementary growth processes are considered from BSM perspective and the crystal growth shape is tackled, the latter resulting from a strong directional kinetic anisotropy in step advancement rates in different crystallographic directions. The most significant surface patterns of growing protein crystals, such as two-dimensional nuclei and growth spiral shapes observed by atomic force microscopy (AFM), are also considered. The activation barrier associated with entering of a protein molecule into the kink site is evaluated and the start of the kinetic roughening is established. Crystal lattice bond energies are estimated (being well above the thermal energy, kBT) from the supersaturation dependence of 2D- into 1D-nuclei transformation.

  5. Convection effects in protein crystal growth

    NASA Technical Reports Server (NTRS)

    Roberts, Glyn O.

    1988-01-01

    Protein crystals for X-ray diffraction study are usually grown resting on the bottom of a hanging drop of a saturated protein solution, with slow evaporation to the air in a small enclosed cell. The evaporation rate is controlled by hanging the drop above a reservoir of water, with its saturation vapor pressure decreased by a low concentration of a passive solute. The drop has a lower solute concentration, and its volume shrinks by evaporation until the molecular concentrations match. Protein crystals can also be grown from a seed crystal suspended or supported in the interior of a supersaturated solution. The main analysis of this report concerns this case because it is less complicated than hanging-drop growth. Convection effects have been suggested as the reason for the apparent cessation of growth at a certain rather small crystal size. It seeems that as the crystal grows, the number of dislocations increases to a point where further growth is hindered. Growth in the microgravity environment of an orbiting space vehicle has been proposed as a method for obtaining larger crystals. Experimental observations of convection effects during the growth of protein crystals have been reported.

  6. Single-crystal superalloy drives turbine advances

    SciTech Connect

    Harris, K.

    1995-04-01

    In searching for ways to improve power-to-weight ratios and fuel efficiency, gas turbine engine manufacturers invest heavily in the development and testing of new alloys. Their goal is to find turbine airfoil materials that can handle the higher operating temperatures, increased component stresses, and faster rotational speeds that are needed to increase turbine performance. Major turbine engine manufacturers find they can achieve these objectives through ultra-high performance, single-crystal superalloys -- a group of nickel-base materials that exhibit outstanding strength and surface stability at temperatures up to 85{percent} of their melting points. One such superalloy is CMSX-4, co-engineered by ingot maker Cannon-Muskegon and turbine engine manufacturers Rolls-Royce and Allison Engine Company. It is currently being used in such applications as Allison`s advanced airfoil programs.

  7. Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation

    SciTech Connect

    Collins, Bernard; Stevens, Raymond C.; Page, Rebecca

    2005-12-01

    It is shown how protein crystallization results can be used to identify buffers that improve protein solubility and, in turn, crystallization success. An optimal solubility screen is described that uses the results of crystallization trials to identify buffers that improve protein solubility and, in turn, crystallization success. This screen is useful not only for standard crystallization experiments, but also can easily be implemented into any high-throughput structure-determination pipeline. As a proof of principle, the predicted novel-fold protein AF2059 from Archaeoglobus fulgidus, which was known to precipitate in most buffers and particularly during concentration experiments, was selected. Using the crystallization results of 192 independent crystallization trials, it was possible to identify a buffer containing 100 mM CHES pH 9.25 that significantly improves its solubility. After transferring AF2059 into this ‘optimum-solubility’ buffer, the protein was rescreened for crystal formation against these same 192 conditions. Instead of extensive precipitation, as observed initially, it was found that 24 separate conditions produced crystals and the exchange of AF2059 into CHES buffer significantly improved crystallization success. Fine-screen optimization of these conditions led to the production of a crystal suitable for high-resolution (2.2 Å) structure determination.

  8. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1993-01-01

    This Final Technical Report for NASA Grant NAG8-774 covers the period from April 27, 1989 through December 31, 1992. It covers five main topics: fluid flow studies, the influence of growth conditions on the morphology of isocitrate lyase crystals, control of nucleation, the growth of lysozyme by the temperature gradient method and graphoepitaxy of protein crystals. The section on fluid flow discusses the limits of detectability in the Schlieren imaging of fluid flows around protein crystals. The isocitrate lyase study compares crystals grown terrestrially under a variety of conditions with those grown in space. The controlling factor governing the morphology of the crystals is the supersaturation. The lack of flow in the interface between the drop and the atmosphere in microgravity causes protein precipitation in the boundary layer and a lowering of the supersaturation in the drop. This lowered supersaturation leads to improved crystal morphology. Preliminary experiments with lysozyme indicated that localized temperature gradients could be used to nucleate crystals in a controlled manner. An apparatus (thermonucleator) was designed to study the controlled nucleation of protein crystals. This apparatus has been used to nucleate crystals of materials with both normal (ice-water, Rochelle salt and lysozyme) and retrograde (horse serum albumin and alpha chymotrypsinogen A) solubility. These studies have lead to the design of an new apparatus that small and more compatible with use in microgravity. Lysozyme crystals were grown by transporting nutrient from a source (lysozyme powder) to the crystal in a temperature gradient. The influence of path length and cross section on the growth rate was demonstrated. This technique can be combined with the thermonucleator to control both nucleation and growth. Graphoepitaxy utilizes a patterned substrate to orient growing crystals. In this study, silicon substrates with 10 micron grooves were used to grow crystals of catalase

  9. Trace fluorescent labeling for protein crystallization

    SciTech Connect

    Pusey, Marc Barcena, Jorge; Morris, Michelle; Singhal, Anuj; Yuan, Qunying; Ng, Joseph

    2015-06-27

    The presence of a covalently bound fluorescent probe at a concentration of <0.5% does not affect the outcome of macromolecule crystallization screening experiments. Additionally, the fluorescence can be used to determine new, not immediately apparent, lead crystallization conditions. Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent.

  10. Crystallization of Membrane protein under Microgravity

    NASA Astrophysics Data System (ADS)

    Henning, C.; Frank, J.; Laubender, G.; Fromme, P.

    2002-01-01

    Proteins are biological molecules which catalyse all essential reactions of cells. The knowledge on the structure of these molecular machines is necessary for the understanding of their function. Many diseases are caused by defects of membrane proteins. In order to develop new medical therapies the construction principle of the proteins must be known. The main difficulty in the determination of the structure of these membrane protein complexes is the crystallisation. Membrane proteins are normally not soluble in water and have therefore to be solubilised from the membranes by use of detergents. The whole protein-detergent micelle must be crystallised to maintain the functional integrity of the protein complexes. These difficulties are the reasons for the fact that crystals of membrane proteins are difficult to grow and most of them are badly ordered, being not appropriate for X-ray structure analysis. The crystallisation of proteins under microgravity leads to the growth of better-ordered crystals by reduction of nucleation rate and the undisturbed growth of the hovering seeds by the absence of sedimentation and convection. The successful crystallistation of a membrane protein under microgravity has been performed during the space shuttle missions USML2 and STS95 in the Space Shuttle with Photosystem I as model protein. Photosystem I is a large membrane protein complex which catalyses one of the first and fundamental steps in oxygen photosynthesis. The crystals of Photosystem I, grown under microgravity were twenty times larger than all Photosystem I crystals which have been grown on earth. They were the basis for the determination of an improved X-ray structure of Photo- system I. These experiments opened the way for the structure enlightenment of more membrane proteins on the basis of microgravity experiments. On board of the International Space Station ideal conditions for the crystallisation of proteins under zero gravity are existing.

  11. Crystallizing Membrane Proteins Using Lipidic Mesophases

    PubMed Central

    Caffrey, Martin; Cherezov, Vadim

    2009-01-01

    A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and α-helical and β-barrel proteins. Its most recent successes are the human engineered β2-adrenergic and adenosine A2A G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase, and for setting up crystallizations in manual mode. Methods for harvesting micro-crystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour. PMID:19390528

  12. Protein Crystal Growth Dynamics and Impurity Incorporation

    NASA Technical Reports Server (NTRS)

    Chernov, Alex A.; Thomas, Bill

    2000-01-01

    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

  13. Crystallization and Structure Analysis of Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Newman, Richard

    In recent years, there has been great progress in the determination of high-resolution three-dimensional (3D) structures of membrane proteins. The first major breakthrough came with the crystallization (1) and X-ray crystallography (2,3) of the bacterial photosynthetic reaction center (see refs. 4 and 5 for reviews). The structure of another, entirely different membrane protein, the bacterial outer membrane porin from Rhodobacter capsulatus, has now been determined by X-ray crystallography (6). Recent results by electron crystallography of two-dimensional (2D) crystals have been most encouraging. The high-resolution 3D structure of bacteriorhodopsin (7) plant light-harvesting complex (8) and projection maps of several other membrane proteins at similar resolutions (9-11) have been obtained by this technique. Electron crystallography seems particularly appropriate for membrane proteins that are prone to form 2D crystals, and it is hoped that many more structures will be determined in this way.

  14. Liquid nitrogen dewar for protein crystal growth

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Gaseous Nitrogen Dewar apparatus developed by Dr. Alex McPherson of the University of California, Irvine for use aboard Mir and the International Space Station allows large quantities of protein samples to be crystallized in orbit. The specimens are contained either in plastic tubing (heat-sealed at each end). Biological samples are prepared with a precipitating agent in either a batch or liquid-liquid diffusion configuration. The samples are then flash-frozen in liquid nitrogen before crystallization can start. On orbit, the Dewar is placed in a quiet area of the station and the nitrogen slowly boils off (it is taken up by the environmental control system), allowing the proteins to thaw to begin crystallization. The Dewar is returned to Earth after one to four months on orbit, depending on Shuttle flight opportunities. The tubes then are analyzed for crystal presence and quality

  15. Nucleation and growth control in protein crystallization

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Nyce, Thomas A.; Meehan, Edward J.; Sowers, Jennifer W.; Monaco, Lisa A.

    1990-01-01

    The five topics summarized in this final report are as follows: (1) a technique for the expedient, semi-automated determination of protein solubilities as a function of temperature and application of this technique to proteins other than lysozyme; (2) a small solution cell with adjustable temperature gradients for the growth of proteins at a predetermined location through temperature programming; (3) a microscopy system with image storage and processing capability for high resolution optical studies of temperature controlled protein growth and etching kinetics; (4) growth experiments with lysozyme in thermosyphon flow ; and (5) a mathematical model for the evolution of evaporation/diffusion induced concentration gradients in the hanging drop protein crystallization technique.

  16. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1992-01-01

    A study is presented of the crystallization of isocitrate lyase (ICL) and the influence of the lack of thermal solutal convection in microgravity on the morphology of ICL crystals is discussed. The latest results of studies with thermonucleation are presented. These include the nucleation of a protein with retrograde solubility and an unknown solubility curve. A new design for a more microgravity compatible thermonuclear is presented.

  17. Magnetic Control of Convection during Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Ramachandran, N.; Leslie, F. W.

    2004-01-01

    An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular Crystals for diffraction analyses has been the central focus for bio-chemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and Sedimentation as is achieved in "microgravity", we have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, f o d o n of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with counteracts on for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility

  18. Monitoring and validating active site redox states in protein crystals.

    PubMed

    Antonyuk, Svetlana V; Hough, Michael A

    2011-06-01

    High resolution protein crystallography using synchrotron radiation is one of the most powerful tools in modern biology. Improvements in resolution have arisen from the use of X-ray beamlines with higher brightness and flux and the development of advanced detectors. However, it is increasingly recognised that the benefits brought by these advances have an associated cost, namely deleterious effects of X-ray radiation on the sample (radiation damage). In particular, X-ray induced reduction and damage to redox centres has been shown to occur much more rapidly than other radiation damage effects, such as loss of resolution or damage to disulphide bridges. Selection of an appropriate combination of in-situ single crystal spectroscopies during crystallographic experiments, such as UV-visible absorption and X-ray absorption spectroscopy (XAFS), allows for effective monitoring of redox states in protein crystals in parallel with structure determination. Such approaches are also essential in cases where catalytic intermediate species are generated by exposure to the X-ray beam. In this article, we provide a number of examples in which multiple single crystal spectroscopies have been key to understanding the redox status of Fe and Cu centres in crystal structures. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

  19. Rotating Vessels for Growing Protein Crystals

    NASA Technical Reports Server (NTRS)

    Cottingham, Paul

    2005-01-01

    Rotating vessels have been proposed as means of growing larger, more nearly uniform protein crystals than would otherwise be possible in the presence of normal Earth gravitation. Heretofore, nonrotating vessels have been used. It is difficult to grow high-quality protein crystals in the terrestrial gravitational field because of convection plumes created by the interaction between gravitation and density gradients in protein-solution depletion layers around growing crystals. The density gradients and the associated convection plumes cause the surfaces of growing crystals to be exposed to nonuniform solution densities, thereby causing the crystals to form in irregular shapes. The microgravitational environment of outer space has been utilized to eliminate gravitation-induced convection, but this approach is generally not favorable because of the high cost and limited availability of space flight. The use of a rotating vessel according to the proposal is intended to ameliorate the effects of gravitation and the resultant convection, relative to the corresponding effects in a non-rotating vessel. The rotation would exert an averaging effect over time, distributing the convective force on the depletion layer. Therefore, the depletion layer would be more nearly uniform and, as a result, the growing crystal would be more nearly perfect. The proposal admits of variations (see figure), including the following: The growing crystal could be rotated about its own central axis or an external axis. The crystal-growth vessel could be of any of various shapes, including cylindrical, hemispherical, conical, and combinations thereof. The crystal-growth vessel could be suspended in a viscous fluid in an outer vessel to isolate the growing crystal from both ambient vibrations and vibrations induced by a mechanism that drives the rotation. The rotation could be coupled to the crystal-growth vessel by viscous or magnetic means. The crystal-growth vessel could be supported within the

  20. Cry Protein Crystals: A Novel Platform for Protein Delivery

    PubMed Central

    Bonnegarde-Bernard, Astrid; Wallace, Julie A.; Dean, Donald H.; Ostrowski, Michael C.; Burry, Richard W.; Boyaka, Prosper N.; Chan, Michael K.

    2015-01-01

    Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues. PMID:26030844

  1. Cry protein crystals: a novel platform for protein delivery.

    PubMed

    Nair, Manoj S; Lee, Marianne M; Bonnegarde-Bernard, Astrid; Wallace, Julie A; Dean, Donald H; Ostrowski, Michael C; Burry, Richard W; Boyaka, Prosper N; Chan, Michael K

    2015-01-01

    Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer's patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues. PMID:26030844

  2. Protein crystal growth - Growth kinetics for tetragonal lysozyme crystals

    NASA Technical Reports Server (NTRS)

    Pusey, M. L.; Snyder, R. S.; Naumann, R.

    1986-01-01

    Results are reported from theoretical and experimental studies of the growth rate of lysozyme as a function of diffusion in earth-gravity conditions. The investigations were carried out to form a comparison database for future studies of protein crystal growth in the microgravity environment of space. A diffusion-convection model is presented for predicting crystal growth rates in the presence of solutal concentration gradients. Techniques used to grow and monitor the growth of hen egg white lysozyme are detailed. The model calculations and experiment data are employed to discuss the effects of transport and interfacial kinetics in the growth of the crystals, which gradually diminished the free energy in the growth solution. Density gradient-driven convection, caused by presence of the gravity field, was a limiting factor in the growth rate.

  3. Advanced single crystal for SSME turbopumps

    NASA Technical Reports Server (NTRS)

    Fritzemeier, L. G.

    1989-01-01

    The objective of this program was to evaluate the influence of high thermal gradient casting, hot isostatic pressing (HIP) and alternate heat treatments on the microstructure and mechanical properties of a single crystal nickel base superalloy. The alloy chosen for the study was PWA 1480, a well characterized, commercial alloy which had previously been chosen as a candidate for the Space Shuttle Main Engine high pressure turbopump turbine blades. Microstructural characterization evaluated the influence of casting thermal gradient on dendrite arm spacing, casting porosity distribution and alloy homogeneity. Hot isostatic pressing was evaluated as a means of eliminating porosity as a preferred fatigue crack initiation site. The alternate heat treatment was chosen to improve hydrogen environment embrittlement resistance and for potential fatigue life improvement. Mechanical property evaluation was aimed primarily at determining improvements in low cycle and high cycle fatigue life due to the advanced processing methods. Statistically significant numbers of tests were conducted to quantitatively demonstrate life differences. High thermal gradient casting improves as-cast homogeneity, which facilitates solution heat treatment of PWA 1480 and provides a decrease in internal pore size, leading to increases in low cycle and high cycle fatigue lives.

  4. Searching for the Best Protein Crystals: Synchrotron Based Measurements of Protein Crystal Quality

    NASA Technical Reports Server (NTRS)

    Borgstahl, Gloria; Snell, Edward H.; Bellamy, Henry; Pangborn, Walter; Nelson, Chris; Arvai, Andy; Ohren, Jeff; Pokross, Matt

    1999-01-01

    We are developing X-ray diffraction methods to quantitatively evaluate the quality of protein crystals. The ultimate use for these crystal quality will be to optimize crystal growth and freezing conditions to obtain the best diffraction data. We have combined super fine-phi slicing with highly monochromatic, low divergence synchrotron radiation and the ADSC Quantum 4 CCD detector at the Stanford Synchrotron Radiation laboratory beamline 1.5 to accurately measure crystal mosaicity. Comparisons of microgravity versus earth-grown insulin crystals using these methods will be presented.

  5. IR laser-induced protein crystal transformation

    SciTech Connect

    Kiefersauer, Reiner Grandl, Brigitte; Krapp, Stephan; Huber, Robert

    2014-05-01

    A novel method and the associated instrumentation for improving crystalline order (higher resolution of X-ray diffraction and reduced mosaicity) of protein crystals by precisely controlled heating is demonstrated. Crystal transformation is optically controlled by a video system. A method and the design of instrumentation, and its preliminary practical realisation, including test experiments, with the object of inducing phase changes of biomolecular crystals by controlled dehydration through heating with infrared (IR) light are described. The aim is to generate and select crystalline phases through transformation in the solid state which have improved order (higher resolution in X-ray diffraction experiments) and reduced mosaic spread (more uniformly aligned mosaic blocks) for diffraction data collection and analysis. The crystal is heated by pulsed and/or constant IR laser irradiation. Loss of crystal water following heating and its reabsorption through equilibration with the environment is measured optically by a video system. Heating proved superior to traditional controlled dehydration by humidity change for the test cases CODH (carbon monoxide dehydrogenase) and CLK2 (a protein kinase). Heating with IR light is experimentally simple and offers an exploration of a much broader parameter space than the traditional method, as it allows the option of varying the rate of phase changes through modification of the IR pulse strength, width and repeat frequency. It impacts the crystal instantaneously, isotropically and homogeneously, and is therefore expected to cause less mechanical stress.

  6. Protein Crystal Growth Apparatus for Microgravity

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor); Dowling, Timothy E. (Inventor)

    1997-01-01

    Apparatus for growing protein crystals under microgravity environment includes a plurality of protein growth assemblies stacked one above the other within a canister. Each of the protein growth assemblies includes a tray having a number of spaced apart growth chambers recessed below an upper surface. the growth chambers each having an upstanding pedestal and an annular reservoir about the pedestal for receiving a wick and precipitating agents. A well is recessed below the top of each pedestal to define a protein crystal growth receptacle. A flexible membrane is positioned on the upper surface of each tray and a sealing plate is positioned above each membrane, each sealing plate having a number of bumpers corresponding in number and alignment to the pedestals for forcing the membrane selectively against the upper end of the respective pedestal to seal the reservoir and the receptacle when the sealing plate is forced down.

  7. Sigmoid kinetics of protein crystal nucleation

    NASA Astrophysics Data System (ADS)

    Nanev, Christo N.; Tonchev, Vesselin D.

    2015-10-01

    A non-linear differential equation expressing the new phase nucleation rate in the different steps of the process (non-stationary and stationary nucleation and in the plateau region) is derived from basic principles of the nucleation theory. It is shown that one and the same sigmoid (logistic) function describes both nucleation scenarios: the one according to the classical theory, and the other according to the modern two-stage mechanism of protein crystal formation. Comparison to experimental data on both insulin crystal nucleation kinetics and on bovine β-lactoglobulin crystallization indicates a good agreement with the sigmoidal prediction. Experimental data for electrochemical nucleation and glass crystallization obey the same sigmoid time dependence, and suggest universality of this nucleation kinetics law.

  8. (PCG) Protein Crystal Growth Human Serum Albumin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Human Serum Albumin. Contributes to many transport and regulatory processes and has multifunctional binding properties which range from various metals, to fatty acids, hormones, and a wide spectrum of therapeutic drugs. The most abundant protein of the circulatory system. It binds and transports an incredible variety of biological and pharmaceutical ligands throughout the blood stream. Principal Investigator on STS-26 was Larry DeLucas.

  9. Convective flow effects on protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Monaco, Lisa A.

    1994-01-01

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

  10. (PCG) Protein Crystal Growth Isocitrate Lyase

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Isocitrate Lyase. Target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast. It regulates the flow of metabolic intermediates required for cell growth. Principal Investigator for STS-26 was Charles Bugg.

  11. (PCG) Protein Crystal Growth Isocitrate Lysase

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Isocitrate Lysase. Target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast. It regulates the flow of metabolic intermediates required for cell growth. Principal Investigator on STS-26 was Charles Bugg.

  12. Convective flow effects on protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Monaco, Lisa A.

    1994-01-01

    A high-resolution microscopic interferometric setup for the monitoring of protein morphologies has been developed. Growth or dissolution of a crystal can be resolved with a long-term depth resolution of 200 A and a lateral resolution of 2 microns. This capability of simultaneously monitoring the interfacial displacement with high local depth resolution has yielded several novel results. We have found with lysozyme that (1) the normal growth rate is oscillatory, and (2) depending on the impurity content of the solution, the growth step density is either greater or lower at the periphery of a facet than in its center. The repartitioning of Na plus and Cl minus ions between lysozyme solutions and crystals was studied for a wide range of crystallization conditions. A nucleation-growth-repartitioning model was developed, to interpret the large body of data in unified way. The results strongly suggest that (1) the ion to lysozyne ratio in the crystal depends mostly on kinetic rather than crystallographic parameters, and (2) lysozyme crystals possess a salt-rich core with a diameter electron microscopy results appear to confirm this finding, which could have far-reaching consequences for x-ray diffraction studies. A computational model for diffusive-convective transport in protein crystallization has been applied to a realistic growth cell geometry, taking into account the findings of the above repartitioning studies and our kinetics data for the growth of lysozyme. The results show that even in the small cell employed, protein concentration nonuniformities and gravity-driven solutal convection can be significant. The calculated convection velocities are of the same order to magnitude as those found in earlier experiments. As expected, convective transport, i.e., at Og, lysozyme crystal growth remains kinetically limited. The salt distribution in the crystal is predicted to be non-uniform at both 1g and 0g, as a consequence of protein depletion in the solution. Static and

  13. Convective flow effects on protein crystal growth

    NASA Astrophysics Data System (ADS)

    Rosenberger, Franz; Monaco, Lisa A.

    A high-resolution microscopic interferometric setup for the monitoring of protein morphologies has been developed. Growth or dissolution of a crystal can be resolved with a long-term depth resolution of 200 A and a lateral resolution of 2 microns. This capability of simultaneously monitoring the interfacial displacement with high local depth resolution has yielded several novel results. We have found with lysozyme that (1) the normal growth rate is oscillatory, and (2) depending on the impurity content of the solution, the growth step density is either greater or lower at the periphery of a facet than in its center. The repartitioning of Na plus and Cl minus ions between lysozyme solutions and crystals was studied for a wide range of crystallization conditions. A nucleation-growth-repartitioning model was developed, to interpret the large body of data in unified way. The results strongly suggest that (1) the ion to lysozyne ratio in the crystal depends mostly on kinetic rather than crystallographic parameters, and (2) lysozyme crystals possess a salt-rich core with a diameter electron microscopy results appear to confirm this finding, which could have far-reaching consequences for x-ray diffraction studies. A computational model for diffusive-convective transport in protein crystallization has been applied to a realistic growth cell geometry, taking into account the findings of the above repartitioning studies and our kinetics data for the growth of lysozyme. The results show that even in the small cell employed, protein concentration nonuniformities and gravity-driven solutal convection can be significant. The calculated convection velocities are of the same order to magnitude as those found in earlier experiments. As expected, convective transport, i.e., at Og, lysozyme crystal growth remains kinetically limited. The salt distribution in the crystal is predicted to be non-uniform at both 1g and 0g, as a consequence of protein depletion in the solution. Static and

  14. Impact of Protein-Metal Ion Interactions on the Crystallization of Silk Fibroin Protein

    NASA Astrophysics Data System (ADS)

    Hu, Xiao; Lu, Qiang; Kaplan, David; Cebe, Peggy

    2009-03-01

    Proteins can easily form bonds with a variety of metal ions, which provides many unique biological functions for the protein structures, and therefore controls the overall structural transformation of proteins. We use advanced thermal analysis methods such as temperature modulated differential scanning calorimetry and quasi-isothermal TMDSC, combined with Fourier transform infrared spectroscopy, and scanning electron microscopy, to investigate the protein-metallic ion interactions in Bombyx mori silk fibroin proteins. Silk samples were mixed with different metal ions (Ca^2+, K^+, Ma^2+, Na^+, Cu^2+, Mn^2+) with different mass ratios, and compared with the physical conditions in the silkworm gland. Results show that all metallic ions can directly affect the crystallization behavior and glass transition of silk fibroin. However, different ions tend to have different structural impact, including their role as plasticizer or anti-plasticizer. Detailed studies reveal important information allowing us better to understand the natural silk spinning and crystallization process.

  15. Convective diffusion in protein crystal growth

    NASA Technical Reports Server (NTRS)

    Baird, J. K.; Meehan, E. J., Jr.; Xidis, A. L.; Howard, S. B.

    1986-01-01

    A protein crystal modeled as a flat plate suspended in the parent solution, with the normal to the largest face perpendicular to gravity and the protein concentration in the solution adjacent to the plate taken to be the equilibrium solubility, is studied. The Navier-Stokes equation and the equation for convective diffusion in the boundary layer next to the plate are solved to calculate the flow velocity and the protein mass flux. The local rate of growth of the plate is shown to vary significantly with depth due to the convection. For an aqueous solution of lysozyme at a concentration of 40 mg/ml, the boundary layer at the top of a 1-mm-high crystal has a thickness of 80 microns at 1 g, and 2570 microns at 10 to the -6th g.

  16. Protein crystallization image classification with elastic net

    NASA Astrophysics Data System (ADS)

    Hung, Jeffrey; Collins, John; Weldetsion, Mehari; Newland, Oliver; Chiang, Eric; Guerrero, Steve; Okada, Kazunori

    2014-03-01

    Protein crystallization plays a crucial role in pharmaceutical research by supporting the investigation of a protein's molecular structure through X-ray diffraction of its crystal. Due to the rare occurrence of crystals, images must be manually inspected, a laborious process. We develop a solution incorporating a regularized, logistic regression model for automatically evaluating these images. Standard image features, such as shape context, Gabor filters and Fourier transforms, are first extracted to represent the heterogeneous appearance of our images. Then the proposed solution utilizes Elastic Net to select relevant features. Its L1-regularization mitigates the effects of our large dataset, and its L2- regularization ensures proper operation when the feature number exceeds the sample number. A two-tier cascade classifier based on naïve Bayes and random forest algorithms categorized the images. In order to validate the proposed method, we experimentally compare it with naïve Bayes, linear discriminant analysis, random forest, and their two-tier cascade classifiers, by 10-fold cross validation. Our experimental results demonstrate a 3-category accuracy of 74%, outperforming other models. In addition, Elastic Net better reduces the false negatives responsible for a high, domain specific risk. To the best of our knowledge, this is the first attempt to apply Elastic Net to classifying protein crystallization images. Performance measured on a large pharmaceutical dataset also fared well in comparison with those presented in the previous studies, while the reduction of the high-risk false negatives is promising.

  17. [Recent advancement of photonic-crystal-based analytical chemistry].

    PubMed

    Chen, Yun; Guo, Zhenpeng; Wang, Jinyi; Chen, Yi

    2014-04-01

    Photonic crystals are a type of novel materials with ordered structure, nanopores/channels and optical band gap. They have hence important applications in physics, chemistry, biological science and engineering fields. This review summarizes the recent advancement of photonic crystals in analytical chemistry applications, with focus on sensing and separating fields happening in the nearest 5 years.

  18. Crystallization of proteins from crude bovine rod outer segments.

    PubMed

    Baker, Bo Y; Gulati, Sahil; Shi, Wuxian; Wang, Benlian; Stewart, Phoebe L; Palczewski, Krzysztof

    2015-01-01

    Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques.

  19. Crystallization of Proteins from Crude Bovine Rod Outer Segments☆

    PubMed Central

    Baker, Bo Y.; Gulati, Sahil; Shi, Wuxian; Wang, Benlian; Stewart, Phoebe L.; Palczewski, Krzysztof

    2015-01-01

    Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques. PMID:25950977

  20. Kinetics of sucrose crystallization in whey protein films.

    PubMed

    Dangaran, Kirsten L; Krochta, John M

    2006-09-20

    The kinetics of sucrose crystallization in whey protein isolate (WPI) films was studied at 25 degrees C in four different relative humidity environments: 23, 33, 44, and 53%. The effects of protein matrix, crystallization inhibitors, and storage environment on the rate constants of sucrose crystallization were determined using the Avrami model of crystallization. It was found that a cross-linked, denatured whey protein (WP) matrix more effectively hindered sucrose crystallization than a protein matrix of native WP. The crystallization inhibitors tested were lactose, raffinose, modified starch (Purity 69), and polyvinylpyrrolidone (Plasdone C15). Raffinose and modified starch were determined to be the more effective inhibitors of sucrose crystallization. At lower relative humidities (23, 33, and 44%), the cross-linked protein matrix played a more important role in sucrose crystallization than the inhibitors. As relative humidity increased (53%), the crystallization inhibitors were more central to controlling sucrose crystallization in WPI films.

  1. The Biological Macromolecule Crystallization Database and NASA Protein Crystal Growth Archive.

    PubMed

    Gilliland, G L; Tung, M; Ladner, J

    1996-01-01

    The NIST/NASA/CARB Biological Macromolecule Crystallization Database (BMCD), NIST Standard Reference Database 21, contains crystal data and crystallization conditions for biological macromolecules. The database entries include data abstracted from published crystallographic reports. Each entry consists of information describing the biological macromolecule crystallized and crystal data and the crystallization conditions for each crystal form. The BMCD serves as the NASA Protein Crystal Growth Archive in that it contains protocols and results of crystallization experiments undertaken in microgravity (space). These database entries report the results, whether successful or not, from NASA-sponsored protein crystal growth experiments in microgravity and from microgravity crystallization studies sponsored by other international organizations. The BMCD was designed as a tool to assist x-ray crystallographers in the development of protocols to crystallize biological macromolecules, those that have previously been crystallized, and those that have not been crystallized.

  2. An automated protein crystal growth facility on the space station

    NASA Technical Reports Server (NTRS)

    Herrmann, Melody

    1988-01-01

    The need is addressed for an automated Protein Crystal Growth experiment on the Space Station and how robotics will be integrated into the system design. This automated laboratory system will enable several hundred protein crystals to grow simultaneously in microgravity and will allow the major variables in protein crystal growth to be monitored and controlled during the experiment. Growing good quality crystals is important in determining the complete structure of the protein by X-ray diffraction. This information is useful in the research and development of medicines and other important medical and biotechnological products. Previous Protein Crystal Growth experiments indicate that the microgravity environment of space allows larger crystals of higher quality to be grown as compared to the same crystals grown on the ground. It is therefore important to have a laboratory in space where protein crystals can be grown under carefully controlled conditions so that a crystal type can be reproduced as needed.

  3. X-ray Microscopic Characterization of Protein Crystals

    NASA Technical Reports Server (NTRS)

    Hu, Z. W.; Holmes, A.; Thomas, B.R.; Chernov, a. A.; Chu, Y. S.; Lai, B.

    2004-01-01

    The microscopic mapping of the variation in degree of perfection and in type of defects in entire protein crystals by x-rays may well be a prerequisite for better understanding causes of lattice imperfections, the growth history, and properties of protein crystals. However, x-ray microscopic characterization of bulk protein crystals, in the as-grown state, is frequently more challenging than that of small molecular crystals due to the experimental difficulties arising largely from the unique features possessed by protein crystals. In this presentation, we will illustrate ssme recent activities in employing coherence-based phase contrast x-ray imaging and high-angular-resolution diffraction techniques for mapping microdefects and the degree of perfection of protein crystals, and demonstrate a correlation between crystal perfection, diffraction phenomena., and crystallization conditions. The observed features and phenomena will be discussed in context to gain insight into the nature of defects, nucleation and growth, and the properties of protein crystals.

  4. Bacillus thuringiensis and Its Pesticidal Crystal Proteins

    PubMed Central

    Schnepf, E.; Crickmore, N.; Van Rie, J.; Lereclus, D.; Baum, J.; Feitelson, J.; Zeigler, D. R.; Dean, D. H.

    1998-01-01

    During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit. PMID:9729609

  5. Optical monitoring of protein crystal growth

    NASA Technical Reports Server (NTRS)

    Choudry, A.

    1988-01-01

    The possibility of using various optical techniques for detecting the onset of nucleation in protein crystal growth was investigated. Direct microscopy, general metrologic techniques, light scattering, ultraviolet absorption, and interferometry are addressed along with techniques for determining pH value. The necessity for collecting basic data on the optical properties of the growth solution as a prerequisite to the evaluation of monitoring techniques is pointed out.

  6. (PCG) Protein Crystal Growth Gamma-Interferon

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Gamma-Interferon. Stimulates the body's immune system and is used clinically in the treatment of cancer. Potential as an anti-tumor agent against solid tumors as well as leukemia's and lymphomas. It has additional utility as an anti-ineffective agent, including antiviral, anti-bacterial, and anti-parasitic activities. Principal Investigator on STS-26 was Charles Bugg.

  7. Lattice Dynamics of a Protein Crystal

    SciTech Connect

    Meinhold, Lars; Merzel, Franci; Smith, Jeremy C.

    2007-09-28

    All-atom lattice-dynamical calculations are reported for a crystalline protein, ribonuclease A. The sound velocities, density of states, heat capacity (C{sub V}) and thermal diffuse scattering are all consistent with available experimental data. C{sub V}{proportional_to}T{sup 1.68} for T<35 K, significantly deviating from a Debye solid. In Bragg peak vicinity, inelastic scattering of x rays by phonons is found to originate from acoustic mode scattering. The results suggest an approach to protein crystal physics combining all-atom lattice-dynamical calculations with experiments on next-generation neutron sources.

  8. Convective flow effects on protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Monaco, Lisa A.

    1995-01-01

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

  9. When proteins are completely hydrated in crystals.

    PubMed

    Carugo, Oliviero

    2016-08-01

    In the crystalline state, protein surface patches that do not form crystal packing contacts are exposed to the solvent and one or more layers of hydration water molecules can be observed. It is well known that these water molecules cannot be observed at very low resolution, when the scarcity of experimental information precludes the observation of several parts of the protein molecule, like for example side-chains at the protein surface. On the contrary, more details are observable at high resolution. Here it is shown that it is necessary to reach a resolution of about 1.5-1.6Å to observe a continuous hydration layer at the protein surface. This contrasts previous estimations, which were more tolerant and according to which a resolution of 2.5Å was sufficient to describe at the atomic level the structure of the hydration layer. These results should prove useful in guiding a more rigorous selection of structural data to study protein hydration and in interpreting new crystal structures. PMID:27112977

  10. Promoting protein crystallization using a plate with simple geometry.

    PubMed

    Chen, Rui-Qing; Yin, Da-Chuan; Liu, Yong-Ming; Lu, Qin-Qin; He, Jin; Liu, Yue

    2014-03-01

    Increasing the probability of obtaining protein crystals in crystallization screening is always an important goal for protein crystallography. In this paper, a new method called the cross-diffusion microbatch (CDM) method is presented, which aims to efficiently promote protein crystallization and increase the chance of obtaining protein crystals. In this method, a very simple crystallization plate was designed in which all crystallization droplets are in one sealed space, so that a variety of volatile components from one droplet can diffuse into any other droplet via vapour diffusion. Crystallization screening and reproducibility tests indicate that this method could be a potentially powerful technique in practical protein crystallization screening. It can help to obtain crystals with higher probability and at a lower cost, while using a simple and easy procedure.

  11. Advances in the directed evolution of proteins

    PubMed Central

    Lane, Michael D.; Seelig, Burckhard

    2014-01-01

    Natural evolution has produced a great diversity of proteins that can be harnessed for numerous applications in biotechnology and pharmaceutical science. Commonly, specific applications require proteins to be tailored by protein engineering. Directed evolution is a type of protein engineering that yields proteins with the desired properties under well-defined conditions and in a practical time frame. While directed evolution has been employed for decades, recent creative developments enable the generation of proteins with previously inaccessible properties. Novel selection strategies, faster techniques, the inclusion of unnatural amino acids or modifications, and the symbiosis of rational design approaches and directed evolution continue to advance protein engineering. PMID:25309990

  12. NMR of Membrane Proteins: Beyond Crystals.

    PubMed

    Rajesh, Sundaresan; Overduin, Michael; Bonev, Boyan B

    2016-01-01

    Membrane proteins are essential for the flow of signals, nutrients and energy between cells and between compartments of the cell. Their mechanisms can only be fully understood once the precise structures, dynamics and interactions involved are defined at atomic resolution. Through advances in solution and solid state NMR spectroscopy, this information is now available, as demonstrated by recent studies of stable peripheral and transmembrane proteins. Here we highlight recent cases of G-protein coupled receptors, outer membrane proteins, such as VDAC, phosphoinositide sensors, such as the FAPP-1 pleckstrin homology domain, and enzymes including the metalloproteinase MMP-12. The studies highlighted have resulted in the determination of the 3D structures, dynamical properties and interaction surfaces for membrane-associated proteins using advanced isotope labelling strategies, solubilisation systems and NMR experiments designed for very high field magnets. Solid state NMR offers further insights into the structure and multimeric assembly of membrane proteins in lipid bilayers, as well as into interactions with ligands and targets. Remaining challenges for wider application of NMR to membrane structural biology include the need for overexpression and purification systems for the production of isotope-labelled proteins with fragile folds, and the availability of only a few expensive perdeuterated detergents.Step changes that may transform the field include polymers, such as styrene maleic acid, which obviate the need for detergent altogether, and allow direct high yield purification from cells or membranes. Broader demand for NMR may be facilitated by MODA software, which instantly predicts membrane interactive residues that can subsequently be validated by NMR. In addition, recent developments in dynamic nuclear polarization NMR instrumentation offer a remarkable sensitivity enhancement from low molarity samples and cell surfaces. These advances illustrate the current

  13. NMR of Membrane Proteins: Beyond Crystals.

    PubMed

    Rajesh, Sundaresan; Overduin, Michael; Bonev, Boyan B

    2016-01-01

    Membrane proteins are essential for the flow of signals, nutrients and energy between cells and between compartments of the cell. Their mechanisms can only be fully understood once the precise structures, dynamics and interactions involved are defined at atomic resolution. Through advances in solution and solid state NMR spectroscopy, this information is now available, as demonstrated by recent studies of stable peripheral and transmembrane proteins. Here we highlight recent cases of G-protein coupled receptors, outer membrane proteins, such as VDAC, phosphoinositide sensors, such as the FAPP-1 pleckstrin homology domain, and enzymes including the metalloproteinase MMP-12. The studies highlighted have resulted in the determination of the 3D structures, dynamical properties and interaction surfaces for membrane-associated proteins using advanced isotope labelling strategies, solubilisation systems and NMR experiments designed for very high field magnets. Solid state NMR offers further insights into the structure and multimeric assembly of membrane proteins in lipid bilayers, as well as into interactions with ligands and targets. Remaining challenges for wider application of NMR to membrane structural biology include the need for overexpression and purification systems for the production of isotope-labelled proteins with fragile folds, and the availability of only a few expensive perdeuterated detergents.Step changes that may transform the field include polymers, such as styrene maleic acid, which obviate the need for detergent altogether, and allow direct high yield purification from cells or membranes. Broader demand for NMR may be facilitated by MODA software, which instantly predicts membrane interactive residues that can subsequently be validated by NMR. In addition, recent developments in dynamic nuclear polarization NMR instrumentation offer a remarkable sensitivity enhancement from low molarity samples and cell surfaces. These advances illustrate the current

  14. Large-scale crystallization of proteins for purification and formulation.

    PubMed

    Hekmat, Dariusch

    2015-07-01

    Since about 170 years, salts were used to create supersaturated solutions and crystallize proteins. The dehydrating effect of salts as well as their kosmotropic or chaotropic character was revealed. Even the suitability of organic solvents for crystallization was already recognized. Interestingly, what was performed during the early times is still practiced today. A lot of effort was put into understanding the underlying physico-chemical interaction mechanisms leading to protein crystallization. However, it was understood that already the solvation of proteins is a highly complex process not to mention the intricate interrelation of electrostatic and hydrophobic interactions taking place. Although many basic questions are still unanswered, preparative protein crystallization was attempted as illustrated in the presented case studies. Due to the highly variable nature of crystallization, individual design of the crystallization process is needed in every single case. It was shown that preparative crystallization from impure protein solutions as a capture step is possible after applying adequate pre-treatment procedures like precipitation or extraction. Protein crystallization can replace one or more chromatography steps. It was further shown that crystallization can serve as an attractive alternative means for formulation of therapeutic proteins. Crystalline proteins can offer enhanced purity and enable highly concentrated doses of the active ingredient. Easy scalability of the proposed protein crystallization processes was shown using the maximum local energy dissipation as a suitable scale-up criterion. Molecular modeling and target-oriented protein engineering may allow protein crystallization to become part of a platform purification process in the near future.

  15. Do protein crystals nucleate within dense liquid clusters?

    PubMed Central

    Maes, Dominique; Vorontsova, Maria A.; Potenza, Marco A. C.; Sanvito, Tiziano; Sleutel, Mike; Giglio, Marzio; Vekilov, Peter G.

    2015-01-01

    Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10−3 of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear following a significant delay after cluster formation. The cDDLS correlation functions follow a Gaussian decay, indicative of nondiffusive motion. A possible explanation is that the crystals are contained inside large clusters and are driven by the elasticity of the cluster surface. Secondly, depolarized oblique illumination dark-field microscopy reveals the evolution from liquid clusters without crystals to newly nucleated crystals contained in the clusters to grown crystals freely diffusing in the solution. Collectively, the observations indicate that the protein-rich clusters in lysozyme and glucose isomerase solutions are locations for crystal nucleation. PMID:26144225

  16. Do protein crystals nucleate within dense liquid clusters?

    PubMed

    Maes, Dominique; Vorontsova, Maria A; Potenza, Marco A C; Sanvito, Tiziano; Sleutel, Mike; Giglio, Marzio; Vekilov, Peter G

    2015-07-01

    Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10(-3) of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear following a significant delay after cluster formation. The cDDLS correlation functions follow a Gaussian decay, indicative of nondiffusive motion. A possible explanation is that the crystals are contained inside large clusters and are driven by the elasticity of the cluster surface. Secondly, depolarized oblique illumination dark-field microscopy reveals the evolution from liquid clusters without crystals to newly nucleated crystals contained in the clusters to grown crystals freely diffusing in the solution. Collectively, the observations indicate that the protein-rich clusters in lysozyme and glucose isomerase solutions are locations for crystal nucleation.

  17. An approach to crystallizing proteins by synthetic symmetrization.

    PubMed

    Banatao, D Rey; Cascio, Duilio; Crowley, Christopher S; Fleissner, Mark R; Tienson, Heather L; Yeates, Todd O

    2006-10-31

    Previous studies of symmetry preferences in protein crystals suggest that symmetric proteins, such as homodimers, might crystallize more readily on average than asymmetric, monomeric proteins. Proteins that are naturally monomeric can be made homodimeric artificially by forming disulfide bonds between individual cysteine residues introduced by mutagenesis. Furthermore, by creating a variety of single-cysteine mutants, a series of distinct synthetic dimers can be generated for a given protein of interest, with each expected to gain advantage from its added symmetry and to exhibit a crystallization behavior distinct from the other constructs. This strategy was tested on phage T4 lysozyme, a protein whose crystallization as a monomer has been studied exhaustively. Experiments on three single-cysteine mutants, each prepared in dimeric form, yielded numerous novel crystal forms that cannot be realized by monomeric lysozyme. Six new crystal forms have been characterized. The results suggest that synthetic symmetrization may be a useful approach for enlarging the search space for crystallizing proteins.

  18. IR laser-induced protein crystal transformation.

    PubMed

    Kiefersauer, Reiner; Grandl, Brigitte; Krapp, Stephan; Huber, Robert

    2014-05-01

    A method and the design of instrumentation, and its preliminary practical realisation, including test experiments, with the object of inducing phase changes of biomolecular crystals by controlled dehydration through heating with infrared (IR) light are described. The aim is to generate and select crystalline phases through transformation in the solid state which have improved order (higher resolution in X-ray diffraction experiments) and reduced mosaic spread (more uniformly aligned mosaic blocks) for diffraction data collection and analysis. The crystal is heated by pulsed and/or constant IR laser irradiation. Loss of crystal water following heating and its reabsorption through equilibration with the environment is measured optically by a video system. Heating proved superior to traditional controlled dehydration by humidity change for the test cases CODH (carbon monoxide dehydrogenase) and CLK2 (a protein kinase). Heating with IR light is experimentally simple and offers an exploration of a much broader parameter space than the traditional method, as it allows the option of varying the rate of phase changes through modification of the IR pulse strength, width and repeat frequency. It impacts the crystal instantaneously, isotropically and homogeneously, and is therefore expected to cause less mechanical stress.

  19. A protein coated piezoelectric crystal detector

    NASA Astrophysics Data System (ADS)

    Suleiman, Ahmad; Pender, Marie; Ngeh-Ngwainbi, Jerome; Lubrano, Glenn; Guilbault, George

    1990-05-01

    The purpose of this project was to develop a protein coated, portable piezoelectric crystal detector for organophosphorus compounds. The performance of acetylcholinesterase, GD-1 anti-soman, anti-DMMP antibody, and bovine serum albumin (BSA) coatings was evaluated. Different immobilization methods were also tested. The responses obtained with the protein coatings immobilized via cross-linking with glutaraldehyde were acceptable, provided that the reference crystal was coated with dextran. The proposed coatings showed good stability and reasonable lifetimes that ranged from approximately three weeks in the case of the antibody coatings to several months in the case of BSA. Although moisture, gasoline, and sulfur are potential interferents, their effects on the sensor were eliminated by using a sodium sulfate scrubber which did not affect the performance of the detector towards organophosphates. A small, battery operated portable instrument capable of real time measurements with alarm function was produced. The instrument can be used in a wide range of applications, depending on the coatings applied to the crystals.

  20. Convective flow effects on protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz; Monaco, Lisa A.

    1993-01-01

    The experimental setup for the in-situ high resolution optical monitoring of protein crystal growth/dissolution morphologies was substantially improved. By augmenting the observation system with a temperature-controlled enclosure, laser illumination for the interferometric microscope, and software for pixel by pixel light intensity recording, a height resolution of about two unit cells for lysozyme can now be obtained. The repartitioning of Na(+) and Cl(-) ions between lysozyme solutions and crystals was studied. Quite unexpectedly, it was found that the longer crystals were in contact with their solution, the lower was their ion content. The development of a model for diffusive-convective transport and resulting distribution of the growth rate on facets was completed. Results obtained for a realistic growth cell geometry show interesting differences between 'growth runs' at 1g and 0g. The kinematic viscosity of lysozyme solutions of various supersaturations and salt concentrations was monitored over time. In contrast to the preliminary finding of other authors, no changes in viscosity were found over four days. The experimental setup for light scattering investigations of aggregation and nucleation in protein solutions was completed, and a computer program for the evaluation of multi-angle light scattering data was acquired.

  1. Convective flow effects on protein crystal growth

    NASA Astrophysics Data System (ADS)

    Rosenberger, Franz; Monaco, Lisa A.

    The experimental setup for the in-situ high resolution optical monitoring of protein crystal growth/dissolution morphologies was substantially improved. By augmenting the observation system with a temperature-controlled enclosure, laser illumination for the interferometric microscope, and software for pixel by pixel light intensity recording, a height resolution of about two unit cells for lysozyme can now be obtained. The repartitioning of Na(+) and Cl(-) ions between lysozyme solutions and crystals was studied. Quite unexpectedly, it was found that the longer crystals were in contact with their solution, the lower was their ion content. The development of a model for diffusive-convective transport and resulting distribution of the growth rate on facets was completed. Results obtained for a realistic growth cell geometry show interesting differences between 'growth runs' at 1g and 0g. The kinematic viscosity of lysozyme solutions of various supersaturations and salt concentrations was monitored over time. In contrast to the preliminary finding of other authors, no changes in viscosity were found over four days. The experimental setup for light scattering investigations of aggregation and nucleation in protein solutions was completed, and a computer program for the evaluation of multi-angle light scattering data was acquired.

  2. Do protein crystals nucleate within dense liquid clusters?

    SciTech Connect

    Maes, Dominique; Vorontsova, Maria A.; Potenza, Marco A. C.; Sanvito, Tiziano; Sleutel, Mike; Giglio, Marzio; Vekilov, Peter G.

    2015-06-27

    The evolution of protein-rich clusters and nucleating crystals were characterized by dynamic light scattering (DLS), confocal depolarized dynamic light scattering (cDDLS) and depolarized oblique illumination dark-field microscopy. Newly nucleated crystals within protein-rich clusters were detected directly. These observations indicate that the protein-rich clusters are locations for crystal nucleation. Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10{sup −3} of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear following a significant delay after cluster formation. The cDDLS correlation functions follow a Gaussian decay, indicative of nondiffusive motion. A possible explanation is that the crystals are contained inside large clusters and are driven by the elasticity of the cluster surface. Secondly, depolarized oblique illumination dark-field microscopy reveals the evolution from liquid clusters without crystals to newly nucleated crystals contained in the clusters to grown crystals freely diffusing in the solution. Collectively, the observations indicate that the protein-rich clusters in

  3. Growth of high-strength protein crystals with nanofibers

    NASA Astrophysics Data System (ADS)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Nakabayashi, Iori; Tsuchikura, Hiroshi; Kuwahara, Atsushi; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-03-01

    Here, we present a novel method of growing protein crystals with nanofibers. Protein crystals were grown by incorporating nanofibers. No obvious differences were observed in diffraction data between fiber-containing and as-grown crystals. The fiber-containing crystals displayed an increased tolerance to osmotic shock caused by soaking in 25% ethanol or 40% dimethyl sulfoxide. This means that the method allowed us to easily increase the crystal mechanical stability. Because the method is very simple, it will provide a variety of possibilities for protein crystallization.

  4. Fluorescence Studies of Protein Crystal Nucleation

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Sumida, John

    2000-01-01

    One of the most powerful and versatile methods for studying molecules in solution is fluorescence. Crystallization typically takes place in a concentrated solution environment, whereas fluorescence typically has an upper concentration limit of approximately 1 x 10(exp -5)M, thus intrinsic fluorescence cannot be employed, but a fluorescent probe must be added to a sub population of the molecules. However the fluorescent species cannot interfere with the self-assembly process. This can be achieved with macromolecules, where fluorescent probes can be covalently attached to a sub population of molecules that are subsequently used to track the system as a whole. We are using fluorescence resonance energy transfer (FRET) to study the initial solution phase self-assembly process of tetragonal lysozyme crystal nucleation, using covalent fluorescent derivatives which crystallize in the characteristic P432121 space group. FRET studies are being carried out between cascade blue (CB-lys, donor, Ex 376 nm, Em 420 nm) and lucifer yellow (LY-lys, acceptor, Ex 425 nm, Em 520 nm) asp101 derivatives. The estimated R0 for this probe pair, the distance where 50% of the donor energy is transferred to the acceptor, is approximately 1.2 nm, compared to 2.2 nm between the side chain carboxyls of adjacent asp101's in the crystalline 43 helix. The short CB-lys lifetime (approximately 5 ns), coupled with the large average distances between the molecules ((sup 3) 50 nm) in solution, ensure that any energy transfer observed is not due to random diffusive interactions. Addition of LY-lys to CB-lys results in the appearance of a second, shorter lifetime (approximately 0.2 ns). Results from these and other ongoing studies will be discussed in conjunction with a model for how tetragonal lysozyme crystals nucleate and grow, and the relevance of that model to microgravity protein crystal growth

  5. Nucleation and Convection Effects in Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    Vekilow, Peter G.

    1998-01-01

    Our work under this grant has significantly contributed to the goals of the NASA supported protein crystallization program. We have achieved the main objectives of the proposed work, as outlined in the original proposal: (1) We have provided important insight into protein nucleation and crystal growth mechanisms to facilitate a rational approach to protein crystallization; (2) We have delineated the factors that currently limit the x-ray diffraction resolution of protein crystals, and their correlation to crystallization conditions; (3) We have developed novel technologies to study and monitor protein crystal nucleation and growth processes, in order to increase the reproducibility and yield of protein crystallization. We have published 17 papers in peer-reviewed scientific journals and books and made more than 15 invited and 9 contributed presentations of our results at international and national scientific meetings.

  6. A new subgroup of lectin-bound biliary proteins binds to cholesterol crystals, modifies crystal morphology, and inhibits cholesterol crystallization.

    PubMed Central

    Busch, N; Lammert, F; Marschall, H U; Matern, S

    1995-01-01

    Biliary proteins inhibiting or promoting cholesterol crystallization are assumed to play a major role in cholesterol gallstone pathogenesis. We now report a new group of biliary proteins that bind to cholesterol crystals, modify crystal morphology, and inhibit cholesterol crystallization. Various glycoprotein mixtures were extracted from abnormal human gallbladder bile using lectin affinity chromatography on concanavalin A, lentil, and Helix pomatia columns and were added to supersaturated model bile. Independent of the protein mixtures added, from the cholesterol crystals harvested, the same four GPs were isolated having molecular masses of 16, 28, 63, and 74 kD, respectively. Each protein was purified using preparative SDS-PAGE, and influence on cholesterol crystallization in model bile was tested at 10 microg/ml. Crystal growth was reduced by 76% (GP63), 65% (GP16), 55% (GP74), and 40% (GP28), respectively. Thus, these glycoproteins are the most potent biliary inhibitors of cholesterol crystallization known so far. Evidence that the inhibiting effect on cholesterol crystallization is mediated via protein-crystal interaction was further provided from scanning electron microscopy studies. Crystals grown in presence of inhibiting proteins showed significantly more ordered structures. Incidence of triclinic crystals and regular aggregates was shifted from 30 to 70% compared with controls. These observations may have important implications for understanding the role of biliary proteins in cholesterol crystallization and gallstone pathogenesis. Images PMID:8675674

  7. JAXA protein crystallization in space: ongoing improvements for growing high-quality crystals.

    PubMed

    Takahashi, Sachiko; Ohta, Kazunori; Furubayashi, Naoki; Yan, Bin; Koga, Misako; Wada, Yoshio; Yamada, Mitsugu; Inaka, Koji; Tanaka, Hiroaki; Miyoshi, Hiroshi; Kobayashi, Tomoyuki; Kamigaichi, Shigeki

    2013-11-01

    The Japan Aerospace Exploration Agency (JAXA) started a high-quality protein crystal growth project, now called JAXA PCG, on the International Space Station (ISS) in 2002. Using the counter-diffusion technique, 14 sessions of experiments have been performed as of 2012 with 580 proteins crystallized in total. Over the course of these experiments, a user-friendly interface framework for high accessibility has been constructed and crystallization techniques improved; devices to maximize the use of the microgravity environment have been designed, resulting in some high-resolution crystal growth. If crystallization conditions were carefully fixed in ground-based experiments, high-quality protein crystals grew in microgravity in many experiments on the ISS, especially when a highly homogeneous protein sample and a viscous crystallization solution were employed. In this article, the current status of JAXA PCG is discussed, and a rational approach to high-quality protein crystal growth in microgravity based on numerical analyses is explained.

  8. JAXA protein crystallization in space: ongoing improvements for growing high-quality crystals.

    PubMed

    Takahashi, Sachiko; Ohta, Kazunori; Furubayashi, Naoki; Yan, Bin; Koga, Misako; Wada, Yoshio; Yamada, Mitsugu; Inaka, Koji; Tanaka, Hiroaki; Miyoshi, Hiroshi; Kobayashi, Tomoyuki; Kamigaichi, Shigeki

    2013-11-01

    The Japan Aerospace Exploration Agency (JAXA) started a high-quality protein crystal growth project, now called JAXA PCG, on the International Space Station (ISS) in 2002. Using the counter-diffusion technique, 14 sessions of experiments have been performed as of 2012 with 580 proteins crystallized in total. Over the course of these experiments, a user-friendly interface framework for high accessibility has been constructed and crystallization techniques improved; devices to maximize the use of the microgravity environment have been designed, resulting in some high-resolution crystal growth. If crystallization conditions were carefully fixed in ground-based experiments, high-quality protein crystals grew in microgravity in many experiments on the ISS, especially when a highly homogeneous protein sample and a viscous crystallization solution were employed. In this article, the current status of JAXA PCG is discussed, and a rational approach to high-quality protein crystal growth in microgravity based on numerical analyses is explained. PMID:24121350

  9. Fluorescence Studies of Protein Crystallization Interactions

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Smith, Lori; Forsythe, Elizabeth

    1999-01-01

    We are investigating protein-protein interactions in under- and over-saturated crystallization solution conditions using fluorescence methods. The use of fluorescence requires fluorescent derivatives where the probe does not markedly affect the crystal packing. A number of chicken egg white lysozyme (CEWL) derivatives have been prepared, with the probes covalently attached to one of two different sites on the protein molecule; the side chain carboxyl of ASP 101, within the active site cleft, and the N-terminal amine. The ASP 101 derivatives crystallize while the N-terminal amine derivatives do not. However, the N-terminal amine is part of the contact region between adjacent 43 helix chains, and blocking this site does would not interfere with formation of these structures in solution. Preliminary FRET data have been obtained at pH 4.6, 0.1M NaAc buffer, at 5 and 7% NaCl, 4 C, using the N-terminal bound pyrene acetic acid (PAA, Ex 340 nm, Em 376 nm) and ASP 101 bound Lucifer Yellow (LY, Ex 425 nm, Em 525 nm) probe combination. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10 (exp 5) M respectively), and all experiments were carried out at approximately Csat or lower total protein concentration. The data at both salt concentrations show a consistent trend of decreasing fluorescence yield of the donor species (PAA) with increasing total protein concentration. This decrease is apparently more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations (reflected in the lower solubility). The estimated average distance between protein molecules at 5 x 10 (exp 6) M is approximately 70 nm, well beyond the range where any FRET can be expected. The calculated RO, where 50% of the donor energy is transferred to the acceptor, for the PAA-CEWL * LY-CEWL system is 3.28 nm, based upon a PAA-CEWL quantum efficiency of 0.41.

  10. Utilization of Protein Crystal Structures in Industry

    NASA Astrophysics Data System (ADS)

    Ishikawa, Kohki

    In industry, protein crystallography is used in mainly two technologies. One is structure-based drug design, and the other is structure-based enzyme engineering. Some successful cases together with recent advances are presented in this article. The cases include the development of an anti-influenza drug, and the introduction of engineered acid phosphatase to the manufacturing process of nucleotides used as umami seasoning.

  11. Dip-pen nanolithography-assisted protein crystallization.

    PubMed

    Ielasi, Francesco S; Hirtz, Michael; Sekula-Neuner, Sylwia; Laue, Thomas; Fuchs, Harald; Willaert, Ronnie G

    2015-01-14

    We demonstrate the use of dip-pen nanolithography (DPN) to crystallize proteins on surface-localized functionalized lipid layer arrays. DOPC lipid layers, containing small amounts of biotin-DOPE lipid molecules, were printed on glass substrates and evaluated in vapor diffusion and batch crystallization screening setups, where streptavidin was used as a model protein for crystallization. Independently of the crystallization system used and the geometry of the lipid layers, nucleation of streptavidin crystals occurred specifically on the DPN-printed biotinylated structures. Protein crystallization on lipid array patches is also demonstrated in a microfluidic chip, which opens the way toward high-throughput screening to find suitable nucleation and crystal growth conditions. The results demonstrate the use of DPN in directing and inducing protein crystallization on specific surface locations.

  12. Analysis of crystallization data in the Protein Data Bank

    PubMed Central

    Kirkwood, Jobie; Hargreaves, David; O’Keefe, Simon; Wilson, Julie

    2015-01-01

    The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored. PMID:26457511

  13. Analysis of crystallization data in the Protein Data Bank.

    PubMed

    Kirkwood, Jobie; Hargreaves, David; O'Keefe, Simon; Wilson, Julie

    2015-10-01

    The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored.

  14. Analysis of crystallization data in the Protein Data Bank.

    PubMed

    Kirkwood, Jobie; Hargreaves, David; O'Keefe, Simon; Wilson, Julie

    2015-10-01

    The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored. PMID:26457511

  15. Protein crystallization: from HTS to kilogram-scale.

    PubMed

    Klyushnichenko, Vadim

    2003-11-01

    The first experiments on protein crystallization started randomly during the 19th century. This technique has been widely used for the determination of the tertiary structure of proteins since the 1950s, when an understanding of the physics of protein crystallization began to emerge. In the 1980s and 1990s, research focused on the study of protein crystal growth processes in microgravity environments, which were created in space shuttle experiments. High-throughput screening (HTS) systems were developed that later found broader laboratory applications. The combination of HTS with an engineering approach opens new opportunities for the protein crystallization process to become a robust, scalable, reproducible and economically viable industrial unit operation.

  16. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1994-01-01

    This research involved (1) using the Atomic Force Microscope (AFM) in a study on the growth of lysozyme crystals and (2) refinement of the design of the Thermonucleator which controls the supersaturation required for the nucleation and growth of protein crystals separately. AFM studies of the (110) tetragonal face confirmed that lysozyme crystals grow by step propagation. There appears to be very little step pile up in the growth regimes which we studied. The step height was measured at = 54A which was equal to the (110) interpane spacing. The AFM images showed areas of step retardation and the formation of pits. These defects ranged in size from 0.1 to 0.4 mu. The source of these defects was not determined. The redesign of the Thermonucleator produced an instrument based on thermoelectric technology which is both easier to use and more amenable to use in a mu g environment. The use of thermoelectric technology resulted in a considerable size reduction which will allow for the design of a multi-unit growth apparatus. The performance of the new apparatus was demonstrated to be the same as the original design.

  17. Fluorescence Studies of Protein Crystal Nucleation

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.

    1999-01-01

    Fluorescence can be used to study protein crystal nucleation through methods such as anisotropy, quenching, and resonance energy transfer (FRET), to follow pH and ionic strength changes, and follow events occurring at the growth interface. We have postulated, based upon a range of experimental evidence that the growth unit of tetragonal hen egg white lysozyme is an octamer. Several fluorescent derivatives of chicken egg white lysozyme have been prepared. The fluorescent probes lucifer yellow (LY), cascade blue, and 5-((2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS), have been covalently attached to ASP 101. All crystallize in the characteristic tetragonal form, indicating that the bound probes are likely laying within the active site cleft. Crystals of the LY and EDANS derivatives have been found to diffract to at least 1.7 A. A second group of derivatives is to the N-terminal amine group, and these do not crystallize as this site is part of the contact region between the adjacent 43 helix chains. However derivatives at these sites would not interfere with formation of the 43 helices in solution. Preliminary FRET studies have been carried out using N-terminal bound pyrene acetic acid (Ex 340 nm, Em 376 nm) lysozyme as a donor and LY (Ex -425 nm, Em 525 nm) labeled lysozyme as an acceptor. FRET data have been obtained at pH 4.6, 0.1 M NaAc buffer, at 5 and 7% NaCl, 4 C. The corresponding Csat values are 0.471 and 0.362 mg/ml (approximately 3.3 and approximately 2.5 x 10(exp -5) M respectively). The data at both salt concentrations show a consistent trend of decreasing fluorescence intensity of the donor species (PAA) with increasing total protein concentration. This decrease is more pronounced at 7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations reflected in the lower solubility. The calculated average distance between any two protein molecules at 5 x 10(exp -6) M is approximately 70nm, well beyond the

  18. Polymer-Induced Heteronucleation for Protein Single Crystal Growth: Structural Elucidation of Bovine Liver Catalase and Concanavalin A Forms

    SciTech Connect

    Foroughi, Leila M.; Kang, You-Na; Matzger, Adam J.

    2012-05-09

    Obtaining single crystals for X-ray diffraction remains a major bottleneck in structural biology; when existing crystal growth methods fail to yield suitable crystals, often the target rather than the crystallization approach is reconsidered. Here we demonstrate that polymer-induced heteronucleation, a powerful technique that has been used for small molecule crystallization form discovery, can be applied to protein crystallization by optimizing the heteronucleant composition and crystallization formats for crystallizing a wide range of protein targets. Applying these advances to two benchmark proteins resulted in dramatically increased crystal size, enabling structure determination, for a half century old form of bovine liver catalase (BLC) that had previously only been characterized by electron microscopy, and the discovery of two new forms of concanavalin A (conA) from the Jack bean and accompanying structural elucidation of one of these forms.

  19. Modeling the SHG activities of diverse protein crystals

    SciTech Connect

    Haupert, Levi M.; DeWalt, Emma L.; Simpson, Garth J.

    2012-11-01

    The origins of the diversity in the SHG signal from protein crystals are investigated and potential protein-crystal coverage by SHG microscopy is assessed. A symmetry-additive ab initio model for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much of the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within the crystal lattice. Two or more orders-of-magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ∼84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices.

  20. 2003 NIH protein structure intiative workshop in protein production and crystallization for structural and functional genomics.

    SciTech Connect

    Adams, M.; Joachimiak, A.; Kim, R.; Montelione, G. T.; Norvell, J.; Biosciences Division; University of Georgia; LBNL; Rutgers Univ.; Robert Wood Johnson Medical School

    2004-03-01

    The United States National Institutes of Health (NIH) Protein Structure Initiative (PSI) is a joint government, university, and industry effort, organized and supported by the National Institute of General Medical Sciences (NIGMS), and aimed at reducing the costs in increasing the speed of protein structure determination. Its long-range goal is to make the three-dimensional atomic-level structures of most proteins in nature easily obtainable from knowledge of their corresponding DNA sequences (http://www.nigms.gov/psi). It is the primary U.S. component of a broad international effort in structural genomics, involving at least 20 projects throughout the world. The PSI is now in its fourth year. Nine PSI pilot research centers have been funded to explore the feasibility and impact of genomic scale protein structure analysis. To date, over 500 3D protein structures, providing the first structural representatives for hundreds of protein domain families, have been completed and deposited by the NIH centers into the public Protein Data Bank. In addition, new technologies for protein sample production, data organization, and structure analysis by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have been developed. These technologies increase the efficiency of protein structure determination both for structural genomics and for the broader structural biology community. Although progress has been substantial, PSI pilot research centers have identified a number of important bottlenecks that need to be solved to meet the goals of the program. For example, it is now accepted that a major challenge to high-throughput protein structure determination is the fact that for some 70% of targeted proteins, it is difficult to produce protein samples and crystals suitable for structural analysis. In an effort to facilitate an effective exchange of developments and advancements between pilot centers, the NIGMS organized a workshop on gene cloning, protein

  1. Analysis of crystallization data in the Protein Data Bank

    SciTech Connect

    Kirkwood, Jobie; Hargreaves, David; O’Keefe, Simon; Wilson, Julie

    2015-09-23

    In a large-scale study using data from the Protein Data Bank, some of the many reported findings regarding the crystallization of proteins were investigated. The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored.

  2. Acoustic Methods to Monitor Protein Crystallization and to Detect Protein Crystals in Suspensions of Agarose and Lipidic Cubic Phase.

    PubMed

    Ericson, Daniel L; Yin, Xingyu; Scalia, Alexander; Samara, Yasmin N; Stearns, Richard; Vlahos, Harry; Ellson, Richard; Sweet, Robert M; Soares, Alexei S

    2016-02-01

    Improvements needed for automated crystallography include crystal detection and crystal harvesting. A technique that uses acoustic droplet ejection to harvest crystals was previously reported. Here a method is described for using the same acoustic instrument to detect protein crystals and to monitor crystal growth. Acoustic pulses were used to monitor the progress of crystallization trials and to detect the presence and location of protein crystals. Crystals were detected, and crystallization was monitored in aqueous solutions and in lipidic cubic phase. Using a commercially available acoustic instrument, crystals measuring ~150 µm or larger were readily detected. Simple laboratory techniques were used to increase the sensitivity to 50 µm by suspending the crystals away from the plastic surface of the crystallization plate. This increased the sensitivity by separating the strong signal generated by the plate bottom that can mask the signal from small protein crystals. It is possible to further boost the acoustic reflection from small crystals by reducing the wavelength of the incident sound pulse, but our current instrumentation does not allow this option. In the future, commercially available sound-emitting transducers with a characteristic frequency near 300 MHz should detect and monitor the growth of individual 3 µm crystals.

  3. Adjustment of Protein Crystal Porosity for Biotemplating: Chemical and Protein Engineering Tools

    NASA Astrophysics Data System (ADS)

    Wine, Yariv; Cohen-Hadar, Noa; Lagziel-Simis, Shira; Dror, Yael; Frolow, Felix; Freeman, Amihay

    2010-05-01

    Protein crystals, routinely prepared for the elucidation of protein 3D structures by X-ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein molecules in the crystal is a complementary 3D array of voids made of interconnected cavities and exhibiting highly ordered porosity. Here we propose and demonstrate feasibility of using chemical and genetic tools to alter protein crystal packing by a series of modifications of targeted sites on protein's surface, enabling the generation of a series of protein crystal biotemplates, all originating from same parent protein.

  4. An ignored variable: solution preparation temperature in protein crystallization.

    PubMed

    Chen, Rui-Qing; Lu, Qin-Qin; Cheng, Qing-Di; Ao, Liang-Bo; Zhang, Chen-Yan; Hou, Hai; Liu, Yong-Ming; Li, Da-Wei; Yin, Da-Chuan

    2015-01-19

    Protein crystallization is affected by many parameters, among which certain parameters have not been well controlled. The temperature at which the protein and precipitant solutions are mixed (i.e., the ambient temperature during mixing) is such a parameter that is typically not well controlled and is often ignored. In this paper, we show that this temperature can influence protein crystallization. The experimental results showed that both higher and lower mixing temperatures can enhance the success of crystallization, which follows a parabolic curve with an increasing ambient temperature. This work illustrates that the crystallization solution preparation temperature is also an important parameter for protein crystallization. Uncontrolled or poorly controlled room temperature may yield poor reproducibility in protein crystallization.

  5. Application of protein engineering to enhance crystallizability and improve crystal properties.

    PubMed

    Derewenda, Zygmunt S

    2010-05-01

    Until recently, protein crystallization has mostly been regarded as a stochastic event over which the investigator has little or no control. With the dramatic technological advances in synchrotron-radiation sources and detectors and the equally impressive progress in crystallographic software, including automated model building and validation, crystallization has increasingly become the rate-limiting step in X-ray diffraction studies of macromolecules. However, with the advent of recombinant methods it has also become possible to engineer target proteins and their complexes for higher propensity to form crystals with desirable X-ray diffraction qualities. As most proteins that are under investigation today are obtained by heterologous overexpression, these techniques hold the promise of becoming routine tools with the potential to transform classical crystallization screening into a more rational high-success-rate approach. This article presents an overview of protein-engineering methods designed to enhance crystallizability and discusses a number of examples of their successful application.

  6. Fluorescence Studies of Protein Crystal Nucleation

    NASA Technical Reports Server (NTRS)

    Pusey, Marc; Sumida, John

    2000-01-01

    -association process is a function of the protein concentration relative to the saturation concentration, and observing it in dilute solution (conc. less than or equal to 10(exp -5)M) requires that the experiments be performed under low solubility conditions, i.e., low temperatures and high salt concentrations. Data from preliminary steady state FRET studies with N-terminal bound pyrene acetic acid (PAA-lys, donor, Ex 340 nm, Em 376 nm) and asp101 LY-lys as an acceptor showed a consistent trend of decreasing donor fluorescence intensity with increasing total protein concentration. The FRET data have been obtained at pH 4.6, 0.1M NaAc buffer, at 5 and 7% NaCl, 4 C. The corresponding C(sub sat) values are 0.471 and 0.362 mg/ml (approx. 3.3 and approx. 2.5 x 10(exp -5)M respectively). The donor fluorescence decrease is more pronounced at7% NaCl, consistent with the expected increased intermolecular interactions at higher salt concentrations as reflected in the lower solubility. Results from these and other ongoing studies will be discussed in conjunction with an emerging model for how tetragonal lysozyme crystals nucleate and the relevance of that model to other proteins.

  7. Electrorheological crystallization of proteins and other molecules

    DOEpatents

    Craig, George D.; Rupp, Bernhard

    1996-01-01

    An electrorheological crystalline mass of a molecule is formed by dispersing the molecule in a dispersion fluid and subjecting the molecule dispersion to a uniform electrical field for a period of time during which time an electrorheological crystalline mass is formed. Molecules that may be used to form an electrorheological crystalline mass include any organic or inorganic molecule which has a permanent dipole and/or which is capable of becoming an induced dipole in the presence of an electric field. The molecules used to form the electrorheological crystalline mass are preferably macromolecules, such as biomolecules, such as proteins, nucleic acids, carbohydrates, lipoproteins and viruses. Molecules are crystallized by a method in which an electric field is maintained for a period of time after the electrorheological crystalline mass has formed during which time at least some of the molecules making up the electrorheological crystalline mass form a crystal lattice. The three dimensional structure of a molecule is determined by a method in which an electrorheological crystalline mass of the molecule is formed, an x-ray diffraction pattern of the electrorheological crystalline mass is obtained and the three dimensional structure of the molecule is calculated from the x-ray diffraction pattern.

  8. Electrorheological crystallization of proteins and other molecules

    DOEpatents

    Craig, G.D.; Rupp, B.

    1996-06-11

    An electrorheological crystalline mass of a molecule is formed by dispersing the molecule in a dispersion fluid and subjecting the molecule dispersion to a uniform electrical field for a period of time during which time an electrorheological crystalline mass is formed. Molecules that may be used to form an electrorheological crystalline mass include any organic or inorganic molecule which has a permanent dipole and/or which is capable of becoming an induced dipole in the presence of an electric field. The molecules used to form the electrorheological crystalline mass are preferably macromolecules, such as biomolecules, such as proteins, nucleic acids, carbohydrates, lipoproteins and viruses. Molecules are crystallized by a method in which an electric field is maintained for a period of time after the electrorheological crystalline mass has formed during which time at least some of the molecules making up the electrorheological crystalline mass form a crystal lattice. The three dimensional structure of a molecule is determined by a method in which an electrorheological crystalline mass of the molecule is formed, an X-ray diffraction pattern of the electrorheological crystalline mass is obtained and the three dimensional structure of the molecule is calculated from the X-ray diffraction pattern. 4 figs.

  9. Detergent-Specific Membrane Protein Crystallization Screens

    NASA Technical Reports Server (NTRS)

    Wiener, Michael

    2007-01-01

    A suite of reagents has been developed for three-dimensional crystallization of integral membranes present in solution as protein-detergent complexes (PDCs). The compositions of these reagents have been determined in part by proximity to the phase boundaries (lower consolute boundaries) of the detergents present in the PDCs. The acquisition of some of the requisite phase-boundary data and the preliminary design of several of the detergent- specific screens was supported by a NASA contract. At the time of expiration of the contract, a partial set of preliminary screens had been developed. This work has since been extended under non-NASA sponsorship, leading to near completion of a set of 20 to 30 different and unique detergent- specific 96-condition screens.

  10. Automating the application of smart materials for protein crystallization

    SciTech Connect

    Khurshid, Sahir; Govada, Lata; EL-Sharif, Hazim F.; Reddy, Subrayal M.; Chayen, Naomi E.

    2015-03-01

    The first semi-liquid, non-protein nucleating agent for automated protein crystallization trials is described. This ‘smart material’ is demonstrated to induce crystal growth and will provide a simple, cost-effective tool for scientists in academia and industry. The fabrication and validation of the first semi-liquid nonprotein nucleating agent to be administered automatically to crystallization trials is reported. This research builds upon prior demonstration of the suitability of molecularly imprinted polymers (MIPs; known as ‘smart materials’) for inducing protein crystal growth. Modified MIPs of altered texture suitable for high-throughput trials are demonstrated to improve crystal quality and to increase the probability of success when screening for suitable crystallization conditions. The application of these materials is simple, time-efficient and will provide a potent tool for structural biologists embarking on crystallization trials.

  11. Growth of protein crystals in hydrogels prevents osmotic shock.

    PubMed

    Sugiyama, Shigeru; Maruyama, Mihoko; Sazaki, Gen; Hirose, Mika; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Matsumura, Hiroyoshi

    2012-04-01

    High-throughput protein X-ray crystallography offers a significant opportunity to facilitate drug discovery. The most reliable approach is to determine the three-dimensional structure of the protein-ligand complex by soaking the ligand in apo crystals. However, protein apo crystals produced by conventional crystallization in a solution are fatally damaged by osmotic shock during soaking. To overcome this difficulty, we present a novel technique for growing protein crystals in a high-concentration hydrogel that is completely gellified and exhibits high strength. This technique allowed us essentially to increase the mechanical stability of the crystals, preventing serious damage to the crystals caused by osmotic shock. Thus, this method may accelerate structure-based drug discoveries.

  12. Some implications of colloid stability theory for protein crystallization

    NASA Technical Reports Server (NTRS)

    Young, C. C.; De Mattei, R. C.; Feigelson, R. S.; Tiller, W. A.

    1988-01-01

    Colloid stability theory has been applied to protein crystallization and predicts a narrow range of conditions under which crystals can be grown without the agglomeration of protein molecules (colloids) in the bulk solution. It also predicts a critical electrolyte concentration above which agglomeration will always occur. Using this theory, the rapid protein agglomeration occurring during Schlieren experiments as well as a terminal crystal size effect in a fixed container were explained. Following this concept, the supposed 'terminal' crystal size has been at least doubled.

  13. Protein crystals as scanned probes for recognition atomic force microscopy.

    PubMed

    Wickremasinghe, Nissanka S; Hafner, Jason H

    2005-12-01

    Lysozyme crystal growth has been localized at the tip of a conventional silicon nitride cantilever through seeded nucleation. After cross-linking with glutaraldehyde, lysozyme protein crystal tips image gold nanoparticles and grating standards with a resolution comparable to that of conventional tips. Force spectra between the lysozyme crystal tips and surfaces covered with antilysozyme reveal an adhesion force that drops significantly upon blocking with free lysozyme, thus confirming that lysozyme crystal tips can detect molecular recognition interactions.

  14. Recent advances in mammalian protein production

    PubMed Central

    Bandaranayake, Ashok D.; Almo, Steven C.

    2014-01-01

    Mammalian protein production platforms have had a profound impact in many areas of basic and applied research, and an increasing number of blockbuster drugs are recombinant mammalian proteins. With global sales of these drugs exceeding US$120 billion per year, both industry and academic research groups continue to develop cost effective methods for producing mammalian proteins to support preclinical and clinical evaluations of potential therapeutics. While a wide range of platforms have been successfully exploited for laboratory use, the bulk of recent biologics have been produced in mammalian cell lines due to the requirement for post translational modification and the biosynthetic complexity of the target proteins. In this review we highlight the range of mammalian expression platforms available for recombinant protein production, as well as advances in technologies for the rapid and efficient selection of highly productive clones. PMID:24316512

  15. Lab-on-a-Chip Based Protein Crystallization

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark J.; Brasseur, Michael M.; Spearing, Scott F.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    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.

  16. Further advances in the production of membrane proteins in Pichia pastoris

    PubMed Central

    Hedfalk, Kristina

    2013-01-01

    Membrane proteins have essential cellular functions and are therefore of high interest in both academia and industry. Many efforts have been made on producing those targets in yields allowing crystallization experiments aiming for high resolution structures and mechanistic understanding. The first step of production provides a crucial barrier to overcome, but what we now see, is great progress in membrane protein structural determination in a relatively short time. Achievements on recombinant protein production have been essential for this development and the yeast Pichia pastoris is the most commonly used host for eukaryotic membrane proteins. High-resolution structures nicely illustrate the successes in protein production, and this is the measure used by Ramón and Marin in their review “Advances in the production of membrane proteins in Pichia pastoris” from 2011. Here, additional advances on production and crystallization of eukaryotic membrane proteins are described and reflected on. PMID:23507631

  17. The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS)

    SciTech Connect

    Gerdts, Cory J.; Elliott, Mark; Lovell, Scott; Mixon, Mark B.; Napuli, Alberto J.; Staker, Bart L.; Nollert, Peter; Stewart, Lance

    2012-02-08

    The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, {approx}10-20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one comprehensive 'hybrid' crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants.

  18. Protein Innovations Advance Drug Treatments, Skin Care

    NASA Technical Reports Server (NTRS)

    2012-01-01

    Dan Carter carefully layered the sheets of tracing paper on the light box. On each sheet were renderings of the atomic components of an essential human protein, one whose structure had long been a mystery. With each layer Carter laid down, a never-before-seen image became clearer. Carter joined NASA s Marshall Space Flight Center in 1985 and began exploring processes of protein crystal growth in space. By bouncing intense X-rays off the crystals, researchers can determine the electron densities around the thousands of atoms forming the protein molecules, unveiling their atomic structures. Cultivating crystals of sufficient quality on Earth was problematic; the microgravity conditions of space were far more accommodating. At the time, only a few hundred protein structures had been mapped, and the methods were time consuming and tedious. Carter hoped his work would help reveal the structure of human serum albumin, a major protein in the human circulatory system responsible for ferrying numerous small molecules in the blood. More was at stake than scientific curiosity. Albumin has a high affinity for most of the world s pharmaceuticals, Carter explains, and its interaction with drugs can change their safety and efficacy. When a medication enters the bloodstream a cancer chemotherapy drug, for example a majority of it can bind with albumin, leaving only a small percentage active for treatment. How a drug interacts with albumin can influence considerations like the necessary effective dosage, playing a significant role in the design and application of therapeutic measures. In spite of numerous difficulties, including having no access to microgravity following the 1986 Space Shuttle Challenger disaster, the image Carter had hoped to see was finally clarifying. In 1988, his lab had acquired specialized X-ray and detection equipment a tipping point. Carter and his colleagues began to piece together albumin s portrait, the formation of its electron densities coalescing on

  19. Ionic strength and intermolecular contacts in protein crystals

    NASA Astrophysics Data System (ADS)

    Iyer, Ganesh H.; Dasgupta, Swagata; Bell, Jeffrey A.

    2000-08-01

    The ionic strengths of crystallization solutions for 206 proteins were observed to form a bimodal distribution. The data was divided into two sets at an ionic strength of 4.4 M, and knowledge-based potentials were calculated to determine contact preferences at intermolecular crystal interfaces. Consistent with previous observations over all ionic strengths, intermolecular crystal contacts tend to exclude nonpolar amino acids; lysine is the least favored polar amino acid at crystal contacts; and arginine and glutamine are the two most favored amino acid at crystal contacts. However, some aspects of intermolecular contact preferences within protein crystals are significantly dependent on ionic strength. Arginine is the most favored residue at low ionic strength, but it takes second place to glutamine at high ionic strength. Other major ionic strength-dependent differences in protein crystal contacts can be explained by the binding of cations or anions. While others have shown the importance of ion binding experimentally in selected protein crystals, these statistical results indicate that intermolecular interface formation must involve ion-mediated contacts in a large number of protein crystals.

  20. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    Li, Fenglei

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition

  1. Transparent Cell for Protein Crystallization under Low Applied Voltage

    NASA Astrophysics Data System (ADS)

    Wakamatsu, Takashi; Ohnishi, Yuuki

    2011-04-01

    A transparent cell with the ability to apply a uniform internal electric field has been designed for protein crystallization. The parallel configuration of two plate electrodes coated with transparent conductive films provides a cell where the growth of protein crystals can be observed. In addition, the electrodes allow the formation of parallel electric fields in the protein solution, which can be applied at a very low voltage so that the electrolysis of the solution does not occur.

  2. Imaging and interferometric analysis of protein crystal growth

    NASA Astrophysics Data System (ADS)

    Raghunandan, Ranjini; Gupta, Anamika Sethia; Muralidhar, K.

    2008-04-01

    Protein crystals are grown under controlled temperature, concentration and vapor pressure conditions, usually by vapor diffusion, liquid-liquid diffusion and dialysis techniques. The present study examines the effects of protein concentration, drop size and reservoir height on the crystal growth of Hen Egg White Lysozyme (HEWL). Crystals are grown by the hanging drop vapor diffusion method using Modular VDX TM Plates. Due to the vapor pressure difference created between the protein drop and the reservoir, evaporation takes place till equilibrium is attained. Crystal formation takes place after a certain level of supersaturation is attained when the protein precipitates out in crystalline form. The observations revealed that the growth is faster for higher lysozyme concentration, smaller drop sizes and larger reservoir heights. The morphology of the crystals is viewed during the growth process using stereomicroscope. The number of crystals formed is the maximum for higher concentrations, drop sizes and reservoir heights. When the number of crystals formed is less, the size of the crystals is comparatively larger. The effect of evaporation of water vapor from the protein drop into the reservoir is studied using Mach-Zehnder interferometry. The recorded interferograms and shadowgraph images indicate the diffusion of condensed water into the reservoir. The radius of the drop is determined using the shadowgraph images of the growth process. The radius decreases with evaporation and the rate of decrease of radius is highest for higher protein concentrations, smaller drop sizes and larger reservoir heights.

  3. Recent Advances in Colloidal and Interfacial Phenomena Involving Liquid Crystals

    PubMed Central

    Bai, Yiqun; Abbott, Nicholas L.

    2011-01-01

    This article describes recent advances in several areas of research involving the interfacial ordering of liquid crystals (LCs). The first advance revolves around the ordering of LCs at bio/chemically functionalized surfaces. Whereas the majority of past studies of surface-induced ordering of LCs have involved surfaces of solids that present a limited diversity of chemical functional groups (surfaces at which van der Waals forces dominate surface-induced ordering), recent studies have moved to investigate the ordering of LCs on chemically complex surfaces. For example, surfaces decorated with biomolecules (e.g. oligopeptides and proteins) and transition metal ions have been investigated, leading to an understanding of the roles that metal-ligand coordination interactions, electrical double-layers, acid-base interactions, and hydrogen bonding can have on the interfacial ordering of LCs. The opportunity to create chemically-responsive LCs capable of undergoing ordering transitions in the presence of targeted molecular events (e.g., ligand exchange around a metal center) has emerged from these fundamental studies. A second advance has focused on investigations of the ordering of LCs at interfaces with immiscible isotropic fluids, particularly water. In contrast to prior studies of surface-induced ordering of LCs on solid surfaces, LC- aqueous interfaces are deformable and molecules at these interfaces exhibit high levels of mobility and thus can reorganize in response to changes in interfacial environment. A range of fundamental investigations involving these LC-aqueous interfaces have revealed that (i) the spatial and temporal characteristics of assemblies formed from biomolecular interactions can be reported by surface-driven ordering transitions in the LCs, (ii) the interfacial phase behaviour of molecules and colloids can be coupled to (and manipulated via) the ordering (and nematic elasticity) of LCs, and (iii) confinement of LCs leads to unanticipated size

  4. CrystPro: Spatiotemporal Analysis of Protein Crystallization Images

    PubMed Central

    2015-01-01

    Thousands of experiments corresponding to different combinations of conditions are set up to determine the relevant conditions for successful protein crystallization. In recent years, high throughput robotic set-ups have been developed to automate the protein crystallization experiments, and imaging techniques are used to monitor the crystallization progress. Images are collected multiple times during the course of an experiment. Huge number of collected images make manual review of images tedious and discouraging. In this paper, utilizing trace fluorescence labeling, we describe an automated system called CrystPro for monitoring the protein crystal growth in crystallization trial images by analyzing the time sequence images. Given the sets of image sequences, the objective is to develop an efficient and reliable system to detect crystal growth changes such as new crystal formation and increase of crystal size. CrystPro consists of three major steps- identification of crystallization trials proper for spatio-temporal analysis, spatio-temporal analysis of identified trials, and crystal growth analysis. We evaluated the performance of our system on 3 crystallization image datasets (PCP-ILopt-11, PCP-ILopt-12, and PCP-ILopt-13) and compared our results with expert scores. Our results indicate a) 98.3% accuracy and .896 sensitivity on identification of trials for spatio-temporal analysis, b) 77.4% accuracy and .986 sensitivity of identifying crystal pairs with new crystal formation, and c) 85.8% accuracy and 0.667 sensitivity on crystal size increase detection. The results show that our method is reliable and efficient for tracking growth of crystals and determining useful image sequences for further review by the crystallographers. PMID:26640418

  5. Automating the application of smart materials for protein crystallization

    PubMed Central

    Khurshid, Sahir; Govada, Lata; EL-Sharif, Hazim F.; Reddy, Subrayal M.; Chayen, Naomi E.

    2015-01-01

    The fabrication and validation of the first semi-liquid nonprotein nucleating agent to be administered automatically to crystallization trials is reported. This research builds upon prior demonstration of the suitability of molecularly imprinted polymers (MIPs; known as ‘smart materials’) for inducing protein crystal growth. Modified MIPs of altered texture suitable for high-throughput trials are demonstrated to improve crystal quality and to increase the probability of success when screening for suitable crystallization conditions. The application of these materials is simple, time-efficient and will provide a potent tool for structural biologists embarking on crystallization trials. PMID:25760603

  6. Counterdiffusion methods applied to protein crystallization.

    PubMed

    Otálora, Fermín; Gavira, José Antonio; Ng, Joseph D; García-Ruiz, Juan Manuel

    2009-11-01

    Accumulated experience during the last years on counterdiffusion crystallization methods shows that they are a convenient and generally applicable way of optimizing solution crystal growth experiments. Irrespective of whether the objective of the experiment is to improve crystal quality or size, many experiments reporting a positive or neutral effect of counterdiffusion exists, but adverse effects are consistently absent. Thus counterdiffusion is viewed as a rational crystallization approach to minimize supersaturation and impurity levels at the crystal growth front and to ensure steadiness of both values. This control of the phase transition state is automatically achieved and sustained by a dynamic equilibrium between mass transport and aggregation kinetics. The course of this function can be implemented in any media permitting diffusive mass transport (gels, capillaries, microfluidic devices or microgravity). The counterdiffusion technique has been exploited in many recent applications revealing interesting effects on nucleation and polymorphic precipitation, hence opening further possibilities for innovative screening of crystallization conditions.

  7. Advances in Homology Protein Structure Modeling

    PubMed Central

    Xiang, Zhexin

    2007-01-01

    Homology modeling plays a central role in determining protein structure in the structural genomics project. The importance of homology modeling has been steadily increasing because of the large gap that exists between the overwhelming number of available protein sequences and experimentally solved protein structures, and also, more importantly, because of the increasing reliability and accuracy of the method. In fact, a protein sequence with over 30% identity to a known structure can often be predicted with an accuracy equivalent to a low-resolution X-ray structure. The recent advances in homology modeling, especially in detecting distant homologues, aligning sequences with template structures, modeling of loops and side chains, as well as detecting errors in a model, have contributed to reliable prediction of protein structure, which was not possible even several years ago. The ongoing efforts in solving protein structures, which can be time-consuming and often difficult, will continue to spur the development of a host of new computational methods that can fill in the gap and further contribute to understanding the relationship between protein structure and function. PMID:16787261

  8. Screening of nucleation conditions using levitated drops for protein crystallization.

    PubMed

    Santesson, Sabina; Cedergren-Zeppezauer, Eila S; Johansson, Thomas; Laurell, Thomas; Nilsson, Johan; Nilsson, Staffan

    2003-04-01

    The growth of suitable protein crystals is an essential step in the structure determination of a protein by X-ray crystallography. At present, crystals are mostly grown using trial-and-error procedures, and protocols that rapidly screen for the crystal nucleation step are rare. Presented here is an approach to minimize the consumption of precious protein material while searching for the nucleation conditions. Acoustically levitated drops of known protein concentration (0.25-1.5-microL volumes) are injected with crystallizing agents using piezoelectric flow-through dispensers (ejecting 50-100-pL droplets at 1-9000 droplets/s). A restricted number of crystallizing agents representing three classes are used: poly(ethylene glycol), salts, and the viscous alcohol 2-methyl 2,4-pentanediol. From a digitized picture of the levitated drop volume, calculations are performed giving the concentrations of all components in the drop at any time during a "precipitation experiment". Supersaturation is the prerequisite for crystal nucleation, and protein precipitation indicates high supersaturation. A light source illuminates the levitated drop, and protein precipitation is monitored using right-angle light scattering. On the basis of these intensity measurements and the volume determination, precipitation diagrams for each crystallizing agent are constructed that give the protein/crystallizing agent concentration boundaries between the minimum and the maximum detectable protein precipitation. Guided by the concentration values obtained from such plots, when approaching the supersaturation region, separate crystallization drops are mixed and allowed to equilibrate under paraffin oil. At conditions in which microcrystals can be observed, the nucleation tendency of the macromolecule is confirmed. Optimization of crystallization conditions can then follow. Proteins tested include alcohol dehydrogenase and D-serine dehydratase. Alcohol dehydrogenase, known to crystallize easily, was

  9. Protein Crystal Movements and Fluid Flows During Microgravity Growth

    NASA Technical Reports Server (NTRS)

    Boggon, Titus J.; Chayen, Naomi E.; Snell, Edward H.; Dong, Jun; Lautenschlager, Peter; Potthast, Lothar; Siddons, D. Peter; Stojanoff, Vivian; Gordon, Elspeth; Thompson, Andrew W.; Zagalsky, Peter F.; Bi, Ru-Chang; Helliwell, John R.

    1997-01-01

    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.

  10. Real-time observation of nonclassical protein crystallization kinetics.

    PubMed

    Sauter, Andrea; Roosen-Runge, Felix; Zhang, Fajun; Lotze, Gudrun; Jacobs, Robert M J; Schreiber, Frank

    2015-02-01

    We present a real-time study of protein crystallization of bovine β-lactoglobulin in the presence of CdCl(2) using small-angle X-ray scattering and optical microscopy. From observing the crystallization kinetics, we propose the following multistep crystallization mechanism that is consistent with our data. In the first step, an intermediate phase is formed, followed by the nucleation of crystals within the intermediate phase. During this period, the number of crystals increases with time, but the crystal growth is slowed down by the surrounding dense intermediate phase due to the low mobility. In the next step, the intermediate phase is consumed by nucleation and slow growth, and the crystals are exposed to the dilute phase. In this stage, the number of crystals becomes nearly constant, whereas the crystals grow rapidly due to access to the free protein molecules in the dilute phase. This real-time study not only provides evidence for a two-step nucleation process for protein crystallization but also elucidates the role and the structural signature of the metastable intermediate phase in this process.

  11. Real-time observation of nonclassical protein crystallization kinetics.

    PubMed

    Sauter, Andrea; Roosen-Runge, Felix; Zhang, Fajun; Lotze, Gudrun; Jacobs, Robert M J; Schreiber, Frank

    2015-02-01

    We present a real-time study of protein crystallization of bovine β-lactoglobulin in the presence of CdCl(2) using small-angle X-ray scattering and optical microscopy. From observing the crystallization kinetics, we propose the following multistep crystallization mechanism that is consistent with our data. In the first step, an intermediate phase is formed, followed by the nucleation of crystals within the intermediate phase. During this period, the number of crystals increases with time, but the crystal growth is slowed down by the surrounding dense intermediate phase due to the low mobility. In the next step, the intermediate phase is consumed by nucleation and slow growth, and the crystals are exposed to the dilute phase. In this stage, the number of crystals becomes nearly constant, whereas the crystals grow rapidly due to access to the free protein molecules in the dilute phase. This real-time study not only provides evidence for a two-step nucleation process for protein crystallization but also elucidates the role and the structural signature of the metastable intermediate phase in this process. PMID:25569484

  12. Membrane proteins, detergents and crystals: what is the state of the art?

    PubMed

    Loll, Patrick J

    2014-12-01

    At the time when the first membrane-protein crystal structure was determined, crystallization of these molecules was widely perceived as extremely arduous. Today, that perception has changed drastically, and the process is regarded as routine (or nearly so). On the occasion of the International Year of Crystallography 2014, this review presents a snapshot of the current state of the art, with an emphasis on the role of detergents in this process. A survey of membrane-protein crystal structures published since 2012 reveals that the direct crystallization of protein-detergent complexes remains the dominant methodology; in addition, lipidic mesophases have proven immensely useful, particularly in specific niches, and bicelles, while perhaps undervalued, have provided important contributions as well. Evolving trends include the addition of lipids to protein-detergent complexes and the gradual incorporation of new detergents into the standard repertoire. Stability has emerged as a critical parameter controlling how a membrane protein behaves in the presence of detergent, and efforts to enhance stability are discussed. Finally, although discovery-based screening approaches continue to dwarf mechanistic efforts to unravel crystallization, recent technical advances offer hope that future experiments might incorporate the rational manipulation of crystallization behaviors.

  13. Rapid protein crystallization by a micro osmotic screening system

    NASA Astrophysics Data System (ADS)

    Chan, Po-Hsiung; Su, Yu-Chuan

    2007-03-01

    This work presents a micro osmotic screening system that grows protein crystals in hours while consuming only micrograms of samples. Throughout the crystallization process, water can be driven in or out of a protein solution (across a semi-permeable membrane) to adjust its concentrations as desired. With the bi-directional and adjustable flow control realized by osmosis, each protein sample can be screened for crystallization conditions over a highly extended range. In the prototype demonstration, 6 × 8 screening arrays having an overall size of 20 × 24 × 2.5 mm3 were fabricated and characterized with crystallization experiments. In these experiments, crystallization conditions for four proteins, including lysozyme, catalase, thaumatin and xylanase, were identified within 2-6 h while consuming less than 20 µl of sample solution for each protein. Furthermore, it was also demonstrated that diffraction-quality crystals may be grown and harvested from the prototype system. As such, this osmotic system pioneers a new class of rapid screening schemes for high-throughput protein crystallization. A portion of this paper was presented at the 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Tokyo, Japan, November 2006.

  14. Large-volume protein crystal growth for neutron macromolecular crystallography

    DOE PAGES

    Ng, Joseph D.; Baird, James K.; Coates, Leighton; Garcia-Ruiz, Juan M.; Hodge, Teresa A.; Huang, Sijay

    2015-03-30

    Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for themore » growth of crystals to significant dimensions that are now relevant to NMC are revisited. We report that these include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.« less

  15. Large-volume protein crystal growth for neutron macromolecular crystallography

    SciTech Connect

    Ng, Joseph D.; Baird, James K.; Coates, Leighton; Garcia-Ruiz, Juan M.; Hodge, Teresa A.; Huang, Sijay

    2015-03-30

    Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for the growth of crystals to significant dimensions that are now relevant to NMC are revisited. We report that these include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.

  16. Diffraction study of protein crystals grown in cryoloops and micromounts.

    PubMed

    Berger, Michael A; Decker, Johannes H; Mathews, Irimpan I

    2010-12-01

    Protein crystals are usually grown in hanging or sitting drops and generally get transferred to a loop or micromount for cryocooling and data collection. This paper describes a method for growing crystals on cryoloops for easier manipulation of the crystals for data collection. This study also investigates the steps for the automation of this process and describes the design of a new tray for the method. The diffraction patterns and the structures of three proteins grown by both the new method and the conventional hanging-drop method are compared. The new setup is optimized for the automation of the crystal mounting process. Researchers could prepare nanolitre drops under ordinary laboratory conditions by growing the crystals directly in loops or micromounts. As has been pointed out before, higher levels of supersaturation can be obtained in very small volumes, and the new method may help in the exploration of additional crystallization conditions.

  17. Large-volume protein crystal growth for neutron macromolecular crystallography.

    PubMed

    Ng, Joseph D; Baird, James K; Coates, Leighton; Garcia-Ruiz, Juan M; Hodge, Teresa A; Huang, Sijay

    2015-04-01

    Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for the growth of crystals to significant dimensions that are now relevant to NMC are revisited. These include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.

  18. A Dominant Factor for Structural Classification of Protein Crystals.

    PubMed

    Qi, Fei; Fudo, Satoshi; Neya, Saburo; Hoshino, Tyuji

    2015-08-24

    With the increasing number of solved protein crystal structures, much information on protein shape and atom geometry has become available. It is of great interest to know the structural diversity for a single kind of protein. Our preliminary study suggested that multiple crystal structures of a single kind of protein can be classified into several groups from the viewpoint of structural similarity. In order to broadly examine this finding, cluster analysis was applied to the crystal structures of hemoglobin (Hb), myoglobin (Mb), human serum albumin (HSA), hen egg-white lysozyme (HEWL), and human immunodeficiency virus type 1 protease (HIV-1 PR), downloaded from the Protein Data Bank (PDB). As a result of classification by cluster analysis, 146 crystal structures of Hb were separated into five groups. The crystal structures of Mb (n = 284), HEWL (n = 336), HSA (n = 63), and HIV-1 PR (n = 488) were separated into six, five, three, and six groups, respectively. It was found that a major factor causing these structural separations is the space group of crystals and that crystallizing agents have an influence on the crystal structures. Amino acid mutation is a minor factor for the separation because no obvious point mutation making a specific cluster group was observed for the five kinds of proteins. In the classification of Hb and Mb, the species of protein source such as humans, rabbits, and mice is another significant factor. When the difference in amino sequence is large among species, the species of protein source is the primary factor causing cluster separation in the classification of crystal structures. PMID:26230289

  19. Nucleation and Crystallization of Globular Proteins: What we Know and What is Missing

    NASA Technical Reports Server (NTRS)

    Rosenberger, F.; Vekilov, P. G.; Muschol, M.; Thomas, B. R.

    1996-01-01

    Recently. much progress has been made in understanding the nucleation and crystallization of globular proteins, including the formation of compositional and structural crystal defects, Insight into the interactions of (screened) protein macro-ions in solution, obtained from light scattering, small angle X-ray scattering and osmotic pressure studies. can guide the search for crystallization conditions. These studies show that the nucleation of globular proteins is governed by the same principles as that of small molecules. However, failure to account for direct and indirect (hydrodynamic) protein interactions in the solutions results in unrealistic aggregation scenarios. Microscopic studies of numerous proteins reveal that crystals grow by the attachment of growth units through the same layer-spreading mechanisms as inorganic crystals. Investigations of the growth kinetics of hen-egg-white lysozyme (HEWL) reveal non-steady behavior under steady external conditions. Long-term variations in growth rates are due to changes in step-originating dislocation groups. Fluctuations on a shorter timescale reflect the non-linear dynamics of layer growth that results from the interplay between interfacial kinetics and bulk transport. Systematic gel electrophoretic analyses suggest that most HEWL crystallization studies have been performed with material containing other proteins at percent levels. Yet, sub-percent levels of protein impurities impede growth step propagation and play a role in the formation of structural/compositional inhomogeneities. In crystal growth from highly purified HEWL solutions, however, such inhomogeneities are much weaker and form only in response to unusually large changes in growth conditions. Equally important for connecting growth conditions to crystal perfection and diffraction resolution are recent advances in structural characterization through high-resolution Bragg reflection profiling and X-ray topography.

  20. Colloidal graphenes as heterogeneous additives to enhance protein crystal yield.

    PubMed

    Gully, Benjamin S; Zou, Jianli; Cadby, Gemma; Passon, Daniel M; Iyer, K Swaminathan; Bond, Charles S

    2012-09-01

    In the structural analysis of proteins via X-ray diffraction, a rate-limiting step is in favourable nucleation, a problematic obstacle in successful generation of protein crystals. Here graphene and graphene oxide were applied to protein crystallisation trials, offering improvements in crystalline output and nucleation.

  1. In vivo protein crystallization opens new routes in structural biology.

    PubMed

    Koopmann, Rudolf; Cupelli, Karolina; Redecke, Lars; Nass, Karol; Deponte, Daniel P; White, Thomas A; Stellato, Francesco; Rehders, Dirk; Liang, Mengning; Andreasson, Jakob; Aquila, Andrew; Bajt, Sasa; Barthelmess, Miriam; Barty, Anton; Bogan, Michael J; Bostedt, Christoph; Boutet, Sébastien; Bozek, John D; Caleman, Carl; Coppola, Nicola; Davidsson, Jan; Doak, R Bruce; Ekeberg, Tomas; Epp, Sascha W; Erk, Benjamin; Fleckenstein, Holger; Foucar, Lutz; Graafsma, Heinz; Gumprecht, Lars; Hajdu, Janos; Hampton, Christina Y; Hartmann, Andreas; Hartmann, Robert; Hauser, Günter; Hirsemann, Helmut; Holl, Peter; Hunter, Mark S; Kassemeyer, Stephan; Kirian, Richard A; Lomb, Lukas; Maia, Filipe R N C; Kimmel, Nils; Martin, Andrew V; Messerschmidt, Marc; Reich, Christian; Rolles, Daniel; Rudek, Benedikt; Rudenko, Artem; Schlichting, Ilme; Schulz, Joachim; Seibert, M Marvin; Shoeman, Robert L; Sierra, Raymond G; Soltau, Heike; Stern, Stephan; Strüder, Lothar; Timneanu, Nicusor; Ullrich, Joachim; Wang, Xiaoyu; Weidenspointner, Georg; Weierstall, Uwe; Williams, Garth J; Wunderer, Cornelia B; Fromme, Petra; Spence, John C H; Stehle, Thilo; Chapman, Henry N; Betzel, Christian; Duszenko, Michael

    2012-03-01

    Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo-grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.

  2. In vivo protein crystallization opens new routes in structural biology

    PubMed Central

    Koopmann, Rudolf; Cupelli, Karolina; Redecke, Lars; Nass, Karol; DePonte, Daniel P; White, Thomas A; Stellato, Francesco; Rehders, Dirk; Liang, Mengning; Andreasson, Jakob; Aquila, Andrew; Bajt, Sasa; Barthelmess, Miriam; Barty, Anton; Bogan, Michael J; Bostedt, Christoph; Boutet, Sébastien; Bozek, John D; Caleman, Carl; Coppola, Nicola; Davidsson, Jan; Doak, R Bruce; Ekeberg, Tomas; Epp, Sascha W; Erk, Benjamin; Fleckenstein, Holger; Foucar, Lutz; Graafsma, Heinz; Gumprecht, Lars; Hajdu, Janos; Hampton, Christina Y; Hartmann, Andreas; Hartmann, Robert; Hauser, Günter; Hirsemann, Helmut; Holl, Peter; Hunter, Mark S; Kassemeyer, Stephan; Kirian, Richard A; Lomb, Lukas; Maia, Filipe R N C; Kimmel, Nils; Martin, Andrew V; Messerschmidt, Marc; Reich, Christian; Rolles, Daniel; Rudek, Benedikt; Rudenko, Artem; Schlichting, Ilme; Schulz, Joachim; Seibert, M Marvin; Shoeman, Robert L; Sierra, Raymond G; Soltau, Heike; Stern, Stephan; Strüder, Lothar; Timneanu, Nicusor; Ullrich, Joachim; Wang, Xiaoyu; Weidenspointner, Georg; Weierstall, Uwe; Williams, Garth J; Wunderer, Cornelia B; Fromme, Petra; Spence, John C H; Stehle, Thilo; Chapman, Henry N; Betzel, Christian; Duszenko, Michael

    2012-01-01

    Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo–grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology. PMID:22286384

  3. In vivo protein crystallization opens new routes in structural biology.

    PubMed

    Koopmann, Rudolf; Cupelli, Karolina; Redecke, Lars; Nass, Karol; Deponte, Daniel P; White, Thomas A; Stellato, Francesco; Rehders, Dirk; Liang, Mengning; Andreasson, Jakob; Aquila, Andrew; Bajt, Sasa; Barthelmess, Miriam; Barty, Anton; Bogan, Michael J; Bostedt, Christoph; Boutet, Sébastien; Bozek, John D; Caleman, Carl; Coppola, Nicola; Davidsson, Jan; Doak, R Bruce; Ekeberg, Tomas; Epp, Sascha W; Erk, Benjamin; Fleckenstein, Holger; Foucar, Lutz; Graafsma, Heinz; Gumprecht, Lars; Hajdu, Janos; Hampton, Christina Y; Hartmann, Andreas; Hartmann, Robert; Hauser, Günter; Hirsemann, Helmut; Holl, Peter; Hunter, Mark S; Kassemeyer, Stephan; Kirian, Richard A; Lomb, Lukas; Maia, Filipe R N C; Kimmel, Nils; Martin, Andrew V; Messerschmidt, Marc; Reich, Christian; Rolles, Daniel; Rudek, Benedikt; Rudenko, Artem; Schlichting, Ilme; Schulz, Joachim; Seibert, M Marvin; Shoeman, Robert L; Sierra, Raymond G; Soltau, Heike; Stern, Stephan; Strüder, Lothar; Timneanu, Nicusor; Ullrich, Joachim; Wang, Xiaoyu; Weidenspointner, Georg; Weierstall, Uwe; Williams, Garth J; Wunderer, Cornelia B; Fromme, Petra; Spence, John C H; Stehle, Thilo; Chapman, Henry N; Betzel, Christian; Duszenko, Michael

    2012-03-01

    Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo-grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology. PMID:22286384

  4. Spatiotemporal development of soaked protein crystal

    NASA Astrophysics Data System (ADS)

    Mizutani, Ryuta; Shimizu, Yusuke; Saiga, Rino; Ueno, Go; Nakamura, Yuki; Takeuchi, Akihisa; Uesugi, Kentaro; Suzuki, Yoshio

    2014-07-01

    Crystal soaking is widely performed in biological crystallography. This paper reports time-resolved X-ray crystallographic and microtomographic analyses of tetragonal crystals of chicken egg-white lysozyme soaked in mother liquor containing potassium hexachloroplatinate. The microtomographic analysis showed that X-ray attenuation spread from the superficial layer of the crystal and then to the crystal core. The crystallographic analyses indicated that platinum sites can be classified into two groups from the temporal development of the electron densities. A soaking process consisting of binding-rate-driven and equilibrium-driven layers is proposed to describe these results. This study suggests that the composition of chemical and structural species resulting from the soaking process varies depending on the position in the crystal.

  5. Statistical Analysis of Crystallization Database Links Protein Physico-Chemical Features with Crystallization Mechanisms

    PubMed Central

    Fusco, Diana; Barnum, Timothy J.; Bruno, Andrew E.; Luft, Joseph R.; Snell, Edward H.; Mukherjee, Sayan; Charbonneau, Patrick

    2014-01-01

    X-ray crystallography is the predominant method for obtaining atomic-scale information about biological macromolecules. Despite the success of the technique, obtaining well diffracting crystals still critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. To help determine relationships between macromolecular properties and their crystallization propensity, we have trained statistical models on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis. PMID:24988076

  6. Statistical analysis of crystallization database links protein physico-chemical features with crystallization mechanisms.

    PubMed

    Fusco, Diana; Barnum, Timothy J; Bruno, Andrew E; Luft, Joseph R; Snell, Edward H; Mukherjee, Sayan; Charbonneau, Patrick

    2014-01-01

    X-ray crystallography is the predominant method for obtaining atomic-scale information about biological macromolecules. Despite the success of the technique, obtaining well diffracting crystals still critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. To help determine relationships between macromolecular properties and their crystallization propensity, we have trained statistical models on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis. PMID:24988076

  7. Containerless protein crystal growth technology: Electrostatic multidrop positioner

    NASA Technical Reports Server (NTRS)

    Rhim, Won-Kyu

    1990-01-01

    A brief discussion of containerless protein crystal growth in space and a diagram of the electrostatic multidrop positioner are presented. A picture of lysome crystals growing in a drop and a graph of levitation voltage versus time (minutes) are also presented.

  8. Chamber Design For Slow Nucleation Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    Pusey, Marc Lee

    1995-01-01

    Multiple-chamber dialysis apparatus grows protein crystals on Earth or in microgravity with minimum of intervention by technician. Use of multiple chambers provides gradation of nucleation and growth rates.

  9. Teaching Protein Crystallization by the Gel Acupuncture Method

    NASA Astrophysics Data System (ADS)

    García-Ruiz, Juan Manuel; Moreno, Abel; Otálora, F.; Rondón, D.; Viedma, C.; Zauscher, F.

    1998-04-01

    This paper provides a detailed description of a simple method to obtain large protein single crystals inside glass capillaries. The method is based upon the properties of gels, which are used to hold capillaries containing the protein solution, and also to act as the mass transport medium for the precipitating agent. Recipes for a set of selected experiments bringing a hands-on experience on the crystallization of different soluble proteins are supplied. These experiments are inexpensive and straightforward enough for teaching at the undergraduate level that large biological macromolecules that are the gate our structural studies and drug design can be crystallized. Using simple equations accounting for the solubility of proteins and for the nucleation process, the experimental results are explained to provide a rational approach to the problem. In addition, because of the nature of the crystallization method which is based on diffusion-reactions systems, the student is introduced to the meaning of self-organization.

  10. Structure of apo acyl carrier protein and a proposal to engineer protein crystallization through metal ions

    SciTech Connect

    Qiu, Xiayang; Janson, Cheryl A.

    2010-11-16

    A topic of current interest is engineering surface mutations in order to improve the success rate of protein crystallization. This report explores the possibility of using metal-ion-mediated crystal-packing interactions to facilitate rational design. Escherichia coli apo acyl carrier protein was chosen as a test case because of its high content of negatively charged carboxylates suitable for metal binding with moderate affinity. The protein was successfully crystallized in the presence of zinc ions. The crystal structure was determined to 1.1 {angstrom} resolution with MAD phasing using anomalous signals from the co-crystallized Zn{sup 2+} ions. The case study suggested an integrated strategy for crystallization and structure solution of proteins via engineering surface Asp and Glu mutants, crystallizing them in the presence of metal ions such as Zn{sup 2+} and solving the structures using anomalous signals.

  11. Inorganic and Protein Crystal Assembly in Solutions

    NASA Technical Reports Server (NTRS)

    Chernov, A. A.

    2005-01-01

    The basic kinetic and thermodynamic concepts of crystal growth will be revisited in view of recent AFM and interferometric findings. These concepts are as follows: 1) The Kossel crystal model that allows only one kink type on the crystal surface. The modern theory is developed overwhelmingly for the Kessel model; 2) Presumption that intensive step fluctuations maintain kink density sufficiently high to allow applicability of Gibbs-Thomson law; 3) Common experience that unlimited step bunching (morphological instability) during layer growth from solutions and supercooled melts always takes place if the step flow direction coincides with that of the fluid.

  12. (PCG) Protein Crystal Growth HIV Reverse Transcriptase

    NASA Technical Reports Server (NTRS)

    1992-01-01

    HIV Reverse Transcriptase crystals grown during the USML-1 (STS-50) mission using Commercial Refrigerator/Incubator Module (CR/IM) at 4 degrees C and the Vapor Diffusion Apparatus (VDA). Reverse transcriptase is an enzyme responsible for copying the nucleic acid genome of the AIDS virus from RNA to DNA. Studies indicated that the space-grown crystals were larger and better ordered (beyond 4 angstroms) than were comparable Earth-grown crystals. Principal Investigators were Charles Bugg and Larry DeLucas.

  13. The role of mass transport in protein crystallization.

    PubMed

    García-Ruiz, Juan Manuel; Otálora, Fermín; García-Caballero, Alfonso

    2016-02-01

    Mass transport takes place within the mesoscopic to macroscopic scale range and plays a key role in crystal growth that may affect the result of the crystallization experiment. The influence of mass transport is different depending on the crystallization technique employed, essentially because each technique reaches supersaturation in its own unique way. In the case of batch experiments, there are some complex phenomena that take place at the interface between solutions upon mixing. These transport instabilities may drastically affect the reproducibility of crystallization experiments, and different outcomes may be obtained depending on whether or not the drop is homogenized. In diffusion experiments with aqueous solutions, evaporation leads to fascinating transport phenomena. When a drop starts to evaporate, there is an increase in concentration near the interface between the drop and the air until a nucleation event eventually takes place. Upon growth, the weight of the floating crystal overcomes the surface tension and the crystal falls to the bottom of the drop. The very growth of the crystal then triggers convective flow and inhomogeneities in supersaturation values in the drop owing to buoyancy of the lighter concentration-depleted solution surrounding the crystal. Finally, the counter-diffusion technique works if, and only if, diffusive mass transport is assured. The technique relies on the propagation of a supersaturation wave that moves across the elongated protein chamber and is the result of the coupling of reaction (crystallization) and diffusion. The goal of this review is to convince protein crystal growers that in spite of the small volume of the typical protein crystallization setup, transport plays a key role in the crystal quality, size and phase in both screening and optimization experiments.

  14. The role of mass transport in protein crystallization.

    PubMed

    García-Ruiz, Juan Manuel; Otálora, Fermín; García-Caballero, Alfonso

    2016-02-01

    Mass transport takes place within the mesoscopic to macroscopic scale range and plays a key role in crystal growth that may affect the result of the crystallization experiment. The influence of mass transport is different depending on the crystallization technique employed, essentially because each technique reaches supersaturation in its own unique way. In the case of batch experiments, there are some complex phenomena that take place at the interface between solutions upon mixing. These transport instabilities may drastically affect the reproducibility of crystallization experiments, and different outcomes may be obtained depending on whether or not the drop is homogenized. In diffusion experiments with aqueous solutions, evaporation leads to fascinating transport phenomena. When a drop starts to evaporate, there is an increase in concentration near the interface between the drop and the air until a nucleation event eventually takes place. Upon growth, the weight of the floating crystal overcomes the surface tension and the crystal falls to the bottom of the drop. The very growth of the crystal then triggers convective flow and inhomogeneities in supersaturation values in the drop owing to buoyancy of the lighter concentration-depleted solution surrounding the crystal. Finally, the counter-diffusion technique works if, and only if, diffusive mass transport is assured. The technique relies on the propagation of a supersaturation wave that moves across the elongated protein chamber and is the result of the coupling of reaction (crystallization) and diffusion. The goal of this review is to convince protein crystal growers that in spite of the small volume of the typical protein crystallization setup, transport plays a key role in the crystal quality, size and phase in both screening and optimization experiments. PMID:26841759

  15. Crystallizing membrane proteins for structure-function studies using lipidic mesophases.

    PubMed

    Caffrey, Martin

    2011-06-01

    The lipidic cubic phase method for crystallizing membrane proteins has posted some high-profile successes recently. This is especially true in the area of G-protein-coupled receptors, with six new crystallographic structures emerging in the last 3½ years. Slowly, it is becoming an accepted method with a proven record and convincing generality. However, it is not a method that is used in every membrane structural biology laboratory and that is unfortunate. The reluctance in adopting it is attributable, in part, to the anticipated difficulties associated with handling the sticky viscous cubic mesophase in which crystals grow. Harvesting and collecting diffraction data with the mesophase-grown crystals is also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. However, over the years, we have worked to make the method user-friendly. To this end, tools for handling the mesophase in the pico- to nano-litre volume range have been developed for efficient crystallization screening in manual and robotic modes. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been implemented for a more rational approach to crystallogenesis, such that the method can now be applied to a wide variety of membrane protein types and sizes. In the present article, these assorted advances are outlined, along with a summary of the membrane proteins that have yielded to the method. The challenges that must be overcome to develop the method further are described.

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

    NASA Technical Reports Server (NTRS)

    Long, Marianna M.; Delucas, Larry J.; Smith, C.; Carson, M.; Moore, K.; Harrington, Michael D.; Pillion, D. J.; Bishop, S. P.; Rosenblum, W. M.; Naumann, R. J.

    1994-01-01

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

  17. Single-drop optimization of protein crystallization.

    PubMed

    Meyer, Arne; Dierks, Karsten; Hilterhaus, Dierk; Klupsch, Thomas; Mühlig, Peter; Kleesiek, Jens; Schöpflin, Robert; Einspahr, Howard; Hilgenfeld, Rolf; Betzel, Christian

    2012-08-01

    A completely new crystal-growth device has been developed that permits charting a course across the phase diagram to produce crystalline samples optimized for diffraction experiments. The utility of the device is demonstrated for the production of crystals for the traditional X-ray diffraction data-collection experiment, of microcrystals optimal for data-collection experiments at a modern microbeam insertion-device synchrotron beamline and of nanocrystals required for data collection on an X-ray laser beamline. PMID:22869140

  18. Single-drop optimization of protein crystallization

    PubMed Central

    Meyer, Arne; Dierks, Karsten; Hilterhaus, Dierk; Klupsch, Thomas; Mühlig, Peter; Kleesiek, Jens; Schöpflin, Robert; Einspahr, Howard; Hilgenfeld, Rolf; Betzel, Christian

    2012-01-01

    A completely new crystal-growth device has been developed that permits charting a course across the phase diagram to produce crystalline samples optimized for diffraction experiments. The utility of the device is demonstrated for the production of crystals for the traditional X-ray diffraction data-collection experiment, of microcrystals optimal for data-collection experiments at a modern microbeam insertion-device synchrotron beamline and of nanocrystals required for data collection on an X-ray laser beamline. PMID:22869140

  19. Single-drop optimization of protein crystallization.

    PubMed

    Meyer, Arne; Dierks, Karsten; Hilterhaus, Dierk; Klupsch, Thomas; Mühlig, Peter; Kleesiek, Jens; Schöpflin, Robert; Einspahr, Howard; Hilgenfeld, Rolf; Betzel, Christian

    2012-08-01

    A completely new crystal-growth device has been developed that permits charting a course across the phase diagram to produce crystalline samples optimized for diffraction experiments. The utility of the device is demonstrated for the production of crystals for the traditional X-ray diffraction data-collection experiment, of microcrystals optimal for data-collection experiments at a modern microbeam insertion-device synchrotron beamline and of nanocrystals required for data collection on an X-ray laser beamline.

  20. Engineering nanoparticle-protein associations for protein crystal nucleation and nanoparticle arrangement

    NASA Astrophysics Data System (ADS)

    Benoit, Denise N.

    Engineering the nanoparticle - protein association offers a new way to form protein crystals as well as new approaches for arrangement of nanoparticles. Central to this control is the nanoparticle surface. By conjugating polymers on the surface with controlled molecular weights many properties of the nanoparticle can be changed including its size, stability in buffers and the association of proteins with its surface. Large molecular weight poly(ethylene glycol) (PEG) coatings allow for weak associations between proteins and nanoparticles. These interactions can lead to changes in how proteins crystallize. In particular, they decrease the time to nucleation and expand the range of conditions over which protein crystals form. Interestingly, when PEG chain lengths are too short then protein association is minimized and these effects are not observed. One important feature of protein crystals nucleated with nanoparticles is that the nanoparticles are incorporated into the crystals. What results are nanoparticles placed at well-defined distances in composite protein-nanoparticle crystals. Crystals on the size scale of 10 - 100 micrometers exhibit optical absorbance, fluorescence and super paramagnetic behavior derivative from the incorporated nanomaterials. The arrangement of nanoparticles into three dimensional arrays also gives rise to new and interesting physical and chemical properties, such as fluorescence enhancement and varied magnetic response. In addition, anisotropic nanomaterials aligned throughout the composite crystal have polarization dependent optical properties.

  1. Advances in membrane protein crystallography: in situ and in meso data collection

    SciTech Connect

    Weyand, Simone; Tate, Christopher G.

    2015-05-23

    Membrane protein structural biology has made tremendous advances over the last decade but there are still many challenges associated with crystallization, data collection and structure determination. Two independent groups, Axford et al. [(2015), Acta Cryst. D71, 1228–1237] and Huang et al. [(2015), Acta Cryst. D71, 1238–1256], have published methods that make a major contribution to addressing these challenges.

  2. Orientation and conformation of lipids in crystals of transmembrane proteins.

    PubMed

    Marsh, Derek; Páli, Tibor

    2013-03-01

    Orientational order parameters and individual dihedral torsion angles are evaluated for phospholipid and glycolipid molecules that are resolved in X-ray structures of integral transmembrane proteins in crystals. The order parameters of the lipid chains and glycerol backbones in protein crystals are characterised by a much wider distribution of orientational order than is found in fluid lipid bilayers and reconstituted lipid-protein membranes. This indicates that the lipids that are resolved in crystals of membrane proteins are mostly not representative of the entire lipid-protein interface. Much of the chain configurational disorder of the membrane-bound lipids in crystals arises from C-C bonds in energetically disallowed skew conformations. This suggests configurational heterogeneity of the lipids at a single binding site: eclipsed conformations occur also in the glycerol backbone torsion angles and the C-C torsion angles of the lipid head groups. Conformations of the lipid glycerol backbone in protein crystals are not restricted to the gauche C1-C2 rotamers found invariably in phospholipid bilayer crystals. Lipid head-group conformations in the protein crystals also do not conform solely to the bent-down conformation, with gauche-gauche configuration of the phosphodiester, that is characteristic of phospholipid bilayer membranes. Stereochemical violations in the protein-bound lipids are evidenced by ester carboxyl groups in non-planar configurations, and even in the cis configuration. Some lipids have the incorrect enantiomeric configuration of the glycerol backbone, and many of the branched methyl groups in the phytanyl chains associated with bacteriorhodopsin have the incorrect S configuration. PMID:22644500

  3. Transport and Growth Kinetics in Microgravity Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    Otalora, F.; Garcia-Ruiz, J. M.; Carotenuto, L.; Castagnolo, D.; Novella, M. L.; Chernov, A. A.

    2002-01-01

    The dynamic coupling between mass transport and incorporation of growth units into the surface of a crystal growing from solution in microgravity is used to derive quantitative information on the crystal growth kinetics. To this end, new procedures for experiment preparation, interferometric data processing and model fitting have been developed. The use of experimental data from the bulk diffusive maw transport together with a model for steady state stagnant crystal growth allows the detailed quantitative understanding of the kinetics of both the concentration depletion zone around the crystal and the growth of the crystal interface. The protein crystal used in the experiment is shown to be growing in the mixed kinetic regime (0.2 x 10(exp -6) centimeters per second less than beta R/D less than 0.9 x 10(exp -6) centimeters per second).

  4. Protein-directed self-assembly of a fullerene crystal.

    PubMed

    Kim, Kook-Han; Ko, Dong-Kyun; Kim, Yong-Tae; Kim, Nam Hyeong; Paul, Jaydeep; Zhang, Shao-Qing; Murray, Christopher B; Acharya, Rudresh; DeGrado, William F; Kim, Yong Ho; Grigoryan, Gevorg

    2016-04-26

    Learning to engineer self-assembly would enable the precise organization of molecules by design to create matter with tailored properties. Here we demonstrate that proteins can direct the self-assembly of buckminsterfullerene (C60) into ordered superstructures. A previously engineered tetrameric helical bundle binds C60 in solution, rendering it water soluble. Two tetramers associate with one C60, promoting further organization revealed in a 1.67-Å crystal structure. Fullerene groups occupy periodic lattice sites, sandwiched between two Tyr residues from adjacent tetramers. Strikingly, the assembly exhibits high charge conductance, whereas both the protein-alone crystal and amorphous C60 are electrically insulating. The affinity of C60 for its crystal-binding site is estimated to be in the nanomolar range, with lattices of known protein crystals geometrically compatible with incorporating the motif. Taken together, these findings suggest a new means of organizing fullerene molecules into a rich variety of lattices to generate new properties by design.

  5. Invariant patterns in crystal lattices: Implications for protein folding algorithms

    SciTech Connect

    HART,WILLIAM E.; ISTRAIL,SORIN

    2000-06-01

    Crystal lattices are infinite periodic graphs that occur naturally in a variety of geometries and which are of fundamental importance in polymer science. Discrete models of protein folding use crystal lattices to define the space of protein conformations. Because various crystal lattices provide discretizations of the same physical phenomenon, it is reasonable to expect that there will exist invariants across lattices related to fundamental properties of the protein folding process. This paper considers whether performance-guaranteed approximability is such an invariant for HP lattice models. The authors define a master approximation algorithm that has provable performance guarantees provided that a specific sublattice exists within a given lattice. They describe a broad class of crystal lattices that are approximable, which further suggests that approximability is a general property of HP lattice models.

  6. Protein-directed self-assembly of a fullerene crystal

    NASA Astrophysics Data System (ADS)

    Kim, Kook-Han; Ko, Dong-Kyun; Kim, Yong-Tae; Kim, Nam Hyeong; Paul, Jaydeep; Zhang, Shao-Qing; Murray, Christopher B.; Acharya, Rudresh; Degrado, William F.; Kim, Yong Ho; Grigoryan, Gevorg

    2016-04-01

    Learning to engineer self-assembly would enable the precise organization of molecules by design to create matter with tailored properties. Here we demonstrate that proteins can direct the self-assembly of buckminsterfullerene (C60) into ordered superstructures. A previously engineered tetrameric helical bundle binds C60 in solution, rendering it water soluble. Two tetramers associate with one C60, promoting further organization revealed in a 1.67-Å crystal structure. Fullerene groups occupy periodic lattice sites, sandwiched between two Tyr residues from adjacent tetramers. Strikingly, the assembly exhibits high charge conductance, whereas both the protein-alone crystal and amorphous C60 are electrically insulating. The affinity of C60 for its crystal-binding site is estimated to be in the nanomolar range, with lattices of known protein crystals geometrically compatible with incorporating the motif. Taken together, these findings suggest a new means of organizing fullerene molecules into a rich variety of lattices to generate new properties by design.

  7. The Stanford Automated Mounter: Enabling High-Throughput Protein Crystal Screening at SSRL

    SciTech Connect

    Smith, C.A.; Cohen, A.E.

    2009-05-26

    The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification, and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening, and data collection. At the Stanford Synchrotron Radiation Laboratory, a National User Facility that provides extremely brilliant X-ray photon beams for use in materials science, environmental science, and structural biology research, the incorporation of advanced robotics has enabled crystals to be screened in a true high-throughput fashion, thus dramatically accelerating the final steps. Up to 288 frozen crystals can be mounted by the beamline robot (the Stanford Auto-Mounting System) and screened for diffraction quality in a matter of hours without intervention. The best quality crystals can then be remounted for the collection of complete X-ray diffraction data sets. Furthermore, the entire screening and data collection experiment can be controlled from the experimenter's home laboratory by means of advanced software tools that enable network-based control of the highly automated beamlines.

  8. Convective flow effects on protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz

    1995-01-01

    During the fifth semi-annual period under this grant we have pursued the following activities: (1) Characterization of the purity and further purification of lysozyme solutions, these efforts are summarized in Section 2; (2) Crystal growth morphology and kinetics studies with tetragonal lysozyme, our observation on the dependence of lysozyme growth kinetics on step sources and impurities has been summarized in a manuscript which was accepted for publication in the Journal of Crystal Growth; (3) Numerical modelling of the interaction between bulk transport and interface kinetics, for a detailed summary of this work see the manuscript which was accepted for publication in the Journal of Crystal Growth; and (4) Light scattering studies, this work has been summarized in a manuscript that has been submitted for publication to the Journal of Chemical Physics.

  9. Characterizing protein crystal contacts and their role in crystallization: rubredoxin as a case study.

    PubMed

    Fusco, Diana; Headd, Jeffrey J; De Simone, Alfonso; Wang, Jun; Charbonneau, Patrick

    2014-01-14

    The fields of structural biology and soft matter have independently sought out fundamental principles to rationalize protein crystallization. Yet the conceptual differences and the limited overlap between the two disciplines have thus far prevented a comprehensive understanding of the phenomenon to emerge. We conduct a computational study of proteins from the rubredoxin family that bridges the two fields. Using atomistic simulations, we characterize the protein crystal contacts, and accordingly parameterize patchy particle models. Comparing the phase diagrams of these schematic models with experimental results enables us to critically examine the assumptions behind the two approaches. The study also reveals features of protein–protein interactions that can be leveraged to crystallize proteins more generally.

  10. A Critical Assessment of Protein Crystal Growth in Microgravity

    NASA Technical Reports Server (NTRS)

    Pusey, Marc

    1997-01-01

    Experiments to grow higher diffraction quality protein crystals in the microgravity environment of an orbiting spacecraft are one of the most frequently flown space experiments. Ground-based research has shown that convective flows occur even about protein crystals growing in the Earth's gravitational field. Further, this research has shown that the resultant flow velocities can cause growth cessation, and probably affect the measured X-ray data quality obtained. How flow deleteriously affects protein crystal growth (PCG) is still not known, and is the subject of ongoing research. Failing a rational method for ameliorating flow effects on Earth, one can, through NASA and other nations space agency sponsored programs, carry out protein crystal growth in the microgravity environment of an orbiting spacecraft. Early first generation PCG hardware was characterized by a very low success rate and a steep design learning curve. Subsequent hardware designs have improved upon their predecessors. Now the crystal grower has a wide variety of hardware configurations and crystal growth protocols to choose from, many of which implement "standard" laboratory protein crystal growth methods. While many of these are first or early second generation hardware the success rate, defined as growing crystals giving data better than has been obtained on Earth, is at least 20% overall and may be considerably higher if one only considers latter experiments. There are a large number of protein crystals grown every year, with hundreds of structures determined. Those crystallized in microgravity represent a small proportion of this total, and there is concern that the costs of the microgravity PCG program(s) do not justify such limited returns. Empirical evidence suggests that optimum crystal growth conditions in microgravity differ from those determined on Earth, further exacerbating the chances of success. Microgravity PCG is probably best suited for "mature" crystallizations, where one has

  11. Simulation of protein association: Kinetic pathways towards crystal contacts.

    PubMed

    Taudt, Aaron; Arnold, Axel; Pleiss, Jürgen

    2015-03-01

    We conducted molecular dynamics simulations combined with distance-based umbrella sampling and forward flux sampling to investigate the early stages of protein crystallization. Formation of contacts with long-range interactions and/or an exposed position on the protein surface was kinetically preferred over more stable hydrophobic contacts with a shorter attractive range, while the thermodynamic stability of the protein crystal was provided by hydrophobic interactions. Contacts with a large interaction area showed complex dissociation pathways that were not detected by distance-based umbrella sampling. Instead, forward flux sampling simulations of contact dissociation identified long-range attractive interactions.

  12. Latest methods of fluorescence-based protein crystal identification

    SciTech Connect

    Meyer, Arne; Betzel, Christian

    2015-01-28

    Fluorescence, whether intrinsic or by using trace fluorescent labeling, can be a powerful aid in macromolecule crystallization. Its use in screening for crystals is discussed here. Successful protein crystallization screening experiments are dependent upon the experimenter being able to identify positive outcomes. The introduction of fluorescence techniques has brought a powerful and versatile tool to the aid of the crystal grower. Trace fluorescent labeling, in which a fluorescent probe is covalently bound to a subpopulation (<0.5%) of the protein, enables the use of visible fluorescence. Alternatively, one can avoid covalent modification and use UV fluorescence, exploiting the intrinsic fluorescent amino acids present in most proteins. By the use of these techniques, crystals that had previously been obscured in the crystallization drop can readily be identified and distinguished from amorphous precipitate or salt crystals. Additionally, lead conditions that may not have been obvious as such under white-light illumination can be identified. In all cases review of the screening plate is considerably accelerated, as the eye can quickly note objects of increased intensity.

  13. (PCG) Protein Crystal Growth Isocitrate Lysase

    NASA Technical Reports Server (NTRS)

    1993-01-01

    Comparison of Earth grown and Space grown Isocitrate Lysase crystals. Target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast. It regulates the flow of metabolic intermediates required for cell growth. Principal Investigator was Charles Bugg.

  14. Protein crystal growth and the International Space Station.

    PubMed

    DeLucas, L J; Moore, K M; Long, M M

    1999-05-01

    Protein structural information plays a key role in understanding biological structure-function relationships and in the development of new pharmaceuticals for both chronic and infectious diseases. The Center for Macromolecular Crystallography (CMC) has devoted considerable effort studying the fundamental processes involved in macromolecular crystal growth both in a 1-g and microgravity environment. Results from experiments performed on more than 35 U.S. space shuttle flights have clearly indicated that microgravity can provide a beneficial environment for macromolecular crystal growth. This research has led to the development of a new generation of pharmaceuticals that are currently in preclinical or clinical trials for diseases such as cutaneous T-cell lymphoma, psoriasis, rheumatoid arthritis, AIDS, influenza, stroke and other cardiovascular complications. The International Space Station (ISS) provides an opportunity to have complete crystallographic capability on orbit, which was previously not possible with the space shuttle orbiter. As envisioned, the x-ray Crystallography Facility (XCF) will be a complete facility for growing protein crystals; selecting, harvesting, and mounting sample crystals for x-ray diffraction; cryo-freezing mounted crystals if necessary; performing x-ray diffraction studies; and downlinking the data for use by crystallographers on the ground. Other advantages of such a facility include crystal characterization so that iterations in the crystal growth conditions can be made, thereby optimizing the final crystals produced in a three month interval on the ISS.

  15. A microfluidic, high throughput protein crystal growth method for microgravity.

    PubMed

    Carruthers, Carl W; Gerdts, Cory; Johnson, Michael D; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3) cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories. PMID:24278480

  16. A Microfluidic, High Throughput Protein Crystal Growth Method for Microgravity

    PubMed Central

    Carruthers Jr, Carl W.; Gerdts, Cory; Johnson, Michael D.; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions’ microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 103 cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories. PMID:24278480

  17. Protein crystal growth and the International Space Station

    NASA Technical Reports Server (NTRS)

    DeLucas, L. J.; Moore, K. M.; Long, M. M.

    1999-01-01

    Protein structural information plays a key role in understanding biological structure-function relationships and in the development of new pharmaceuticals for both chronic and infectious diseases. The Center for Macromolecular Crystallography (CMC) has devoted considerable effort studying the fundamental processes involved in macromolecular crystal growth both in a 1-g and microgravity environment. Results from experiments performed on more than 35 U.S. space shuttle flights have clearly indicated that microgravity can provide a beneficial environment for macromolecular crystal growth. This research has led to the development of a new generation of pharmaceuticals that are currently in preclinical or clinical trials for diseases such as cutaneous T-cell lymphoma, psoriasis, rheumatoid arthritis, AIDS, influenza, stroke and other cardiovascular complications. The International Space Station (ISS) provides an opportunity to have complete crystallographic capability on orbit, which was previously not possible with the space shuttle orbiter. As envisioned, the x-ray Crystallography Facility (XCF) will be a complete facility for growing protein crystals; selecting, harvesting, and mounting sample crystals for x-ray diffraction; cryo-freezing mounted crystals if necessary; performing x-ray diffraction studies; and downlinking the data for use by crystallographers on the ground. Other advantages of such a facility include crystal characterization so that iterations in the crystal growth conditions can be made, thereby optimizing the final crystals produced in a three month interval on the ISS.

  18. A microfluidic, high throughput protein crystal growth method for microgravity.

    PubMed

    Carruthers, Carl W; Gerdts, Cory; Johnson, Michael D; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3) cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories.

  19. Investigations on gravity influence upon protein crystallization by the gel acupuncture technique

    NASA Astrophysics Data System (ADS)

    Moreno, Abel; Antonio González-Ramírez, Luis; de los Angeles Hernández-Hernández, María.; Oliver-Salvador, Carmen; Soriano-García, Manuel; Rodríguez-Romero, Adela

    1999-01-01

    Most investigations on biological macromolecules are important for the knowledge of the functions in living organisms. Nowadays it is well known that the three-dimensional structure of proteins is obtained either by NMR or X-ray crystallographic methods. The crucial part in the latter is the availability of high-quality crystals in order to perform structural characterization. Nevertheless, there are some approaches to overcome this problem from the statistical [Jancarik and Kim, J. Appl. Crystallogr. 24 (1991) 409] and physico-chemical point of view [Riès-Kautt and Ducruix, Methods in Enzymology 276 Part A, Ch. 3, 1997, p. 23]. Once the crystals are obtained, the following part of the research must be focused on growing the crystal in order to have an appropriate size for X-ray analysis. There are additional advances in the methods for crystallizing, growing and determining what kind of biophysical or biochemical parameters have to be taken into account in order to obtain a high quality protein crystal, these advances have been already published elsewhere [Ducruix and Giegé, Crystallization of Nucleic Acids and Proteins. A Practical Approach, IRL Press, Oxford, 1991; McPherson, The Preparation and Analysis of Protein Crystals, Wiley, New York, 1982]. In order to evaluate these parameters, we have developed a new technique, called the gel acupuncture technique for crystallizing proteins inside an X-ray capillary tube as well as for trying to study the "in situ" crystal growth phenomena [Garcı´a-Ruiz et al., Mater. Res. Bull. 28 (1993) 541; Garcı´a-Ruiz and Moreno, Acta Crystallogr. D 50 (1994) 484]. In this work, we present our recent investigations on the influence of the gravity vector upon protein crystallization. Three proteins were chosen in order to test this possible influence, taking into account the size of each: satellite tobacco mosaic virus (1000 kDa) and two proteins of "low molecular weight", thaumatin I (22 kDa) and concanavalin A (200 kda

  20. CRYSTALP2: sequence-based protein crystallization propensity prediction

    PubMed Central

    Kurgan, Lukasz; Razib, Ali A; Aghakhani, Sara; Dick, Scott; Mizianty, Marcin; Jahandideh, Samad

    2009-01-01

    Background Current protocols yield crystals for <30% of known proteins, indicating that automatically identifying crystallizable proteins may improve high-throughput structural genomics efforts. We introduce CRYSTALP2, a kernel-based method that predicts the propensity of a given protein sequence to produce diffraction-quality crystals. This method utilizes the composition and collocation of amino acids, isoelectric point, and hydrophobicity, as estimated from the primary sequence, to generate predictions. CRYSTALP2 extends its predecessor, CRYSTALP, by enabling predictions for sequences of unrestricted size and provides improved prediction quality. Results A significant majority of the collocations used by CRYSTALP2 include residues with high conformational entropy, or low entropy and high potential to mediate crystal contacts; notably, such residues are utilized by surface entropy reduction methods. We show that the collocations provide complementary information to the hydrophobicity and isoelectric point. Tests on four datasets show that CRYSTALP2 outperforms several existing sequence-based predictors (CRYSTALP, OB-score, and SECRET). CRYSTALP2's accuracy, MCC, and AROC range between 69.3 and 77.5%, 0.39 and 0.55, and 0.72 and 0.79, respectively. Our predictions are similar in quality and are complementary to the predictions of the most recent ParCrys and XtalPred methods. Our results also suggest that, as work in protein crystallization continues (thereby enlarging the population of proteins with known crystallization propensities), the prediction quality of the CRYSTALP2 method should increase. The prediction model and the datasets used in this contribution can be downloaded from . Conclusion CRYSTALP2 provides relatively accurate crystallization propensity predictions for a given protein chain that either outperform or complement the existing approaches. The proposed method can be used to support current efforts towards improving the success rate in obtaining

  1. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography Using Surface Acoustic Waves.

    PubMed

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela H; French, Jarrod B; Huang, Tony Jun

    2015-06-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming, a surface acoustic wave-based method for manipulating and patterning crystals is developed. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and submicrometer-sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but will also make it possible to collect data on samples that were previously intractable.

  2. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography using Surface Acoustic Waves

    PubMed Central

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela; French, Jarrod B.; Jun Huang, Tony

    2015-01-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming we have developed a surface acoustic wave-based method for manipulating and patterning crystals. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and sub-micrometer sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but also will make it possible to collect data on samples that were previously intractable. PMID:25641793

  3. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    SciTech Connect

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  4. Single crystal piezoelectric composites for advanced NDT ultrasound

    NASA Astrophysics Data System (ADS)

    Jiang, Xiaoning; Snook, Kevin; Hackenberger, Wesley S.; Geng, Xuecang

    2007-04-01

    In this paper, the design, fabrication and characterization of PMN-PT single crystal/epoxy composites are reported for NDT ultrasound transducers. Specifically, 1-3 PMN-PT/epoxy composites with center frequencies of 5 MHz - 40 MHz were designed and fabricated using either the dice-and-fill method or a photolithography based micromachining process. The measured electromechanical coefficients for composites with frequency of 5 MHz - 15 MHz were about 0.78-0.83, and the coupling coefficients for composites with frequencies of 25 MHz- 40 MHz were about 0.71-0.72. The dielectric loss remains low (< 0.05). These properties hold promise for advanced NDT ultrasound applications.

  5. Membrane protein crystallization: Current trends and future perspectives

    PubMed Central

    Parker, Joanne L.; Newstead, Simon

    2016-01-01

    Alpha helical membrane proteins are the targets for many pharmaceutical drugs and play important roles in physiology and disease processes. In recent years substantial progress has been made in determining their atomic structure using X-ray crystallography. However, a major bottleneck still remains; the identification of conditions that give crystals that are suitable for structure determination. Over the past 10 years we have been analyzing the crystallization conditions reported for alpha helical membrane proteins with the aim to facilitate a rational approach to the design and implementation of successful crystallization screens. The result has been the development of MemGold, MemGoldII and the additive screen MemAdvantage. The associated analysis, summarized and updated in this chapter, has revealed a number of surprisingly successfully strategies for crystallization and detergent selection. PMID:27553235

  6. Protein adsorption on surfaces: dynamic contact-angle (DCA) and quartz-crystal microbalance (QCM) measurements.

    PubMed

    Stadler, H; Mondon, M; Ziegler, C

    2003-01-01

    Adsorption of the protein bovine serum albumin (BSA) on gold has been tested at various concentrations in aqueous solution by dynamic contact-angle analysis (DCA) and quartz-crystal microbalance (QCM) measurements. With the Wilhelmy plate technique advancing and receding contact angles and the corresponding hysteresis were measured and correlated with the hydrophilicity and the homogeneity of the surface. With electrical admittance measurements of a gold-coated piezoelectrical quartz crystal, layer mass and viscoelastic contributions to the resonator's frequency shift during adsorption could be separated. A correlation was found between the adsorbed mass and the homogeneity and hydrophilicity of the adsorbed film.

  7. Growing protein crystals in microgravity - The NASA Microgravity Science and Applications Division (MSAD) Protein Crystal Growth (PCG) program

    NASA Technical Reports Server (NTRS)

    Herren, B.

    1992-01-01

    In collaboration with a medical researcher at the University of Alabama at Birmingham, NASA's Marshall Space Flight Center in Huntsville, Alabama, under the sponsorship of the Microgravity Science and Applications Division (MSAD) at NASA Headquarters, is continuing a series of space experiments in protein crystal growth which could lead to innovative new drugs as well as basic science data on protein molecular structures. From 1985 through 1992, Protein Crystal Growth (PCG) experiments will have been flown on the Space Shuttle a total of 14 times. The first four hand-held experiments were used to test hardware concepts; later flights incorporated these concepts for vapor diffusion protein crystal growth with temperature control. This article provides an overview of the PCG program: its evolution, objectives, and plans for future experiments on NASA's Space Shuttle and Space Station Freedom.

  8. Liquid Between Macromolecules in Protein Crystals: Static Versus Dynamics

    NASA Technical Reports Server (NTRS)

    Chernov, A. A.

    2005-01-01

    Protein crystals are so fragile that they often can not be handled by tweezers. Indeed, measurements of the Young modulus, E, of lysozyme crystals resulted in E approx. equals 0.1 - 1 GPa, the lower figures, 0.1 - 0.5 GPa, being obtained from triple point bending of as-grown and not cross-linked crystals sitting in solution. The bending strength was found to be approx.10(exp -2) E. On the other hand, ultrasound speed and Mandelstam-Raman-Brilloin light scattering experiments led to much higher figures, E approx. equals 2.7 GPa. The lower figures for E were found from static or low frequency crystal deformations measurements, while the higher moduli are based on high frequency lattice vibrations, 10(exp 7) - 10(exp 10) 1/s. The physical reason for the about an order of magnitude discrepancy is in different behavior of water filling space between protein molecules. At slow lattice deformation, the not-bound intermolecular water has enough time to flow from the compressed to expanded regions of the deformed crystal. At high deformation frequencies in the ultra- and hypersound waves, the water is confined in the intermolecular space and, on that scale, behaves like a solid, thus contributing to the elastic crystal moduli. In this case, the reciprocal crystal modulus is expected to be an average of the water protein and water compressibilities (reciprocal compressibilities): the bulk modulus for lysozyme is 26 GPa, for water it is 7 GPa. Anisotropy of the crystal moduli comes from intermolecular contacts within the lattice while the high frequency hardness comes from the bulk of protein molecules and water bulk moduli. These conclusions are based on the analysis of liquid flow in porous medium to be presented.

  9. Novel protein crystal growth technology: Proof of concept

    NASA Technical Reports Server (NTRS)

    Nyce, Thomas A.; Rosenberger, Franz

    1989-01-01

    A technology for crystal growth, which overcomes certain shortcomings of other techniques, is developed and its applicability to proteins is examined. There were several unknowns to be determined: the design of the apparatus for suspension of crystals of varying (growing) diameter, control of the temperature and supersaturation, the methods for seeding and/or controlling nucleation, the effect on protein solutions of the temperature oscillations arising from the circulation, and the effect of the fluid shear on the suspended crystals. Extensive effort was put forth to grow lysozyme crystals. Under conditions favorable to the growth of tetragonal lysozyme, spontaneous nucleation could be produced but the number of nuclei could not be controlled. Seed transfer techniques were developed and implemented. When conditions for the orthorhombic form were tried, a single crystal 1.5 x 0.5 x 0.2 mm was grown (after in situ nucleation) and successfully extracted. A mathematical model was developed to predict the flow velocity as a function of the geometry and the operating temperatures. The model can also be used to scaleup the apparatus for growing larger crystals of other materials such as water soluble non-linear optical materials. This crystal suspension technology also shows promise for high quality solution growth of optical materials such as TGS and KDP.

  10. Thermal Optimization of Growth and Quality in Protein Crystals

    NASA Technical Reports Server (NTRS)

    Wiencek, John M.

    1996-01-01

    Experimental evidence suggests that larger and higher quality crystals can be attained in the microgravity of space; however, the effect of growth rate on protein crystal quality is not well documented. This research is the first step towards providing strategies to grow crystals under constant rates of growth. Controlling growth rates at a constant value allows for direct one-to-one comparison of results obtained in microgravity and on earth. The overall goal of the project was to control supersaturation at a constant value during protein crystal growth by varying temperature in a predetermined manner. Applying appropriate theory requires knowledge of specific physicochemical properties of the protein solution including the effect of supersaturation on growth rates and the effect of temperature on protein solubility. Such measurements typically require gram quantities of protein and many months of data acquisition. A second goal of the project applied microcalorimetry for the rapid determination of these physicochemical properties using a minimum amount of protein. These two goals were successfully implemented on hen egg-white lysozyme. Results of these studies are described in the attached reprints.

  11. Applications of the second virial coefficient: protein crystallization and solubility

    SciTech Connect

    Wilson, William W.; DeLucas, Lawrence J.

    2014-04-30

    This article highlights some of the ground-based studies emanating from NASA’s Microgravity Protein Crystal Growth (PCG) program, and includes a more detailed discussion of the history and the progress made in one of the NASA-funded PCG investigations involving the use of measured second virial coefficients (B values) as a diagnostic indicator of solution conditions conducive to protein crystallization. This article begins by highlighting some of the ground-based studies emanating from NASA’s Microgravity Protein Crystal Growth (PCG) program. This is followed by a more detailed discussion of the history of and the progress made in one of the NASA-funded PCG investigations involving the use of measured second virial coefficients (B values) as a diagnostic indicator of solution conditions conducive to protein crystallization. A second application of measured B values involves the determination of solution conditions that improve or maximize the solubility of aqueous and membrane proteins. These two important applications have led to several technological improvements that simplify the experimental expertise required, enable the measurement of membrane proteins and improve the diagnostic capability and measurement throughput.

  12. Liquid drop stability for protein crystal growth in microgravity

    NASA Technical Reports Server (NTRS)

    Owen, Robert B.; Broom, Beth H.; Snyder, Robert S.; Daniel, Ron

    1987-01-01

    It is possible to grow protein crystals for biomedical research in microgravity by deploying a protein-rich solution from a syringe, forming a drop in which crystallization can occur with the proper degree of supersaturation. Drop stability is critical to the success of this research, due to the large drop sizes which can be achieved in space. In order to determine the type of syringe tips most suitable to support these large drops, tests were performed during brief periods of weightlessness onboard the NASA KC-135 low-gravity simulation aircraft. The drops were analyzed using three simple models in which the samples were approximated by modified pendulum and spring systems. It was concluded that the higher frequency systems were the most stable, indicating that of the syringes utilized, a disk-shaped configuration provided the most stable environment of low-gravity protein crystal growth.

  13. Nucleation and Convection Effects in Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz

    1997-01-01

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

  14. Split green fluorescent protein as a modular binding partner for protein crystallization

    SciTech Connect

    Nguyen, Hau B.; Hung, Li-Wei; Yeates, Todd O.; Terwilliger, Thomas C. Waldo, Geoffrey S.

    2013-12-01

    A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization.

  15. THz Microscopy of Anisotropy and Correlated Motions in Protein Crystals

    NASA Astrophysics Data System (ADS)

    Niessen, Katherine; Acbas, Gheorghe; Snell, Edward; Markelz, Andrea

    2013-03-01

    We introduce a new technique, Crystal Anisotropy Terahertz Microscopy (CATM) which can directly measure correlated intra-molecular protein vibrations. The terahertz (THz) frequency range (5-100 cm-1) corresponds to global correlated protein motions, proposed to be essential to protein function [1, 2]. CATM accesses these motions by removal of the relaxational background of the solvent and residue side chain librational motions. We demonstrate narrowband features in the anisotropic absorbance for hen egg-white lysozyme (HEWL) single crystals as well as HEWL with triacetylglucosamine (HEWL-3NAG) inhibitor single crystals. The most prominent features for the HEWL crystals appear at 45 cm-1, 69 cm-1, and 78 cm-1 and the strength of the absorption varies with crystal orientation relative to the THz polarization. Calculations show similar anisotropic features, suggesting specific correlated mode identification is possible. 1. Hammes-Schiffer, S. and S.J. Benkovic, Relating Protein Motion to Catalysis. Annu. Rev. Biochem., 2006. 75: p. 519-41. 2. Henzler-Wildman, K.A., et al., Intrinsic motions along an enzymatic reaction trajectory. Nature, 2007. 450(7171): p. 838-U13. This work supported by NSF MRI2 grant DBI295998.

  16. Mechanisms of protein and virus crystal growth: An atomic force microscopy study of Canavalin crystallization

    SciTech Connect

    Land, T.A.; De Yoreo, J.J.; Malkin, A.J.; Kutznesov, Y.G.; McPherson, A.

    1995-03-10

    The evolution of surface morphology and step dynamics during growth of single crystals of the protein Canavalin and of the cubic satellite tobacco mosaic virus crystals (STMV) have been investigated by in situ atomic force microscopy. These two crystals were observed to grow by very different mechanisms. Growth of Canavalin occurs on complex vicinal hillocks formed by multiple, independently acting screw dislocations. Small cluster were observed on the terraces. STMV on the other hand, was observed to grow by 2D nucleation of islands. No dislocations were found on the crystal. The results are used to determine the growth mechanisms and estimate fundamental materials parameters. The images also illustrate the important mechanism of defect incorporation and provide insight to the processes that limit the growth rate and uniformity of these crystals.

  17. Rigidity analysis of protein biological assemblies and periodic crystal structures

    PubMed Central

    2013-01-01

    Background We initiate in silico rigidity-theoretical studies of biological assemblies and small crystals for protein structures. The goal is to determine if, and how, the interactions among neighboring cells and subchains affect the flexibility of a molecule in its crystallized state. We use experimental X-ray crystallography data from the Protein Data Bank (PDB). The analysis relies on an effcient graph-based algorithm. Computational experiments were performed using new protein rigidity analysis tools available in the new release of our KINARI-Web server http://kinari.cs.umass.edu. Results We provide two types of results: on biological assemblies and on crystals. We found that when only isolated subchains are considered, structural and functional information may be missed. Indeed, the rigidity of biological assemblies is sometimes dependent on the count and placement of hydrogen bonds and other interactions among the individual subchains of the biological unit. Similarly, the rigidity of small crystals may be affected by the interactions between atoms belonging to different unit cells. We have analyzed a dataset of approximately 300 proteins, from which we generated 982 crystals (some of which are biological assemblies). We identified two types of behaviors. (a) Some crystals and/or biological assemblies will aggregate into rigid bodies that span multiple unit cells/asymmetric units. Some of them create substantially larger rigid cluster in the crystal/biological assembly form, while in other cases, the aggregation has a smaller effect just at the interface between the units. (b) In other cases, the rigidity properties of the asymmetric units are retained, because the rigid bodies did not combine. We also identified two interesting cases where rigidity analysis may be correlated with the functional behavior of the protein. This type of information, identified here for the first time, depends critically on the ability to create crystals and biological assemblies

  18. Mesoscale crystallization of calcium phosphate nanostructures in protein (casein) micelles

    NASA Astrophysics Data System (ADS)

    Thachepan, Surachai; Li, Mei; Mann, Stephen

    2010-11-01

    Aqueous micelles of the multi-protein calcium phosphate complex, casein, were treated at 60 °C and pH 7 over several months. Although partial dissociation of the micelles into 12 nm sized amorphous calcium phosphate (ACP)/protein nanoparticles occurred within a period of 14 days, crystallization of the ACP nanoclusters into bundles of hydroxyapatite (HAP) nanofilaments was not observed until after 12 weeks. The HAP nanofilaments were formed specifically within the partially disrupted protein micelles suggesting a micelle-mediated pathway of mesoscale crystallization. Similar experiments using ACP-containing synthetic micelles prepared from β-casein protein alone indicated that co-aligned bundles of HAP nanofilaments were produced within the protein micelle interior after 24 hours at temperatures as low as 35 °C. The presence of Mg2+ ions in the casein micelles, as well as a possible synergistic effect associated with the multi-protein nature of the native aggregates, could account for the marked inhibition in mesoscale crystallization observed in the casein micelles compared with the single-component β-casein constructs.Aqueous micelles of the multi-protein calcium phosphate complex, casein, were treated at 60 °C and pH 7 over several months. Although partial dissociation of the micelles into 12 nm sized amorphous calcium phosphate (ACP)/protein nanoparticles occurred within a period of 14 days, crystallization of the ACP nanoclusters into bundles of hydroxyapatite (HAP) nanofilaments was not observed until after 12 weeks. The HAP nanofilaments were formed specifically within the partially disrupted protein micelles suggesting a micelle-mediated pathway of mesoscale crystallization. Similar experiments using ACP-containing synthetic micelles prepared from β-casein protein alone indicated that co-aligned bundles of HAP nanofilaments were produced within the protein micelle interior after 24 hours at temperatures as low as 35 °C. The presence of Mg2+ ions in

  19. Protein crystal screening and characterization for serial femtosecond nanocrystallography

    PubMed Central

    Darmanin, Connie; Strachan, Jamie; Adda, Christopher G.; Ve, Thomas; Kobe, Bostjan; Abbey, Brian

    2016-01-01

    The recent development of X-ray free electron lasers (XFELs) has spurred the development of serial femtosecond nanocrystallography (SFX) which, for the first time, is enabling structure retrieval from sub-micron protein crystals. Although there are already a growing number of structures published using SFX, the technology is still very new and presents a number of unique challenges as well as opportunities for structural biologists. One of the biggest barriers to the success of SFX experiments is the preparation and selection of suitable protein crystal samples. Here we outline a protocol for preparing and screening for suitable XFEL targets. PMID:27139248

  20. Antibody fragments for stabilization and crystallization of G protein-coupled receptors and their signaling complexes.

    PubMed

    Shukla, Arun K; Gupta, Charu; Srivastava, Ashish; Jaiman, Deepika

    2015-01-01

    G protein-coupled receptors (GPCRs) are one of the key players in extracellular signal recognition and their subsequent communications with cellular signaling machinery. Crystallization and high-resolution structure determination of GPCRs has been one of the major advances in the area of GPCR biology over the last 7-8 years. There have primarily been three approaches to GPCR crystallization till date. These are fusion protein strategy, thermostabilization, and antibody fragment-mediated crystallization. Of these, antibody fragment-mediated crystallization has not only provided the first breakthrough in structure determination of a non-rhodopsin GPCR but it has also assisted in obtaining structures of fully active conformations of GPCRs. Antibody fragment approach has also been crucial in obtaining structural information on GPCR signaling complexes. Here, we highlight the specific examples of GPCR crystal structures that have utilized antibody fragments for promoting crystallogenesis and structure solution. We also discuss emerging powerful technologies such as the nanobody technology and the synthetic phage display libraries in the context of GPCR crystallization and underline how these tools are likely to propel key GPCR structural studies in future.

  1. Development of compartment for studies on the growth of protein crystals in space.

    PubMed

    Yamazaki, T; Tsukamoto, K; Yoshizaki, I; Fukuyama, S; Miura, H; Shimaoka, T; Maki, T; Oshi, K; Kimura, Y

    2016-03-01

    To clarify the growth mechanism of a protein crystal, it is essential to measure its growth rate with respect to the supersaturation. We developed a compartment (growth cell) for measuring the growth rate (<0.1 nm s(-1)) of the face of a protein crystal at a controlled supersaturation by interferometry over a period of half a year in space. The growth cell mainly consists of quartz glass, in which the growth solution and a seed crystal are enclosed by capillaries, the screw sample holder, and a helical insert. To avoid the destruction of the cell and the evaporation of the water from the solution inside the cell, we selected the materials for these components with care. The equipment was successfully used to examine the growth of a lysozyme crystal at a controlled supersaturation in space, where convection is negligible because of the microgravity environment, thereby advancing our understanding of the mechanism of protein crystal growth from solution. The technique used to develop the growth cell is useful not only for space experiments but also for kinetic studies of materials with very slow growth and dissolution rates (<10(-3) nm s(-1)).

  2. Development of compartment for studies on the growth of protein crystals in space

    NASA Astrophysics Data System (ADS)

    Yamazaki, T.; Tsukamoto, K.; Yoshizaki, I.; Fukuyama, S.; Miura, H.; Shimaoka, T.; Maki, T.; Oshi, K.; Kimura, Y.

    2016-03-01

    To clarify the growth mechanism of a protein crystal, it is essential to measure its growth rate with respect to the supersaturation. We developed a compartment (growth cell) for measuring the growth rate (<0.1 nm s-1) of the face of a protein crystal at a controlled supersaturation by interferometry over a period of half a year in space. The growth cell mainly consists of quartz glass, in which the growth solution and a seed crystal are enclosed by capillaries, the screw sample holder, and a helical insert. To avoid the destruction of the cell and the evaporation of the water from the solution inside the cell, we selected the materials for these components with care. The equipment was successfully used to examine the growth of a lysozyme crystal at a controlled supersaturation in space, where convection is negligible because of the microgravity environment, thereby advancing our understanding of the mechanism of protein crystal growth from solution. The technique used to develop the growth cell is useful not only for space experiments but also for kinetic studies of materials with very slow growth and dissolution rates (<10-3 nm s-1).

  3. Development of compartment for studies on the growth of protein crystals in space.

    PubMed

    Yamazaki, T; Tsukamoto, K; Yoshizaki, I; Fukuyama, S; Miura, H; Shimaoka, T; Maki, T; Oshi, K; Kimura, Y

    2016-03-01

    To clarify the growth mechanism of a protein crystal, it is essential to measure its growth rate with respect to the supersaturation. We developed a compartment (growth cell) for measuring the growth rate (<0.1 nm s(-1)) of the face of a protein crystal at a controlled supersaturation by interferometry over a period of half a year in space. The growth cell mainly consists of quartz glass, in which the growth solution and a seed crystal are enclosed by capillaries, the screw sample holder, and a helical insert. To avoid the destruction of the cell and the evaporation of the water from the solution inside the cell, we selected the materials for these components with care. The equipment was successfully used to examine the growth of a lysozyme crystal at a controlled supersaturation in space, where convection is negligible because of the microgravity environment, thereby advancing our understanding of the mechanism of protein crystal growth from solution. The technique used to develop the growth cell is useful not only for space experiments but also for kinetic studies of materials with very slow growth and dissolution rates (<10(-3) nm s(-1)). PMID:27036758

  4. Understanding the fabric of protein crystals: computational classification of biological interfaces and crystal contacts.

    PubMed

    Capitani, Guido; Duarte, Jose M; Baskaran, Kumaran; Bliven, Spencer; Somody, Joseph C

    2016-02-15

    Modern structural biology still draws the vast majority of information from crystallography, a technique where the objects being investigated are embedded in a crystal lattice. Given the complexity and variety of those objects, it becomes fundamental to computationally assess which of the interfaces in the lattice are biologically relevant and which are simply crystal contacts. Since the mid-1990s, several approaches have been applied to obtain high-accuracy classification of crystal contacts and biological protein-protein interfaces. This review provides an overview of the concepts and main approaches to protein interface classification: thermodynamic estimation of interface stability, evolutionary approaches based on conservation of interface residues, and co-occurrence of the interface across different crystal forms. Among the three categories, evolutionary approaches offer the strongest promise for improvement, thanks to the incessant growth in sequence knowledge. Importantly, protein interface classification algorithms can also be used on multimeric structures obtained using other high-resolution techniques or for protein assembly design or validation purposes. A key issue linked to protein interface classification is the identification of the biological assembly of a crystal structure and the analysis of its symmetry. Here, we highlight the most important concepts and problems to be overcome in assembly prediction. Over the next few years, tools and concepts of interface classification will probably become more frequently used and integrated in several areas of structural biology and structural bioinformatics. Among the main challenges for the future are better addressing of weak interfaces and the application of interface classification concepts to prediction problems like protein-protein docking.

  5. Fusion proteins as alternate crystallization paths to difficult structure problems

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C.; Rueker, Florian; Ho, Joseph X.; Lim, Kap; Keeling, Kim; Gilliland, Gary; Ji, Xinhua

    1994-01-01

    The three-dimensional structure of a peptide fusion product with glutathione transferase from Schistosoma japonicum (SjGST) has been solved by crystallographic methods to 2.5 A resolution. Peptides or proteins can be fused to SjGST and expressed in a plasmid for rapid synthesis in Escherichia coli. Fusion proteins created by this commercial method can be purified rapidly by chromatography on immobilized glutathione. The potential utility of using SjGST fusion proteins as alternate paths to the crystallization and structure determination of proteins is demonstrated.

  6. Morphology and the Strength of Intermolecular Contact in Protein Crystals

    NASA Technical Reports Server (NTRS)

    Matsuura, Yoshiki; Chernov, Alexander A.

    2002-01-01

    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.

  7. Applications of the second virial coefficient: protein crystallization and solubility

    PubMed Central

    Wilson, William W.; DeLucas, Lawrence J.

    2014-01-01

    This article begins by highlighting some of the ground-based studies emanating from NASA’s Microgravity Protein Crystal Growth (PCG) program. This is followed by a more detailed discussion of the history of and the progress made in one of the NASA-funded PCG investigations involving the use of measured second virial coefficients (B values) as a diagnostic indicator of solution conditions conducive to protein crystallization. A second application of measured B values involves the determination of solution conditions that improve or maximize the solubility of aqueous and membrane proteins. These two important applications have led to several technological improvements that simplify the experimental expertise required, enable the measurement of membrane proteins and improve the diagnostic capability and measurement throughput. PMID:24817708

  8. Can proteins and crystals self-catalyze methyl rotations?

    SciTech Connect

    Smith, Jeremy C; Baudry, Jerome

    2005-10-01

    The {chi} (C{sub {alpha}}-C{sub {beta}}) torsional barrier in the dipeptide alanine (N-methyl-l-alanyl-N-methylamide) crystal was investigated using ab initio calculations at various levels of theory, molecular mechanics, and molecular dynamics. For one of the two molecules in the asymmetric unit the calculations suggest that rotation around the ? dihedral angle is catalyzed by the crystal environment, reducing by up to 2kT the torsional barrier in the crystal with respect to that in the gas phase. This catalytic effect is present at both low and room temperature and originates from a van der Waals destabilization of the minima in the methyl dihedral potential coming from the nonbonded environment of the side chain. Screening of a subset of the Protein Data Bank with a pharmacophore model reproducing the crystal environment around this side chain methyl identified a protein containing an alanine residue with an environment similar to that in the crystal. Calculations indicate that this ? torsional barrier is also reduced in the protein at low temperature but not at room temperature. This suggests that environment-catalyzed rotation of methyl groups can occur both in the solid phase and in native biological structures, though this effect might be temperature-dependent. The relevance of this catalytic effect is discussed in terms of its natural occurrence and its possible contribution to the low-frequency vibrational modes of molecules.

  9. Application of protein engineering to enhance crystallizability and improve crystal properties

    PubMed Central

    Derewenda, Zygmunt S.

    2010-01-01

    Until recently, protein crystallization has mostly been regarded as a stochastic event over which the investigator has little or no control. With the dramatic technological advances in synchrotron-radiation sources and detectors and the equally impressive progress in crystallographic software, including automated model building and validation, crystallization has increasingly become the rate-limiting step in X-ray diffraction studies of macromolecules. However, with the advent of recombinant methods it has also become possible to engineer target proteins and their complexes for higher propensity to form crystals with desirable X-ray diffraction qualities. As most proteins that are under investigation today are obtained by heterologous overexpression, these tech­niques hold the promise of becoming routine tools with the potential to transform classical crystallization screening into a more rational high-success-rate approach. This article presents an overview of protein-engineering methods designed to enhance crystallizability and discusses a number of examples of their successful application. PMID:20445236

  10. Growth of protein crystals suspended in a closed loop thermosyphon

    NASA Astrophysics Data System (ADS)

    Nyce, Thomas A.; Rosenberger, Franz

    1991-03-01

    The quality of protein crystals often suffers from their growth at a liquid or solid surface. A novel solution growth method was developed to alleviate this problem. A growing crystal is suspended in a specially configured upflow of supersaturated nutrient, which is provided by the effect of fluid buoyancy in a closed loop thermosyphon. The flow rate and supersaturation are controlled by the temperature distribution in the thermosyphon, while contact of the crystal with the wall during growth is practically eliminated. The method was applied to the growth of lysozyme single crystals, with surprising results. While the orthorhombic form of lysozyme grew readily to the suspension limit of this particular apparatus (1.5 mm), the tetragonal form grew only to a maximum size less than 0.1 mm. Seed crystals of tetragonal lysozyme introduced into stagnant batch controls did not experience the growth cessation that the suspended crystals did. A likely cause of this growth cessation is the fluid shear forces on the suspended crystals.

  11. Protein adsorption at calcium oxalate monohydrate crystal surfaces.

    NASA Astrophysics Data System (ADS)

    Wesson, J.; Sheng, X.; Rimer, J.; Jung, T.; Ward, M.

    2008-03-01

    Calcium oxalate monohydrate (COM) crystals are the dominant inorganic phase in most kidney stones, and kidney stones form as aggregates of COM crystals and organic material, principally proteins, but little is known about the molecular level events at COM surfaces that regulate COM aggregation. We have examined the influence of polyelectrolytes on the force of adhesion between chemically modified atomic force microscopy (AFM) tips and selected COM crystal faces in saturated solution. In general, we found that polyanions bind to COM surfaces and block adhesion of a carboxylate functionalized AFM tip, while polycations had no measureable effect on adhesion force under the same conditions. We did observe a unique absence of interaction between poly(glutamic acid) and the COM (100) face compared to other synthetic polyanions, and some native urinary protein structures also exhibited unique face selective interactions, suggesting that simple electrostatic models will not completely explain the data.

  12. The effect of microgravity on protein crystal growth

    NASA Technical Reports Server (NTRS)

    Mcpherson, Alexander; Greenwood, Aaron; Day, John

    1991-01-01

    Based on the results of microgravity crystallization experiments using the protein canavalin aboard four separate U.S. Space Shuttle missions, visual observations and diffraction data are presented that support the contention that protein crystals of improved quality can be obtained in a microgravity environment. With canavalin, no significant increase in resolution was noted, but an overall improvement in diffraction quality, as judged by statistical analyses of the data, was clear. This improvement in quality may be due primarily to the elimination of defects and dislocations rather than an overall enhancement of order. The mechanism for this improvement may be microgravity-stabilized depletion zones that develop around growing crystals that establish and maintain optimal growth conditions more rapidly following nucleation. Such zones would be destroyed by convective flow effects in earth's gravity.

  13. X-ray transparent Microfluidics for Protein Crystallization and Biomineralization

    NASA Astrophysics Data System (ADS)

    Opathalage, Achini

    Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions. In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 A. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3.

  14. Exploring Carbon Nanomaterial Diversity for Nucleation of Protein Crystals

    NASA Astrophysics Data System (ADS)

    Govada, Lata; Leese, Hannah S.; Saridakis, Emmanuel; Kassen, Sean; Chain, Benny; Khurshid, Sahir; Menzel, Robert; Hu, Sheng; Shaffer, Milo S. P.; Chayen, Naomi E.

    2016-02-01

    Controlling crystal nucleation is a crucial step in obtaining high quality protein crystals for structure determination by X-ray crystallography. Carbon nanomaterials (CNMs) including carbon nanotubes, graphene oxide, and carbon black provide a range of surface topographies, porosities and length scales; functionalisation with two different approaches, gas phase radical grafting and liquid phase reductive grafting, provide routes to a range of oligomer functionalised products. These grafted materials, combined with a range of controls, were used in a large-scale assessment of the effectiveness for protein crystal nucleation of 20 different carbon nanomaterials on five proteins. This study has allowed a direct comparison of the key characteristics of carbon-based nucleants: appropriate surface chemistry, porosity and/or roughness are required. The most effective solid system tested in this study, carbon black nanoparticles functionalised with poly(ethylene glycol) methyl ether of mean molecular weight 5000, provides a novel highly effective nucleant, that was able to induce crystal nucleation of four out of the five proteins tested at metastable conditions.

  15. Exploring Carbon Nanomaterial Diversity for Nucleation of Protein Crystals.

    PubMed

    Govada, Lata; Leese, Hannah S; Saridakis, Emmanuel; Kassen, Sean; Chain, Benny; Khurshid, Sahir; Menzel, Robert; Hu, Sheng; Shaffer, Milo S P; Chayen, Naomi E

    2016-02-04

    Controlling crystal nucleation is a crucial step in obtaining high quality protein crystals for structure determination by X-ray crystallography. Carbon nanomaterials (CNMs) including carbon nanotubes, graphene oxide, and carbon black provide a range of surface topographies, porosities and length scales; functionalisation with two different approaches, gas phase radical grafting and liquid phase reductive grafting, provide routes to a range of oligomer functionalised products. These grafted materials, combined with a range of controls, were used in a large-scale assessment of the effectiveness for protein crystal nucleation of 20 different carbon nanomaterials on five proteins. This study has allowed a direct comparison of the key characteristics of carbon-based nucleants: appropriate surface chemistry, porosity and/or roughness are required. The most effective solid system tested in this study, carbon black nanoparticles functionalised with poly(ethylene glycol) methyl ether of mean molecular weight 5000, provides a novel highly effective nucleant, that was able to induce crystal nucleation of four out of the five proteins tested at metastable conditions.

  16. Exploring Carbon Nanomaterial Diversity for Nucleation of Protein Crystals

    PubMed Central

    Govada, Lata; Leese, Hannah S.; Saridakis, Emmanuel; Kassen, Sean; Chain, Benny; Khurshid, Sahir; Menzel, Robert; Hu, Sheng; Shaffer, Milo S. P.; Chayen, Naomi E.

    2016-01-01

    Controlling crystal nucleation is a crucial step in obtaining high quality protein crystals for structure determination by X-ray crystallography. Carbon nanomaterials (CNMs) including carbon nanotubes, graphene oxide, and carbon black provide a range of surface topographies, porosities and length scales; functionalisation with two different approaches, gas phase radical grafting and liquid phase reductive grafting, provide routes to a range of oligomer functionalised products. These grafted materials, combined with a range of controls, were used in a large-scale assessment of the effectiveness for protein crystal nucleation of 20 different carbon nanomaterials on five proteins. This study has allowed a direct comparison of the key characteristics of carbon-based nucleants: appropriate surface chemistry, porosity and/or roughness are required. The most effective solid system tested in this study, carbon black nanoparticles functionalised with poly(ethylene glycol) methyl ether of mean molecular weight 5000, provides a novel highly effective nucleant, that was able to induce crystal nucleation of four out of the five proteins tested at metastable conditions. PMID:26843366

  17. The Growth of Protein Crystals Using McDUCK

    NASA Technical Reports Server (NTRS)

    Ewing, Felicia; Wilson, Lori; Nadarajah, Arunan; Pusey, Marc

    1998-01-01

    Most of the current microgravity crystal growth hardware is optimized to produce crystals within the limited time available on orbit. This often results in the actual nucleation and growth process being rushed or the system not coming to equilibrium within the limited time available. Longer duration hardware exists, but one cannot readily pick out crystals grown early versus those which nucleated and grew more slowly. We have devised a long duration apparatus, the Multi-chamber Dialysis Unit for Crystallization Kinetics, or McDUCK. This apparatus-is a series of protein chambers, stacked upon a precipitant reservoir chamber. All chambers are separated by a dialysis membrane, which serves to pass small molecules while retaining the protein. The volume of the Precipitant chamber is equal to the sum of the volumes of the protein chamber. In operation, the appropriate chambers are filled with precipitant solution or protein solution, and the McDUCK is placed standing upright, with the precipitant chamber on the bottom. The precipitant diffuses upwards over time, with the time to reach equilibration a function of the diffusivity of the precipitant and the overall length of the diffusion pathway. Typical equilibration times are approximately 2-4 months, and one can readily separate rapid from slow nucleation and growth crystals. An advantage on Earth is that the vertical precipitant concentration gradient dominates that of the solute, thus dampening out solute density gradient driven convective flows. However, large Earth-grown crystals have so far tended to be more two dimensional. Preliminary X-ray diffraction analysis of lysozyme crystals grown in McDUCK have indicated that the best, and largest, come from the middle chambers, suggesting that there is an optimal growth rate. Further, the improvements in diffraction resolution have been better signal to noise ratios in the low resolution data, not an increase in resolution overall. Due to the persistently large crystals

  18. Understanding the fabric of protein crystals: computational classification of biological interfaces and crystal contacts

    PubMed Central

    Capitani, Guido; Duarte, Jose M.; Baskaran, Kumaran; Bliven, Spencer; Somody, Joseph C.

    2016-01-01

    Modern structural biology still draws the vast majority of information from crystallography, a technique where the objects being investigated are embedded in a crystal lattice. Given the complexity and variety of those objects, it becomes fundamental to computationally assess which of the interfaces in the lattice are biologically relevant and which are simply crystal contacts. Since the mid-1990s, several approaches have been applied to obtain high-accuracy classification of crystal contacts and biological protein–protein interfaces. This review provides an overview of the concepts and main approaches to protein interface classification: thermodynamic estimation of interface stability, evolutionary approaches based on conservation of interface residues, and co-occurrence of the interface across different crystal forms. Among the three categories, evolutionary approaches offer the strongest promise for improvement, thanks to the incessant growth in sequence knowledge. Importantly, protein interface classification algorithms can also be used on multimeric structures obtained using other high-resolution techniques or for protein assembly design or validation purposes. A key issue linked to protein interface classification is the identification of the biological assembly of a crystal structure and the analysis of its symmetry. Here, we highlight the most important concepts and problems to be overcome in assembly prediction. Over the next few years, tools and concepts of interface classification will probably become more frequently used and integrated in several areas of structural biology and structural bioinformatics. Among the main challenges for the future are better addressing of weak interfaces and the application of interface classification concepts to prediction problems like protein–protein docking. Supplementary information: Supplementary data are available at Bioinformatics online. Contact: guido.capitani@psi.ch PMID:26508758

  19. Heterogeneous distribution of dye-labelled biomineralizaiton proteins in calcite crystals

    NASA Astrophysics Data System (ADS)

    Liu, Chuang; Xie, Liping; Zhang, Rongqing

    2015-12-01

    Biominerals are highly ordered crystals mediated by organic matters especially proteins in organisms. However, how specific proteins are distributed inside biominerals are not well understood. In the present study, we use fluorescein isothiocyanate (FITC) to label extracted proteins from the shells of bivalve Pinctada fucata. By confocal laser scanning microscopy (CLSM), we observe a heterogeneous distribution of dye-labelled proteins inside synthetic calcite at the microscale. Proteins from the prismatic calcite layers accumulate at the edge of crystals while proteins from the nacreous aragonite layers accumulate at the center of crystals. Raman and X-ray powder diffraction show that both the proteins cannot alter the crystal phase. Scanning electron microscope demonstrates both proteins are able to affect the crystal morphology. This study may provide a direct approach for the visualization of protein distributions in crystals by small-molecule dye-labelled proteins as the additives in the crystallization process and improve our understanding of intracrystalline proteins distribution in biogenic calcites.

  20. Protein purification and crystallization artifacts: The tale usually not told.

    PubMed

    Niedzialkowska, Ewa; Gasiorowska, Olga; Handing, Katarzyna B; Majorek, Karolina A; Porebski, Przemyslaw J; Shabalin, Ivan G; Zasadzinska, Ewelina; Cymborowski, Marcin; Minor, Wladek

    2016-03-01

    The misidentification of a protein sample, or contamination of a sample with the wrong protein, may be a potential reason for the non-reproducibility of experiments. This problem may occur in the process of heterologous overexpression and purification of recombinant proteins, as well as purification of proteins from natural sources. If the contaminated or misidentified sample is used for crystallization, in many cases the problem may not be detected until structures are determined. In the case of functional studies, the problem may not be detected for years. Here several procedures that can be successfully used for the identification of crystallized protein contaminants, including: (i) a lattice parameter search against known structures, (ii) sequence or fold identification from partially built models, and (iii) molecular replacement with common contaminants as search templates have been presented. A list of common contaminant structures to be used as alternative search models was provided. These methods were used to identify four cases of purification and crystallization artifacts. This report provides troubleshooting pointers for researchers facing difficulties in phasing or model building. PMID:26660914

  1. Generation of recombinant antibody fragments for membrane protein crystallization.

    PubMed

    Mir, Syed H; Escher, Claudia; Kao, Wei-Chun; Birth, Dominic; Wirth, Christophe; Hunte, Carola

    2015-01-01

    Membrane proteins are challenging targets for crystallization and structure determination by X-ray crystallography. Hurdles can be overcome by antibody-mediated crystallization. More than 25 unique structures of membrane protein:antibody complexes have already been determined. In the majority of cases, hybridoma-derived antibody fragments either in Fab or Fv fragment format were employed for these complexes. We will briefly introduce the background and current status of the strategy and describe in detail the current protocols of well-established methods for the immunization, the selection, and the characterization of antibodies, as well as the cloning, the production, and the purification of recombinant antibodies useful for structural analysis of membrane proteins.

  2. Four crystal forms of a Bence-Jones protein

    SciTech Connect

    Makino, Debora L.; Henschen-Edman, Agnes H.; McPherson, Alexander

    2005-01-01

    Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 y ago. The trigonal crystal form may shed some light on the formation of fibrils common to certain storage diseases. Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 years ago. Closely related tetragonal and orthorhombic forms belonging to space groups P4{sub 3}2{sub 1}2 and P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = b = 68.7, c = 182.1 and a = 67.7, b = 69.4, c = 87.3 Å, diffract to 1.5 and 1.9 Å, respectively. Two closely related trigonal forms, both belonging to space group P3{sub 1}21 with unit-cell parameters a = b = 154.3 Å but differing by a doubling of the c axis, one 46.9 Å and the other 94.0 Å, diffract to 2.9 and 2.6 Å resolution, respectively. The trigonal crystal of short c-axis length shows a positive indication of twinning. The trigonal crystal of longer c axis, which appeared only after eight months of incubation at room temperature, is likely to be composed of proteolytically degraded molecules and unlike the other crystal forms contains two entire Bence-Jones dimers in the asymmetric unit. This latter crystal form may shed some light on the formation of fibrils common to certain storage diseases.

  3. Protein-directed self-assembly of a fullerene crystal

    PubMed Central

    Kim, Kook-Han; Ko, Dong-Kyun; Kim, Yong-Tae; Kim, Nam Hyeong; Paul, Jaydeep; Zhang, Shao-Qing; Murray, Christopher B.; Acharya, Rudresh; DeGrado, William F.; Kim, Yong Ho; Grigoryan, Gevorg

    2016-01-01

    Learning to engineer self-assembly would enable the precise organization of molecules by design to create matter with tailored properties. Here we demonstrate that proteins can direct the self-assembly of buckminsterfullerene (C60) into ordered superstructures. A previously engineered tetrameric helical bundle binds C60 in solution, rendering it water soluble. Two tetramers associate with one C60, promoting further organization revealed in a 1.67-Å crystal structure. Fullerene groups occupy periodic lattice sites, sandwiched between two Tyr residues from adjacent tetramers. Strikingly, the assembly exhibits high charge conductance, whereas both the protein-alone crystal and amorphous C60 are electrically insulating. The affinity of C60 for its crystal-binding site is estimated to be in the nanomolar range, with lattices of known protein crystals geometrically compatible with incorporating the motif. Taken together, these findings suggest a new means of organizing fullerene molecules into a rich variety of lattices to generate new properties by design. PMID:27113637

  4. Protein-directed self-assembly of a fullerene crystal.

    PubMed

    Kim, Kook-Han; Ko, Dong-Kyun; Kim, Yong-Tae; Kim, Nam Hyeong; Paul, Jaydeep; Zhang, Shao-Qing; Murray, Christopher B; Acharya, Rudresh; DeGrado, William F; Kim, Yong Ho; Grigoryan, Gevorg

    2016-01-01

    Learning to engineer self-assembly would enable the precise organization of molecules by design to create matter with tailored properties. Here we demonstrate that proteins can direct the self-assembly of buckminsterfullerene (C60) into ordered superstructures. A previously engineered tetrameric helical bundle binds C60 in solution, rendering it water soluble. Two tetramers associate with one C60, promoting further organization revealed in a 1.67-Å crystal structure. Fullerene groups occupy periodic lattice sites, sandwiched between two Tyr residues from adjacent tetramers. Strikingly, the assembly exhibits high charge conductance, whereas both the protein-alone crystal and amorphous C60 are electrically insulating. The affinity of C60 for its crystal-binding site is estimated to be in the nanomolar range, with lattices of known protein crystals geometrically compatible with incorporating the motif. Taken together, these findings suggest a new means of organizing fullerene molecules into a rich variety of lattices to generate new properties by design. PMID:27113637

  5. Protein Dielectrophoresis: Advances, Challenges and Applications

    PubMed Central

    Nakano, Asuka; Ros, Alexandra

    2013-01-01

    Protein dielectrophoresis (DEP) has the potential to play an important role as a manipulation, fractionation, pre-concentration and separation method in bioanalysis and as manipulation tool for nanotechnological applications. The first demonstrations of protein DEP have been reported almost twenty years ago. Since then various experimental realizations to manipulate proteins by DEP as well as more targeted applications employing protein DEP have been demonstrated. This review summarizes the experimental studies in the field of protein DEP trapping and focusing as well as specific applications in separation, molecular patterning, on bioprobes and biosensors. While a comprehensive theoretical model describing protein DEP is still lacking we also attempt to provide an overview of the factors influencing protein DEP and relate to currently available theoretical models. We further point out the variations in experimental conditions used in the past to study the somewhat 20 proteins as well as the implications of protein molecular structure to the DEP response. PMID:23400789

  6. Protein crystal growth; Proceedings of the First International Conference, Stanford University, CA, August 14-16, 1985

    NASA Technical Reports Server (NTRS)

    Feigelson, R. S. (Editor)

    1986-01-01

    Papers are presented on mechanisms of nucleation and growth of protein crystals, the role of purification in the crystallization of proteins and nucleic acids, and the effect of chemical impurities in polyethylene glycol on macromolecular crystallization. Also considered are growth kinetics of tetragonal lysozyme crystals, thermodynamic and kinetic considerations for crystal growth of complex molecules from solution, protein single-crystal growth under microgravity, and growth of organic crystals in a microgravity environment. Papers are also presented on preliminary investigations of protein crystal growth using the Space Shuttle, convective diffusion in protein crystal growth, and the growth and characterization of membrane protein crystals.

  7. An overview of heavy-atom derivatization of protein crystals

    PubMed Central

    Pike, Ashley C. W.; Garman, Elspeth F.; Krojer, Tobias; von Delft, Frank; Carpenter, Elisabeth P.

    2016-01-01

    Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in low-resolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative. PMID:26960118

  8. Split green fluorescent protein as a modular binding partner for protein crystallization.

    PubMed

    Nguyen, Hau B; Hung, Li-Wei; Yeates, Todd O; Terwilliger, Thomas C; Waldo, Geoffrey S

    2013-12-01

    A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10-11) hairpin in complex with GFP(1-9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10-11) hairpin with a variety of GFP(1-9) mutants engineered for favorable crystallization.

  9. Split green fluorescent protein as a modular binding partner for protein crystallization

    PubMed Central

    Nguyen, Hau B.; Hung, Li-Wei; Yeates, Todd O.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2013-01-01

    A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-­strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization. PMID:24311592

  10. Screening and Crystallization Plates for Manual and High-throughput Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    Thorne, Robert E. (Inventor); Berejnov, Viatcheslav (Inventor); Kalinin, Yevgeniy (Inventor)

    2010-01-01

    In one embodiment, a crystallization and screening plate comprises a plurality of cells open at a top and a bottom, a frame that defines the cells in the plate, and at least two films. The first film seals a top of the plate and the second film seals a bottom of the plate. At least one of the films is patterned to strongly pin the contact lines of drops dispensed onto it, fixing their position and shape. The present invention also includes methods and other devices for manual and high-throughput protein crystal growth.

  11. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  12. Modification of protein crystal packing by systematic mutations of surface residues: implications on biotemplating and crystal porosity.

    PubMed

    Wine, Yariv; Cohen-Hadar, Noa; Lamed, Raphael; Freeman, Amihay; Frolow, Felix

    2009-10-15

    Bioinspired nano-scale biotemplating for the development of novel composite materials has recently culminated in several demonstrations of nano-structured hybrid materials. Protein crystals, routinely prepared for the elucidation of protein 3D structures by X-ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein "building blocks" in the crystal, a complementary 3D array of interconnected cavities--voids array, exhibiting highly ordered porosity is formed. The porous arrays of protein crystal may serve as a nano-structured, accurate biotemplate by a "filling" process. These cavities arrays are shaped by the mode of protein packing throughout the crystallization process. Here we propose and demonstrate feasibility of targeting site specific mutations to modify protein's surface to affect protein crystal packing, enabling the generation of a series of protein crystal "biotemplates" all originating from same parent protein. The selection of these modification sites was based on in silico analysis of protein-protein interface contact areas in the parent crystal. The model protein selected for this study was the N-terminal type II cohesin from the cellulosomal scaffold in ScaB subunit of Acetivibrio cellulolyticus and mutations were focused on lysine residues involved in protein packing as prime target. The impact of systematically mutating these lysine residues on protein packing and its resulting interconnected cavities array were found to be most significant when surface lysine residues were substituted to tryptophan residues. Our results demonstrate the feasibility of using pre-designed site directed mutations for the generation of a series of protein crystal biotemplates from a "parent" protein.

  13. Effects of impurities on membrane-protein crystallization in different systems

    SciTech Connect

    Kors, Christopher A.; Wallace, Ellen; Davies, Douglas R.; Li, Liang; Laible, Philip D.; Nollert, Peter

    2009-10-01

    The effects of commonly encountered impurities on various membrane-protein crystallization regimes are investigated and it is found that the lipidic cubic phase crystallization methodology is the most robust, tolerating protein contamination levels of up to 50%, with little effect on crystal quality. If generally applicable, this tolerance may be exploited (i) in initial crystallization trials to determine the ‘crystallizability’ of a given membrane-protein and (ii) to subject partially pure membrane-protein samples to crystallization trials. When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP

  14. Nanoliter-scale protein crystallization and screening with a microfluidic droplet robot.

    PubMed

    Zhu, Ying; Zhu, Li-Na; Guo, Rui; Cui, Heng-Jun; Ye, Sheng; Fang, Qun

    2014-05-23

    Large-scale screening of hundreds or even thousands of crystallization conditions while with low sample consumption is in urgent need, in current structural biology research. Here we describe a fully-automated droplet robot for nanoliter-scale crystallization screening that combines the advantages of both automated robotics technique for protein crystallization screening and the droplet-based microfluidic technique. A semi-contact dispensing method was developed to achieve flexible, programmable and reliable liquid-handling operations for nanoliter-scale protein crystallization experiments. We applied the droplet robot in large-scale screening of crystallization conditions of five soluble proteins and one membrane protein with 35-96 different crystallization conditions, study of volume effects on protein crystallization, and determination of phase diagrams of two proteins. The volume for each droplet reactor is only ca. 4-8 nL. The protein consumption significantly reduces 50-500 fold compared with current crystallization stations.

  15. Recent results and new hardware developments for protein crystal growth in microactivity

    NASA Technical Reports Server (NTRS)

    Delucas, L. J.; Long, M. M.; Moore, K. M.; Smith, C.; Carson, M.; Narayana, S. V. L.; Carter, D.; Clark, A. D., Jr.; Nanni, R. G.; Ding, J.

    1993-01-01

    Protein crystal growth experiments have been performed on 16 space shuttle missions since April, 1985. The initial experiments utilized vapor diffusion crystallization techniques similar to those used in laboratories for earth-based experiments. More recent experiments have utilized temperature induced crystallization as an alternative method for growing high quality protein crystals in microgravity. Results from both vapor diffusion and temperature induced crystallization experiments indicate that proteins grown in microgravity may be larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth.

  16. Crystallizing Conditions, Developmental Advance and Education. First Annual Report.

    ERIC Educational Resources Information Center

    Feldman, David

    This research report outlines progress made in the development of a conceptual framework (called "crystallization") which is intended to explain the conditions found to be critical to the child's potential for developmental change. The research completed and proposed on crystallization has centered around four main areas of activity: (1) a field…

  17. From protein structure to function via single crystal optical spectroscopy

    PubMed Central

    Ronda, Luca; Bruno, Stefano; Bettati, Stefano; Storici, Paola; Mozzarelli, Andrea

    2015-01-01

    The more than 100,000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic “artifacts,” including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density maps, thus limiting the relevance of structure determinations. Moreover, for most of these structures, no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in infereing protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman, and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5′-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms. PMID:25988179

  18. Crystal structure of Homo sapiens protein LOC79017

    SciTech Connect

    Bae, Euiyoung; Bingman, Craig A.; Aceti, David J.; Phillips, Jr., George N.

    2010-02-08

    LOC79017 (MW 21.0 kDa, residues 1-188) was annotated as a hypothetical protein encoded by Homo sapiens chromosome 7 open reading frame 24. It was selected as a target by the Center for Eukaryotic Structural Genomics (CESG) because it did not share more than 30% sequence identity with any protein for which the three-dimensional structure is known. The biological function of the protein has not been established yet. Parts of LOC79017 were identified as members of uncharacterized Pfam families (residues 1-95 as PB006073 and residues 104-180 as PB031696). BLAST searches revealed homologues of LOC79017 in many eukaryotes, but none of them have been functionally characterized. Here, we report the crystal structure of H. sapiens protein LOC79017 (UniGene code Hs.530024, UniProt code O75223, CESG target number go.35223).

  19. The first crystal structure of an archaeal helical repeat protein

    PubMed Central

    Yoneda, Kazunari; Sakuraba, Haruhiko; Tsuge, Hideaki; Katunuma, Nobuhiko; Kuramitsu, Seiki; Kawabata, Takeshi; Ohshima, Toshihisa

    2005-01-01

    The crystal structure of ST1625p, a protein encoded by a hypothetical open reading frame ST1625 in the genome of the hyperthermophilic archaeon Sulfolobus tokodaii, was determined at 2.2 Å resolution. The only sequence similarity exhibited by the amino-acid sequence of ST1625p was a 33% identity with the sequence of SSO0983p from S. solfataricus. The 19 kDa monomeric protein was observed to consist of a right-handed superhelix assembled from a tandem repeat of ten α-­helices. A structural homology search using the DALI and MATRAS algorithms indicates that this protein can be classified as a helical repeat protein. PMID:16511116

  20. Experiment and theory for heterogeneous nucleation of protein crystals in a porous medium

    NASA Astrophysics Data System (ADS)

    Chayen, Naomi E.; Saridakis, Emmanuel; Sear, Richard P.

    2006-01-01

    The determination of high-resolution structures of proteins requires crystals of suitable quality. Because of the new impetus given to structural biology by structural genomics/proteomics, the problem of crystallizing proteins is becoming increasingly acute. There is therefore an urgent requirement for the development of new efficient methods to aid crystal growth. Nucleation is the crucial step that determines the entire crystallization process. Hence, the holy grail is to design a "universal nucleant," a substrate that induces the nucleation of crystals of any protein. We report a theory for nucleation on disordered porous media and its experimental testing and validation using a mesoporous bioactive gel-glass. This material induced the crystallization of the largest number of proteins ever crystallized using a single nucleant. The combination of the model and the experimental results opens up the scope for the rational design of nucleants, leading to alternative means of controlling crystallization. protein crystallization | phase diagram | microbatch | vapor diffusion

  1. Detection of Membrane Protein Two-Dimensional Crystals in Living Cells

    PubMed Central

    Gualtieri, E.J.; Guo, F.; Kissick, D.J.; Jose, J.; Kuhn, R.J.; Jiang, W.; Simpson, G.J.

    2011-01-01

    It is notoriously difficult to grow membrane protein crystals and solve membrane protein structures. Improved detection and screening of membrane protein crystals are needed. We have shown here that second-order nonlinear optical imaging of chiral crystals based on second harmonic generation can provide sensitive and selective detection of two-dimensional protein crystalline arrays with sufficiently low background to enable crystal detection within the membranes of live cells. The method was validated using bacteriorhodopsin crystals generated in live Halobacterium halobium bacteria and confirmed by electron microscopy from the isolated crystals. Additional studies of alphavirus glycoproteins indicated the presence of localized crystalline domains associated with virus budding from mammalian cells. These results suggest that in vivo crystallization may provide a means for expediting membrane protein structure determination for proteins exhibiting propensities for two-dimensional crystal formation. PMID:21190673

  2. Conformational Exchange in a Membrane Transport Protein Is Altered in Protein Crystals

    SciTech Connect

    D Freed; P Horanyi; M Wiener; D Cafiso

    2011-12-31

    Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B{sub 12}. Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.

  3. Conformational Exchange in a Membrane Transport Protein Is Altered in Protein Crystals

    SciTech Connect

    Freed, Daniel M.; Horanyi, Peter S.; Wiener, Michael C.; Cafiso, David S.

    2010-09-27

    Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B{sub 12}. Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.

  4. Effects of impurities on membrane-protein crystallization in different systems

    PubMed Central

    Kors, Christopher A.; Wallace, Ellen; Davies, Douglas R.; Li, Liang; Laible, Philip D.; Nollert, Peter

    2009-01-01

    When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP-based crystallization screens. If generally applicable, this tolerance for impurities may avoid the need for samples of ultrahigh purity when undertaking initial crystallization screening trials to determine preliminary crystallization conditions that can be optimized for a given target protein. PMID:19770503

  5. Protein crystal growth results from shuttle flight 51-F

    NASA Technical Reports Server (NTRS)

    Bugg, C. E.

    1985-01-01

    The protein crystal growth (PCG) experiments run on 51-F were analyzed. It was found that: (1) sample stability is increased over that observed during the experiments on flight 51-D; (2) the dialysis experiments produced lysozyme crystals that were significantly larger than those obtained in our identical ground-based studies; (3) temperature fluctuations apparently caused problems during the crystallization experiments on 51-F; (4) it is indicated that teflon tape stabilizes droplets on the syringe tips; (5) samples survived during the reentry and landing in glass tips that were not stoppered with plungers; (6) from the ground-based studies, it was expected that equilibration should be complete within 2 to 4 days for all of these vapor-diffusion experiments, thus it appears that the vapor diffusion rates are somewhat slower under microgravity conditions; (7) drop tethering was highly successful, all four of the tethered drops were stable, even though they contained MPD solutions; (8) the PCG experiments on 51-F were done to assess the hardware and experimental procedures that are developed for future flights, when temperature control will be available. Lysozyme crystals obtained by microdialysis are considerably larger than those obtained on the ground, using the identical apparatus and procedures.

  6. Organic crystal-binding peptides: morphology control and one-pot formation of protein-displaying organic crystals

    NASA Astrophysics Data System (ADS)

    Niide, Teppei; Ozawa, Kyohei; Nakazawa, Hikaru; Oliveira, Daniel; Kasai, Hitoshi; Onodera, Mari; Asano, Ryutaro; Kumagai, Izumi; Umetsu, Mitsuo

    2015-11-01

    Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar

  7. Correlated Protein Motion Measurements of Dihydrofolate Reductase Crystals

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2014-03-01

    We report the first direct measurements of the long range structural vibrational modes in dihydrofolate reductase (DHFR). DHFR is a universal housekeeping enzyme that catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetra-hydrofolate, with the aid of coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). This crucial enzymatic role as the target for anti-cancer [methotrexate (MTX)], and other clinically useful drugs, has made DHFR a long-standing target of enzymological studies. The terahertz (THz) frequency range (5-100 cm-1), corresponds to global correlated protein motions. In our lab we have developed Crystal Anisotropy Terahertz Microscopy (CATM), which directly measures these large scale intra-molecular protein vibrations, by removing the relaxational background of the solvent and residue side chain librational motions. We demonstrate narrowband features in the anisotropic absorbance for mouse DHFR with the ligand binding of NADPH and MTX single crystals as well as Escherichia coli DHFR with the ligand binding of NADPH and MTX single crystals. This work is supported by NSF grant MRI2 grant DBI2959989.

  8. Protein secretion in Pichia pastoris and advances in protein production.

    PubMed

    Damasceno, Leonardo M; Huang, Chung-Jr; Batt, Carl A

    2012-01-01

    Yeast expression systems have been successfully used for over 20 years for the production of recombinant proteins. With the growing interest in recombinant protein expression for various uses, yeast expression systems, such as the popular Pichia pastoris, are becoming increasingly important. Although P. pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, there is still room for improvement of this expression system. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. Therefore, endoplasmic reticulum protein folding, correct glycosylation, vesicular transport to the plasma membrane, gene dosage, secretion signal sequences, and secretome studies are important considerations for improved recombinant protein production. PMID:22057543

  9. Photonic Crystal Hydrogel Enhanced Plasmonic Staining for Multiplexed Protein Analysis.

    PubMed

    Mu, Zhongde; Zhao, Xiangwei; Huang, Yin; Lu, Meng; Gu, Zhongze

    2015-12-01

    Plasmonic nanoparticles are commonly used as optical transducers in sensing applications. The optical signals resulting from the interaction of analytes and plamsonic nanoparticles are influenced by surrounding physical structures where the nanoparticles are located. This paper proposes inverse opal photonic crystal hydrogel as 3D structure to improve Raman signals from plasmonic staining. By hybridization of the plasmonic nanoparticles and photonic crystal, surface-enhanced Raman spectroscopy (SERS) analysis of multiplexed protein is realized. It benefits the Raman analysis by providing high-density "hot spots" in 3D and extra enhancement of local electromagnetic field at the band edge of PhC with periodic refractive index distribution. The strong interaction of light and the hybrid 3D nanostructure offers new insights into plasmonic nanoparticle applications and biosensor design. PMID:26436833

  10. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    SciTech Connect

    Timmins, P.A.; Pebay-Peyroula, E.

    1994-12-31

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H{sub 2}O/D{sub 2}O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

  11. Recent advances in protein profiling of tissues and tissue fluids.

    PubMed

    Yang, Shi; Huang, Chun-Ming

    2007-08-01

    Creating protein profiles of tissues and tissue fluids, which contain secreted proteins and peptides released from various cells, is critical for biomarker discovery as well as drug and vaccine target selection. It is extremely difficult to obtain pure samples from tissues or tissue fluids, however, and identification of complex protein mixtures is still a challenge for mass spectrometry analysis. Here, we summarize recent advances in techniques for extracting proteins from tissues for mass spectrometry profiling and imaging. We also introduce a novel technique using a capillary ultrafiltration (CUF) probe to enable in vivo collection of proteins from the tissue microenvironment. The CUF probe technique is compared with existing sampling techniques, including perfusion, saline wash, fine-needle aspiration and microdialysis. In this review, we also highlight quantitative mass spectrometric proteomic approaches with, and without, stable-isotope labels. Advances in quantitative proteomics will significantly improve protein profiling of tissue and tissue fluid samples collected by CUF probes.

  12. Crystallization of the large membrane protein complex photosystem I in a microfluidic channel.

    PubMed

    Abdallah, Bahige G; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

    2013-12-23

    Traditional macroscale protein crystallization is accomplished nontrivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time-consuming and resource-intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these "difficult to crystallize" proteins such as femtosecond nanocrystallography are being developed yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via second-order nonlinear imaging of chiral crystals (SONICC) was applied for the detection of nanometer- and micrometer-sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date.

  13. Technological advances in site-directed spin labeling of proteins.

    PubMed

    Hubbell, Wayne L; López, Carlos J; Altenbach, Christian; Yang, Zhongyu

    2013-10-01

    Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion.

  14. Crystal structure of the petal death protein from carnation flower.

    PubMed

    Teplyakov, Alexey; Liu, Sijiu; Lu, Zhibing; Howard, Andrew; Dunaway-Mariano, Debra; Herzberg, Osnat

    2005-12-20

    Expression of the PSR132 protein from Dianthus caryophyllus (carnation, clover pink) is induced in response to ethylene production associated with petal senescence, and thus the protein is named petal death protein (PDP). Recent work has established that despite the annotation of PDP in sequence databases as carboxyphosphoenolpyruvate mutase, the enzyme is actually a C-C bond cleaving lyase exhibiting a broad substrate profile. The crystal structure of PDP has been determined at 2.7 A resolution, revealing a dimer-of-dimers oligomeric association. Consistent with sequence homology, the overall alpha/beta barrel fold of PDP is the same as that of other isocitrate lyase/PEP mutase superfamily members, including a swapped eighth helix within a dimer. Moreover, Mg(2+) binds in the active site of PDP with a coordination pattern similar to that seen in other superfamily members. A compound, covalently bound to the catalytic residue, Cys144, was interpreted as a thiohemiacetal adduct resulting from the reaction of glutaraldehyde used to cross-link the crystals. The Cys144-carrying flexible loop that gates access to the active site is in the closed conformation. Models of bound substrates and comparison with the closed conformation of isocitrate lyase and 2-methylisocitrate lyase revealed the structural basis for the broad substrate profile of PDP.

  15. Crystallizing Membrane Proteins in Lipidic Mesophases. A Host Lipid Screen

    SciTech Connect

    Li, Dianfan; Lee, Jean; Caffrey, Martin

    2011-11-30

    The default lipid for the bulk of the crystallogenesis studies performed to date using the cubic mesophase method is monoolein. There is no good reason, however, why this 18-carbon, cis-monounsaturated monoacylglycerol should be the preferred lipid for all target membrane proteins. The latter come from an array of biomembrane types with varying properties that include hydrophobic thickness, intrinsic curvature, lateral pressure profile, lipid and protein makeup, and compositional asymmetry. Thus, it seems reasonable that screening for crystallizability based on the identity of the lipid creating the hosting mesophase would be worthwhile. For this, monoacylglycerols with differing acyl chain characteristics, such as length and olefinic bond position, must be available. A lipid synthesis and purification program is in place in the author's laboratory to serve this need. In the current study with the outer membrane sugar transporter, OprB, we demonstrate the utility of host lipid screening as a means for generating diffraction-quality crystals. Host lipid screening is likely to prove a generally useful strategy for mesophase-based crystallization of membrane proteins.

  16. Three-dimensional Raman spectroscopic imaging of protein crystals deposited on a nanodroplet.

    PubMed

    Nitahara, Satoshi; Maeki, Masatoshi; Yamaguchi, Hiroshi; Yamashita, Kenichi; Miyazaki, Masaya; Maeda, Hideaki

    2012-12-21

    Confocal Raman spectroscopic imaging has been used to find the location of protein crystals deposited in a nanodroplet. The depth of the protein crystal has been clearly identified by comparing the three-dimensional Raman spectroscopic images of the protein with those of water. Additionally, the low concentration region around a growing protein crystal in the nanodroplet was visualized using two-dimensional Raman spectroscopic imaging.

  17. Nucleation and convection effects in protein crystal growth

    NASA Technical Reports Server (NTRS)

    Rosenberger, Franz (Principal Investigator)

    1996-01-01

    The following activities are reported on: repartitioning of NaCl and protein impurities in lysozyme crystallization; dependence of lysozyme growth kinetics on step sources and impurities; facet morphology response to nonuniformities in nutrient and impurity supply; interactions in undersaturated and supersaturated lysozyme solutions; heterogeneity determination and purification of commercial hen egg white lysozyme; nonlinear response of layer growth dynamics in the mixed kinetics-bulk transport regime; development of a simultaneous multiangle light scattering technique; and x-ray topography of tetragonal lysozyme grown by the temperature-control technique.

  18. Determination of protein and solvent volumes in protein crystals from contrast variation data

    SciTech Connect

    Badger, J.

    1994-12-31

    By varying the relative values of protein and solvent scattering densities in a crystal, it is possible to obtain information on the shape and dimensions of protein molecular envelopes. Neutron diffraction methods are ideally suited to these contrast variation experiments because H/D exchange leads to large differential changes in the protein and solvent scattering densities and is structurally non-perturbing. Low resolution structure factors have been measured from cubic insulin crystals with differing H/D contents. Structure factors calculated from a simple binary density model, in which uniform scattering densities represent the protein and solvent volumes in the crystals, were compared with these data. The contrast variation differences in the sets of measured structure factors were found to be accurately fitted by this simple model. Trial applications to two problems in crystal structure determination illustrate how this fact may be exploited. (1) A translation function that employs contrast variation data gave a sharp minimum within 1-9{Angstrom} of the correctly positioned insulin molecule and is relatively insensitive to errors in the atomic model. (2) An ab initio phasing method for the contrast variation data, based on analyzing histograms of the density distributions in trial maps, was found to recover the correct molecular envelope.

  19. Protein products obtained by site-preferred partial crosslinking in protein crystals and "liberated" by redissolution.

    PubMed

    Buch, Michal; Wine, Yariv; Dror, Yael; Rosenheck, Sonia; Lebendiker, Mario; Giordano, Rita; Leal, Ricardo M F; Popov, Alexander N; Freeman, Amihay; Frolow, Felix

    2014-07-01

    The use of protein crystals as a source of nanoscale biotemplates has attracted growing interest in recent years owing to their inherent internal order. As these crystals are vulnerable to environmental changes, potential applications require their stabilization by chemical crosslinking. We have previously shown that such intermolecular chemical crosslinking reactions occurring within protein crystals are not random events, but start at preferred crosslinking sites imposed by the alignment of protein molecules and their packing within the crystalline lattice. Here we propose a new working hypothesis and demonstrate its feasibility in enabling us to extricate homogeneous populations of single protein molecules that display chemical point mutations or of dimers that show homogeneous chemical crosslinking, and that have the potential for isolation of higher structures. Characterization of the crosslinking mechanism and its end products opens the way to the potential retrieval of such specific modified/intermolecular crosslinked products simply by effecting partial crosslinking at identified preferred sites, followed by time-controlled arrest of the crosslinking reaction and dissolution of the crystals by medium exchange complemented by chromatographic purification.

  20. Membrane Protein Structure Determination Using Crystallography and Lipidic Mesophases - Recent Advances and Successes

    PubMed Central

    Caffrey, Martin; Li, Dianfan; Dukkipati, Abhiram

    2012-01-01

    activity of membrane proteins reconstituted into the bilayer of the cubic phase as a prelude to crystallogenesis. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been designed for a more rational approach to crystallogenesis such that the method can now be applied to an even wider variety of membrane protein types. In this Current Topics article, these assorted advances are outlined along with a summary of the membrane proteins that have yielded to the method. The prospects for and the challenges that must be overcome to further develop the method are described. PMID:22783824

  1. Silicon and germanium crystallization techniques for advanced device applications

    NASA Astrophysics Data System (ADS)

    Liu, Yaocheng

    Three-dimensional architectures are believed to be one of the possible approaches to reduce interconnect delay in integrated circuits. Metal-induced crystallization (MIC) can produce reasonably high-quality Si crystals with low-temperature processing, enabling the monolithic integration of multilevel devices and circuits. A two-step MIC process was developed to make single-crystal Si pillars on insulator by forming a single-grain NiSi2 template in the first step and crystallizing the amorphous Si by NiSi2-mediated solid-phase epitaxy (SPE) in the second step. A transmission electron microscopy study clearly showed the quality improvement over the traditional MIC process. Another crystallization technique developed is rapid melt growth (RMG) for the fabrication of Ge crystals and Ge-on-insulator (GeOI) substrates. Ge is an important semiconductor with high carrier mobility and excellent optoelectronic properties. GeOI substrates are particularly desired to achieve high device performances and to solve the process problems traditionally associated with bulk Ge wafers. High-quality Ge crystals and GeOI structures were grown on Si substrates using the novel rapid melt growth technique that integrates the key elements in Czochralski growth---seeding, melting, epitaxy and defect necking. Growth velocity and nucleation rate were calculated to determine the RMG process window. Self-aligned microcrucibles were created to hold the Ge liquid during the RMG annealing. Material characterization showed a very low defect density in the RMG GeOI structures. The Ge films are relaxed, with their orientations controlled by the Si substrates. P-channel MOSFETs and p-i-n photodetectors were fabricated with the GeOI substrates. The device properties are comparable to those obtained with bulk Ge wafers, indicating that the RMG GeOI substrates are well suited for device fabrication. A new theory, growth-induced barrier lowering (GIBL), is proposed to understand the defect generation in

  2. Challenges and Opportunities for New Protein Crystallization Strategies in Structure-Based Drug Design

    PubMed Central

    Grey, Jessica; Thompson, David

    2010-01-01

    Structure-based drug design (SBDD) has emerged as a valuable pharmaceutical lead discovery tool, showing potential for accelerating the discovery process, while reducing developmental costs and boosting potencies of the drug that is ultimately selected. SBDD is a iterative, rational, lead compound sculpting process that involves both the synthesis of new derivatives and the evaluation of their binding to the target structure either through computational docking or elucidation of the target structure as a complex with the lead compound. This method heavily relies on the production of high-resolution (< 2Å) three-dimensional structures of the drug target, obtained through X-ray crystallographic analysis, in the presence or absence of the drug candidate. The lack of generalized methods for high quality crystal production is still a major bottleneck in the process of macromolecular crystallization. This review provides a brief introduction to SBDD and describes several macromolecular crystallization strategies, with an emphasis on advances and challenges facing researchers in the field today. Recent trends in the development of more universal macromolecular crystallization techniques, particularly nucleation-based techniques that are applicable to both soluble and integral membrane proteins, are also discussed. PMID:21116481

  3. Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel

    PubMed Central

    Abdallah, Bahige G.; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

    2014-01-01

    Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these ‘difficult to crystallize’ proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. PMID:24191698

  4. Superheating of ice crystals in antifreeze protein solutions.

    PubMed

    Celik, Yeliz; Graham, Laurie A; Mok, Yee-Foong; Bar, Maya; Davies, Peter L; Braslavsky, Ido

    2010-03-23

    It has been argued that for antifreeze proteins (AFPs) to stop ice crystal growth, they must irreversibly bind to the ice surface. Surface-adsorbed AFPs should also prevent ice from melting, but to date this has been demonstrated only in a qualitative manner. Here we present the first quantitative measurements of superheating of ice in AFP solutions. Superheated ice crystals were stable for hours above their equilibrium melting point, and the maximum superheating obtained was 0.44 degrees C. When melting commenced in this superheated regime, rapid melting of the crystals from a point on the surface was observed. This increase in melting temperature was more appreciable for hyperactive AFPs compared to the AFPs with moderate antifreeze activity. For each of the AFP solutions that exhibited superheating, the enhancement of the melting temperature was far smaller than the depression of the freezing temperature. The present findings clearly show that AFPs adsorb to ice surfaces as part of their mechanism of action, and this absorption leads to protection of ice against melting as well as freezing. PMID:20215465

  5. Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins

    SciTech Connect

    Leung, Daisy W.; Borek, Dominika; Farahbakhsh, Mina; Ramanan, Parameshwaran; Nix, Jay C.; Wang, Tianjiao; Prins, Kathleen C.; Otwinowski, Zbyszek; Honzatko, Richard B.; Helgeson, Luke A.; Basler, Christopher F.; Amarasinghe, Gaya K.

    2010-06-21

    VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6{sub 1}22, P2{sub 1}2{sub 1}2{sub 1} and P2{sub 1}, respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source.

  6. A soft, mean-field potential derived from crystal contacts for predicting protein-protein interactions.

    PubMed

    Robert, C H; Janin, J

    1998-11-13

    We derive a series of novel mean-field potentials from statistical analyses of protein-protein contact regions in crystal structures. These potentials are parameterized in terms of the number of contacts made by an atom in an interface region. Such an explicit number dependence avoids the pairwise assumption and is intrinsically softer than distance-based approaches. It appears well suited to protein-protein docking applications, for which detailed interface geometry is generally lacking. In tests including protein complex reconstitution and docking of independently determined protein structures, we show that a hydrophobic potential of this type performs remarkably well, identifying native-like complexes by their favourable potential energies and in several cases demonstrating a recognition energy gap of 4-8 kcal/mol according to the system.

  7. Analysis of zinc binding sites in protein crystal structures.

    PubMed Central

    Alberts, I. L.; Nadassy, K.; Wodak, S. J.

    1998-01-01

    The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations. PMID:10082367

  8. Brain protein deciphered at Advanced Light Source

    SciTech Connect

    2010-01-01

    This computer-generated model of a rat glutamate receptor is the first complete portrait of this important link in the nervous system. At the top of the Y-shaped protein, a pair of molecules splay outward like diverging prongs. The bottom section, which is embedded in a neuronal membrane, houses the ion channel. The resolution of this image is 3.6 angstroms per pixel, or just under four ten-billionths of a meter per image unit. http://newscenter.lbl.gov/feature-stories/2010/01/21/glutamate-receptor/

  9. Re-structuring protein crystals porosity for biotemplating by chemical modification of lysine residues.

    PubMed

    Cohen-Hadar, Noa; Lagziel-Simis, Shira; Wine, Yariv; Frolow, Felix; Freeman, Amihay

    2011-01-01

    Protein crystals are routinely prepared for the elucidation of protein structure by X-ray crystallography. These crystals present an highly accurate periodical array of protein molecules with accompanying highly ordered porosity made of interconnected voids. The permeability of the porous protein crystals to a wide range of solutes has recently triggered attempts to explore their potential application as biotemplates by a controlled "filling" process for the fabrication of novel, nano-structured composite materials. Gaining control of the porosity of a given protein crystal may lead to the preparation of a series of "biotemplates" enabling different 'filler'/protein content ratios, resulting in different nanostructured composites. One way to gain such control is to produce a series of polymorphic forms of a given "parent-protein" crystal. As protein packing throughout crystallization is primarily dominated by the chemical composition of the surface of protein molecules and its impact on protein-protein interactions, modification of residues exposed on the surface will affect protein packing, leading to modified porosity. Here we propose to provide influence on the porosity of protein crystals for biotemplating by pre-crystallization chemical modification of lysine residues exposed on protein's surface. The feasibility of this approach was demonstrated by the serial application of chemical "modifiers" leading to protein derivatives exhibiting altered porosity by affecting protein "packing" throughout protein crystallization. Screening of a series of modifying agents for lysine modification of hen egg white lysozyme revealed that pre-crystallization modification preserving their positive charge did not affect crystal porosity, while modification resulting in their conversion to negatively charged groups induced dramatic change in protein crystal's packing and porosity. Furthermore, we demonstrate that chemical modification of lysine residues affecting modified

  10. Protein-protein interactions as druggable targets: recent technological advances.

    PubMed

    Higueruelo, Alicia P; Jubb, Harry; Blundell, Tom L

    2013-10-01

    Classical target-based drug discovery, where large chemical libraries are screened using inhibitory assays for a single target, has struggled to find ligands that inhibit protein-protein interactions (PPI). Nevertheless, in the past decade there have been successes that have demonstrated that PPI can be useful drug targets, and the field is now evolving fast. This review focuses on the new approaches and concepts that are being developed to tackle these challenging targets: the use of fragment based methods to explore the chemical space, stapled peptides to regulate intracellular PPI, alternatives to competitive inhibition and the use of antibodies to enable small molecule discovery for these targets.

  11. A Proposed Model for Protein Crystal Nucleation and Growth

    NASA Technical Reports Server (NTRS)

    Pusey, Marc; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    How does one take a molecule, strongly asymmetric in both shape and charge distribution, and assemble it into a crystal? We propose a model for the nucleation and crystal growth process for tetragonal lysozyme, based upon fluorescence, light, neutron, and X-ray scattering data, size exclusion chromatography experiments, dialysis kinetics, AFM, and modeling of growth rate data, from this and other laboratories. The first species formed is postulated to be a 'head to side' dimer. Through repeating associations involving the same intermolecular interactions this grows to a 4(sub 3) helix structure, that in turn serves as the basic unit for nucleation and subsequent crystal growth. High salt attenuates surface charges while promoting hydrophobic interactions. Symmetry facilitates subsequent helix-helix self-association. Assembly stability is enhanced when a four helix structure is obtained, with each bound to two neighbors. Only two unique interactions are required. The first are those for helix formation, where the dominant interaction is the intermolecular bridging anion. The second is the anti-parallel side-by-side helix-helix interaction, guided by alternating pairs of symmetry related salt bridges along each side. At this stage all eight unique positions of the P4(sub3)2(sub 1),2(sub 1) unit cell are filled. The process is one of a) attenuating the most strongly interacting groups, such that b) the molecules begin to self-associate in defined patterns, so that c) symmetry is obtained, which d) propagates as a growing crystal. Simple and conceptually obvious in hindsight, this tells much about what we are empirically doing when we crystallize macromolecules. By adjusting the growth parameters we are empirically balancing the intermolecular interactions, preferentially attenuating the dominant strong (for lysozyme the charged groups) while strengthening the lesser strong (hydrophobic) interactions. In the general case for proteins the lack of a singularly defined

  12. A simple apparatus for controlling nucleation and size in protein crystal growth

    NASA Technical Reports Server (NTRS)

    Gernert, Kim M.; Smith, Robert; Carter, Daniel C.

    1988-01-01

    A simple device is described for controlling vapor equilibrium in macromolecular crystallization as applied to the protein crystal growth technique commonly referred to as the 'hanging drop' method. Crystal growth experiments with hen egg white lysozyme have demonstrated control of the nucleation rate. Nucleation rate and final crystal size have been found to be highly dependent upon the rate at which critical supersaturation is approached. Slower approaches show a marked decrease in the nucleation rate and an increase in crystal size.

  13. Crystal Structure of the Japanese Encephalitis Virus Envelope Protein

    SciTech Connect

    Luca, Vincent C.; AbiMansour, Jad; Nelson, Christopher A.; Fremont, Daved H.

    2012-03-13

    Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-{angstrom} resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.

  14. Stabilization of protein crystals by electrostatic interactions as revealed by a numerical approach.

    PubMed

    Takahashi, T; Endo, S; Nagayama, K

    1993-11-20

    We developed a novel algorithm to solve numerically the Poisson-Boltzmann equations under a periodic boundary condition. By employing this algorithm to calculate the electrostatic potentials in two different types of protein crystals, a bovine pancreatic trypsin inhibitor (BPTI) orthorhombic crystal and a pig-insulin cubic crystal, the energy contributions of the electrostatic interactions to the crystals' stability were evaluated. At a high ionic strength, the condensed state of proteins in the crystal was stabilized electrostatically compared with that isolated in dilute solution because the attractive electrostatic interactions between neighboring protein molecules overcame the repulsive forces that originated from the same net charges of the equivalent protein molecules. On the other hand, at a low ionic strength the electrostatic interactions destabilized the crystalline state of both proteins, although a different dependence on the ionic strength was found between them. Here, the insulin crystal was more stable than the BPTI one because of the higher charge density in the BPTI crystal. In all of the solvent ionic strengths investigated, the attractive electrostatic interactions between charge pairs separated by less than 5 A on the respective protein molecules prominently stabilize the protein crystals. Therefore, two protein molecules in the crystals are oriented to compensate each other for their opposite charges on the surfaces. We also found a specific role for bound phosphate ions in the stabilization of the BPTI crystal, based on comparison of the electrostatic energies of the two crystals with and without the ions. By determining the contribution of each atomic charge in the crystals to the electrostatic energy, it was revealed that several electrostatic pairs specifically contributed to the crystal's stability. On the basis of our numerical calculation results, we propose a new method to design protein molecules that adopt stable crystals by replacing

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

    NASA Technical Reports Server (NTRS)

    Achari, Aniruddha; Roeber, Dana F.; Barnes, Cindy L.; Kundrot, Craig E.; Stinson, Thomas N. (Technical Monitor)

    2002-01-01

    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.

  16. Organic crystal-binding peptides: morphology control and one-pot formation of protein-displaying organic crystals.

    PubMed

    Niide, Teppei; Ozawa, Kyohei; Nakazawa, Hikaru; Oliveira, Daniel; Kasai, Hitoshi; Onodera, Mari; Asano, Ryutaro; Kumagai, Izumi; Umetsu, Mitsuo

    2015-12-21

    Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.

  17. Advances in EXAFS Studies of Thermal Properties of Crystals

    SciTech Connect

    Fornasini, Paolo

    2007-02-02

    Thanks to the peculiar sensitivity to correlation of vibrational motion, EXAFS contains original information on thermal properties of crystals. The thermal expansions measured by EXAFS and by Bragg scattering have a different physical meaning. They can be experimentally distinguished by accurate temperature dependent measurements, leading to the evaluation of the perpendicular mean square relative displacement (MSRD). These results, besides giving a deeper insight on the connection between EXAFS and lattice dynamics, open new perspectives for studying the local origin of negative thermal expansion.

  18. Crysalis: an integrated server for computational analysis and design of protein crystallization

    PubMed Central

    Wang, Huilin; Feng, Liubin; Zhang, Ziding; Webb, Geoffrey I.; Lin, Donghai; Song, Jiangning

    2016-01-01

    The failure of multi-step experimental procedures to yield diffraction-quality crystals is a major bottleneck in protein structure determination. Accordingly, several bioinformatics methods have been successfully developed and employed to select crystallizable proteins. Unfortunately, the majority of existing in silico methods only allow the prediction of crystallization propensity, seldom enabling computational design of protein mutants that can be targeted for enhancing protein crystallizability. Here, we present Crysalis, an integrated crystallization analysis tool that builds on support-vector regression (SVR) models to facilitate computational protein crystallization prediction, analysis, and design. More specifically, the functionality of this new tool includes: (1) rapid selection of target crystallizable proteins at the proteome level, (2) identification of site non-optimality for protein crystallization and systematic analysis of all potential single-point mutations that might enhance protein crystallization propensity, and (3) annotation of target protein based on predicted structural properties. We applied the design mode of Crysalis to identify site non-optimality for protein crystallization on a proteome-scale, focusing on proteins currently classified as non-crystallizable. Our results revealed that site non-optimality is based on biases related to residues, predicted structures, physicochemical properties, and sequence loci, which provides in-depth understanding of the features influencing protein crystallization. Crysalis is freely available at http://nmrcen.xmu.edu.cn/crysalis/. PMID:26906024

  19. Two-dimensional crystals of carboxysome shell proteins recapitulate the hexagonal packing of three-dimensional crystals

    PubMed Central

    Dryden, Kelly A; Crowley, Christopher S; Tanaka, Shiho; Yeates, Todd O; Yeager, Mark

    2009-01-01

    Bacterial microcompartments (BMCs) are large intracellular bodies that serve as simple organelles in many bacteria. They are proteinaceous structures composed of key enzymes encapsulated by a polyhedral protein shell. In previous studies, the organization of these large shells has been inferred from the conserved packing of the component shell proteins in two-dimensional (2D) layers within the context of three-dimensional (3D) crystals. Here, we show that well-ordered, 2D crystals of carboxysome shell proteins assemble spontaneously when His-tagged proteins bind to a monolayer of nickelated lipid molecules at an air–water interface. The molecular packing within the 2D crystals recapitulates the layered hexagonal sheets observed in 3D crystals. The results reinforce current models for the molecular design of BMC shells. PMID:19844993

  20. Microphase Separation Controlled beta-Sheet Crystallization Kinetics in Fibrous Proteins

    SciTech Connect

    Hu, X.; Lu, Q; Kaplan, D; Cebe, P

    2009-01-01

    Silk is a naturally occurring fibrous protein with a multiblock chain architecture. As such, it has many similarities with synthetic block copolymers, including the possibility for e-sheet crystallization restricted within the crystallizable blocks. The mechanism of isothermal crystallization kinetics of e-sheet crystals in silk multiblock fibrous proteins is reported in this study. Kinetics theories, such as Avrami analysis which was established for studies of synthetic polymer crystal growth, are for the first time extended to investigate protein self-assembly in e-sheet rich Bombyx mori silk fibroin samples, using time-resolved Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and synchrotron real-time wide-angle X-ray scattering (WAXS). The Avrami exponent, n, was close to 2 for all methods and crystallization temperatures, indicating formation of e-sheet crystals in silk proteins is different from the 3-D spherulitic crystal growth found in synthetic polymers. Observations by scanning electron microscopy support the view that the protein structures vary during the different stages of crystal growth, and show a microphase separation pattern after chymotrypsin enzyme biodegradation. We present a model to explain the crystallization of the multiblock silk fibroin protein, by analogy to block copolymers: crystallization of e-sheets occurs under conditions of geometrical restriction caused by phase separation of the crystallizable and uncrystallizable blocks. This crystallization model could be widely applicable in other proteins with multiblock (i.e., crystallizable and noncrystallizable) domains.

  1. Toward rational protein crystallization: A Web server for the design of crystallizable protein variants

    PubMed Central

    Goldschmidt, Lukasz; Cooper, David R.; Derewenda, Zygmunt S.; Eisenberg, David

    2007-01-01

    Growing well-diffracting crystals constitutes a serious bottleneck in structural biology. A recently proposed crystallization methodology for “stubborn crystallizers” is to engineer surface sequence variants designed to form intermolecular contacts that could support a crystal lattice. This approach relies on the concept of surface entropy reduction (SER), i.e., the replacement of clusters of flexible, solvent-exposed residues with residues with lower conformational entropy. This strategy minimizes the loss of conformational entropy upon crystallization and renders crystallization thermodynamically favorable. The method has been successfully used to crystallize more than 15 novel proteins, all stubborn crystallizers. But the choice of suitable sites for mutagenesis is not trivial. Herein, we announce a Web server, the surface entropy reduction prediction server (SERp server), designed to identify mutations that may facilitate crystallization. Suggested mutations are predicted based on an algorithm incorporating a conformational entropy profile, a secondary structure prediction, and sequence conservation. Minor considerations include the nature of flanking residues and gaps between mutation candidates. While designed to be used with default values, the server has many user-controlled parameters allowing for considerable flexibility. Within, we discuss (1) the methodology of the server, (2) how to interpret the results, and (3) factors that must be considered when selecting mutations. We also attempt to benchmark the server by comparing the server's predictions with successful SER structures. In most cases, the structure yielding mutations were easily identified by the SERp server. The server can be accessed at http://www.doe-mbi.ucla.edu/Services/SER. PMID:17656576

  2. The Feasibility of Bulk Crystallization as an Industrial Purification and Production Technique for Proteins

    NASA Technical Reports Server (NTRS)

    Judge, Russell A.; Forsythe, Elizabeth L.; Johns, Michael R.; Pusey, Marc L.; White, Edward T.

    1998-01-01

    Bulk crystallization in stirred vessels is used industrially for the recovery and purification of many inorganic and organic materials. Although much has been written on the crystallization of proteins for X-ray diffraction analysis, very little has been reported on the application of bulk crystallization in stirred vessels. In this study, a 1-liter, seeded, stirred, batch crystallizer was used with ovalbumin as a model protein to test the feasibility of this crystallization method as a recovery and purification process for proteins. Results were obtained for ovalbumin solubility, nucleation thresholds, crystal breakage and crystal growth kinetics in bulk solution under a range of operating conditions of pH and ammonium sulphate concentration (Judge et al., 1996). Experiments were also performed to determine the degree of purification that can be achieved by the crystallization of ovalbumin from a mixture of proteins. The effect of the presence of these proteins upon the ovalbumin crystal growth kinetics was also investigated (Judge et al., 1995). All of these aspects are essential for the design of bulk crystallization processes which have not previously been reported for proteins. Results from a second study that investigated the effect of structurally different proteins on the solubility, crystal growth rates and crystal purity of chicken egg white lysozyme are also presented (Judge et al., 1997). In this case face growth rates were measured using lysozyme purified by liquid chromatography and the effect of the addition of specific protein impurities were observed on the (110) and (101) crystal faces. In these two studies the results are presented to show the feasibility and purifying ability of crystallization as a production process for proteins.

  3. Growth of Diffraction-Quality Protein Crystals Using a Harvestable Microfluidic Device

    PubMed Central

    2015-01-01

    Protein crystallization is the major bottleneck in the entire process of protein crystallography, and obtaining diffraction-quality crystals can be unpredictable and sometimes exceptionally difficult, requiring many rounds of high-throughput screening. Recently, a more time- and cost-saving strategy to use the commercially available microfluidic devices called Crystal Formers has emerged. Herein we show the application of such a device using a protein from Legionella pneumophila called LidL that is predicted to be involved in the ability to efficiently manipulate host cell trafficking events once internalized by the host cell. After setting up just one 96-channel Crystal Former tray, we were able to obtain a diffraction-quality crystal that diffracted to 2.76 Å. These results show that Crystal Formers can be used to screen and optimize crystals to directly produce crystals for structure determination. PMID:25013386

  4. Byonic: Advanced Peptide and Protein Identification Software

    PubMed Central

    Bern, Marshall; Kil, Yong J.; Becker, Christopher

    2013-01-01

    Byonic™ is the name of a software package for peptide and protein identification by tandem mass spectrometry. This software, which has only recently become commercially available, facilitates a much wider range of search possibilities than previous search software such as SEQUEST and Mascot. Byonic allows the user to define an essentially unlimited number of variable modification types. Byonic also allows the user to set a separate limit on the number of occurrences of each modification type, so that a search may consider only one or two chance modifications such as oxidations and deamidations per peptide, yet allow three or four biological modifications such as phosphorylations, which tend to cluster together. Hence Byonic can search for 10s or even 100s of modification types simultaneously without a prohibitively large combinatorial explosion. Byonic’s Wildcard Search™ allows the user to search for unanticipated or even unknown modifications alongside known modifications. Finally, Byonic’s Glycopeptide Search allows the user to identify glycopeptides without prior knowledge of glycan masses or glycosylation sites. PMID:23255153

  5. High-throughput method for optimum solubility screening for homogeneity and crystallization of proteins

    DOEpatents

    Kim, Sung-Hou; Kim, Rosalind; Jancarik, Jamila

    2012-01-31

    An optimum solubility screen in which a panel of buffers and many additives are provided in order to obtain the most homogeneous and monodisperse protein condition for protein crystallization. The present methods are useful for proteins that aggregate and cannot be concentrated prior to setting up crystallization screens. A high-throughput method using the hanging-drop method and vapor diffusion equilibrium and a panel of twenty-four buffers is further provided. Using the present methods, 14 poorly behaving proteins have been screened, resulting in 11 of the proteins having highly improved dynamic light scattering results allowing concentration of the proteins, and 9 were crystallized.

  6. Advances in flow visualization using liquid-crystal coatings

    NASA Technical Reports Server (NTRS)

    Holmes, Bruce J.; Obara, Clifford J.

    1987-01-01

    This paper discusses a new four-part mixing method for visualizing boundary layer flows, including transitions, separation, and shock locations, by the use of liquid-crystal coatings. The method controls the event temperature and color-play bandwidth best suited to specific experimental conditions, and is easily learned. The method is applicable almost throughout the altitude and speed ranges for subsonic aircraft flight envelopes, and is also applicable to supersonic flow visualization and for general use in high- and low-speed wind tunnel and water tunnel testing.

  7. Crystal structure of a mutant of archaeal ribosomal protein L1 from Methanococcus jannaschii

    NASA Astrophysics Data System (ADS)

    Sarskikh, A. V.; Gabdulkhakov, A. G.; Kostareva, O. S.; Shklyaeva, A. A.; Tishchenko, S. V.

    2014-05-01

    The crystal structure of a mutant of archaeal ribosomal protein L1 from Methanococcus jannaschii with the deletion of a nonconserved positively charged cluster consisting of eight C-terminal amino acid residues is determined by the molecular replacement method at 1.75 Å resolution. This mutant is shown to form more stable and ordered crystals belonging to a space group other than that of the wild-type protein crystals. The positively charged C-terminal region has only a slight effect on the interaction between protein L1 and RNA molecules. Hence, this mutant can be used to prepare protein-RNA complexes and obtain their crystals.

  8. Use of layer silicate for protein crystallization: effects of Micromica and chlorite powders in hanging drops.

    PubMed

    Takehara, Masahide; Ino, Keita; Takakusagi, Yoichi; Oshikane, Hiroyuki; Nureki, Osamu; Ebina, Takeo; Mizukami, Fujio; Sakaguchi, Kengo

    2008-02-15

    Two kinds of layer silicate powder, Micromica and chlorite, were used to aid protein crystallization by the addition to hanging drops. Using appropriate crystallization buffers, Micromica powder facilitated crystal growth speed for most proteins tested in this study. Furthermore, the addition of Micromica powder to hanging drops allowed the successful crystallization of lysozyme, catalase, concanavalin A, and trypsin even at low protein concentrations and under buffer conditions that otherwise would not generate protein crystals. Except for threonine synthase and apoferritin, the presence of chlorite delayed crystallization but induced the formation of large crystals. X-ray analysis of thaumatin crystals generated by our novel procedure gave better quality data than did that of crystals obtained by a conventional hanging drop method. Our results suggest that the speed of crystal growth and the quality of the corresponding X-ray data may be inversely related, at least for the formation of thaumatin crystals. The effect of Micromica and chlorite powders and the application of layer silicate powder for protein crystallization are discussed.

  9. Imatinib (Gleevec@) conformations observed in single crystals, protein-Imatinib co-crystals and molecular dynamics: Implications for drug selectivity

    NASA Astrophysics Data System (ADS)

    Golzarroshan, B.; Siddegowda, M. S.; Li, Hong qi; Yathirajan, H. S.; Narayana, B.; Rathore, R. S.

    2012-06-01

    Structure and dynamics of the Leukemia drug, Imatinib, were examined using X-ray crystallography and molecular dynamics studies. Comparison of conformations observed in single crystals with several reported co-crystals of protein-drug complexes suggests existence of two conserved conformations of Imatinib, extended and compact (or folded), corresponding to two binding modes of interaction with the receptor. Furthermore, these conformations are conserved throughout a dynamics simulation. The present study attempts to draw a parallel on conformations and binding patterns of interactions, obtained from small-molecule single-crystal and macromolecule co-crystal studies, and provides structural insights for understanding the high selectivity of this drug molecule.

  10. Isolation, purification, crystallization, and preliminary X-ray diffraction study of the crystals of HU protein from M. gallisepticum

    NASA Astrophysics Data System (ADS)

    Nikolaeva, A. Yu.; Timofeev, V. I.; Boiko, K. M.; Korzhenevskii, D. A.; Rakitina, T. V.; Dorovatovskii, P. V.; Lipkin, A. V.

    2015-11-01

    HU proteins are involved in bacterial DNA and RNA repair. Since these proteins are absent in cells of higher organisms, inhibitors of HU proteins can be used as effective and safe antibiotics. The crystallization conditions for the M. gallisepticum HU protein were found and optimized by the vapor-diffusion method. The X-ray diffraction data set was collected to 2.91 Å resolution from the crystals grown by the vapor-diffusion method on a synchrotron source. The crystals of the HU protein belong to sp. gr. P41212 and have the following unit-cell parameters: a = b = 97.94 Å, c = 77.92 Å, α = β = γ = 90°.

  11. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    SciTech Connect

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

    2015-01-01

    An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s{sup −1}) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

  12. Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization.

    PubMed

    Lasala, R; Coudray, N; Abdine, A; Zhang, Z; Lopez-Redondo, M; Kirshenbaum, R; Alexopoulos, J; Zolnai, Z; Stokes, D L; Ubarretxena-Belandia, I

    2015-02-01

    Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 β-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization.

  13. Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization

    PubMed Central

    Lasala, R.; Coudray, N.; Abdine, A.; Zhang, Z.; Lopez-Redondo, M.; Kirshenbaum, R.; Alexopoulos, J.; Zolnai, Z.; Stokes, D.L.; Ubarretxena-Belandia, I.

    2014-01-01

    Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 β-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1,300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization. PMID:25478971

  14. Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization.

    PubMed

    Lasala, R; Coudray, N; Abdine, A; Zhang, Z; Lopez-Redondo, M; Kirshenbaum, R; Alexopoulos, J; Zolnai, Z; Stokes, D L; Ubarretxena-Belandia, I

    2015-02-01

    Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 β-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization. PMID:25478971

  15. Enhancement of nucleation of protein crystals on nano-wrinkled surfaces.

    PubMed

    Bommineni, Praveen K; Punnathanam, Sudeep N

    2016-01-01

    The synthesis of high quality protein crystals is essential for determining their structure. Hence the development of strategies to facilitate the nucleation of protein crystals is of prime importance. Recently, Ghatak and Ghatak [Langmuir 2013, 29, 4373] reported heterogeneous nucleation of protein crystals on nano-wrinkled surfaces. Through a series of experiments on different proteins, they were able to obtain high quality protein crystals even at low protein concentrations and sometimes without the addition of a precipitant. In this study, the mechanism of protein crystal nucleation on nano-wrinkled surfaces is studied through Monte Carlo simulations. The wrinkled surface is modeled by a sinusoidal surface. Free-energy barriers for heterogeneous crystal nucleation on flat and wrinkled surfaces are computed and compared. The study reveals that the enhancement of nucleation is closely related to the two step nucleation process seen during protein crystallization. There is an enhancement of protein concentration near the trough of the sinusoidal surface which aids in nucleation. However, the high curvature at the trough acts as a deterrent to crystal nucleus formation. Hence, significant lowering of the free-energy barrier is seen only if the increase in the protein concentration at the trough is very high.

  16. A Photonic Crystal Protein Hydrogel Sensor for Candida albicans.

    PubMed

    Cai, Zhongyu; Kwak, Daniel H; Punihaole, David; Hong, Zhenmin; Velankar, Sachin S; Liu, Xinyu; Asher, Sanford A

    2015-10-26

    We report two-dimensional (2D) photonic crystal (PC) sensing materials that selectively detect Candida albicans (C. albicans). These sensors utilize Concanavalin A (Con A) protein hydrogels with a 2D PC embedded on the Con A protein hydrogel surface, that multivalently and selectively bind to mannan on the C. albicans cell surface to form crosslinks. The resulting crosslinks shrink the Con A protein hydrogel, reduce the 2D PC particle spacing, and blue-shift the light diffracted from the PC. The diffraction shifts can be visually monitored, measured with a spectrometer, or determined from the Debye diffraction ring diameter. Our unoptimized hydrogel sensor has a detection limit of around 32 CFU/mL for C. albicans. This sensor distinguishes between C. albicans and those microbes devoid of cell-surface mannan such as the gram-negative bacterium E. coli. This sensor provides a proof-of-concept for utilizing recognition between lectins and microbial cell surface carbohydrates to detect microorganisms in aqueous environments. PMID:26480336

  17. Towards long-wavelength protein crystallography: keeping a protein crystal frozen in vacuum

    NASA Astrophysics Data System (ADS)

    Mykhaylyk, Vitaliy; Wagner, Armin

    2013-03-01

    There is growing interest to explore the long-wavelength X-ray domain for macromolecular crystallography (MX) experiments but there are a number of practical issues that make these experiments difficult to perform. In this article we study several aspects related to cooling a protein crystal in a vacuum environment. We investigated thermal contact conductance (TCC) of copper-copper joints and designed a demountable sample holder assembly with a magnetic joint that facilitates good thermal conductivity and reliability over a long time period. The thermal conductivity of amorphous ice formed by a 20% solution of ethylene glycol was studied. It is concluded that the ice thickness is the factor that can compromise the cooling of protein crystals and therefore it should be carefully controlled.

  18. Crystal Structure of a Lipid G Protein-Coupled Receptor

    SciTech Connect

    Hanson, Michael A; Roth, Christopher B; Jo, Euijung; Griffith, Mark T; Scott, Fiona L; Reinhart, Greg; Desale, Hans; Clemons, Bryan; Cahalan, Stuart M; Schuerer, Stephan C; Sanna, M Germana; Han, Gye Won; Kuhn, Peter; Rosen, Hugh; Stevens, Raymond C

    2012-03-01

    The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P1-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P1, resulting in the modulation of immune and stromal cell responses.

  19. High Pressure Cryocooling of Protein Crystals: The Enigma of Water

    NASA Astrophysics Data System (ADS)

    Gruner, Sol M.

    2010-03-01

    A novel high-pressure cryocooling technique for preparation biological samples for x-ray analysis is described. The method, high-pressure cryocooling, involves cooling samples to cryogenic temperatures (e.g., 100 K) in high-pressure Helium gas (up to 200 MPa). It bears both similarities and differences to high-pressure cooling methods that have been used to prepare samples for electron microscopy, and has been especially useful for cryocooling of macromolecular crystals for x-ray diffraction. Examples will be given where the method has been effective in providing high quality crystallographic data for difficult samples, such as cases where ligands needed to be stabilized in binding sites to be visualized, or where very high resolution data were required. The talk concludes with a discussion of data obtained by high-pressure cryocooling that pertains to two of the most important problems in modern science: the enigma of water and how water affects the activity of proteins.

  20. Use of dye to distinguish salt and protein crystals under microcrystallization conditions

    NASA Technical Reports Server (NTRS)

    Cosenza, Larry (Inventor); Bray, Terry L. (Inventor); DeLucas, Lawrence J. (Inventor); Gester, Thomas E. (Inventor); Hamrick, David T. (Inventor)

    2007-01-01

    An improved method of screening crystal growth conditions is provided wherein molecules are crystallized from solutions containing dyes. These dyes are selectively incorporated or associated with crystals of particular character thereby rendering crystals of particular character colored and improving detection of the dyed crystals. A preferred method involves use of dyes in protein solutions overlayed by oil. Use of oil allows the use of small volumes of solution and facilitates the screening of large numbers of crystallization conditions in arrays using automated devices that dispense appropriate solutions to generate crystallization trials, overlay crystallization trials with an oil, provide appropriate conditions conducive to crystallization and enhance detection of dyed (colored) or undyed (uncolored) crystals that result.

  1. Direct observation of protein microcrystals in crystallization buffer by atmospheric scanning electron microscopy.

    PubMed

    Maruyama, Yuusuke; Ebihara, Tatsuhiko; Nishiyama, Hidetoshi; Konyuba, Yuji; Senda, Miki; Numaga-Tomita, Takuro; Senda, Toshiya; Suga, Mitsuo; Sato, Chikara

    2012-01-01

    X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called "crystallization bottleneck". Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iβ in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few μm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals.

  2. Direct Observation of Protein Microcrystals in Crystallization Buffer by Atmospheric Scanning Electron Microscopy

    PubMed Central

    Maruyama, Yuusuke; Ebihara, Tatsuhiko; Nishiyama, Hidetoshi; Konyuba, Yuji; Senda, Miki; Numaga-Tomita, Takuro; Senda, Toshiya; Suga, Mitsuo; Sato, Chikara

    2012-01-01

    X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called “crystallization bottleneck”. Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iβ in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few μm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals. PMID:22949879

  3. Direct observation of protein microcrystals in crystallization buffer by atmospheric scanning electron microscopy.

    PubMed

    Maruyama, Yuusuke; Ebihara, Tatsuhiko; Nishiyama, Hidetoshi; Konyuba, Yuji; Senda, Miki; Numaga-Tomita, Takuro; Senda, Toshiya; Suga, Mitsuo; Sato, Chikara

    2012-01-01

    X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called "crystallization bottleneck". Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iβ in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few μm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals. PMID:22949879

  4. Heterogeneous distribution of dye-labelled biomineralizaiton proteins in calcite crystals

    PubMed Central

    Liu, Chuang; Xie, Liping; Zhang, Rongqing

    2015-01-01

    Biominerals are highly ordered crystals mediated by organic matters especially proteins in organisms. However, how specific proteins are distributed inside biominerals are not well understood. In the present study, we use fluorescein isothiocyanate (FITC) to label extracted proteins from the shells of bivalve Pinctada fucata. By confocal laser scanning microscopy (CLSM), we observe a heterogeneous distribution of dye-labelled proteins inside synthetic calcite at the microscale. Proteins from the prismatic calcite layers accumulate at the edge of crystals while proteins from the nacreous aragonite layers accumulate at the center of crystals. Raman and X-ray powder diffraction show that both the proteins cannot alter the crystal phase. Scanning electron microscope demonstrates both proteins are able to affect the crystal morphology. This study may provide a direct approach for the visualization of protein distributions in crystals by small-molecule dye-labelled proteins as the additives in the crystallization process and improve our understanding of intracrystalline proteins distribution in biogenic calcites. PMID:26675363

  5. Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy.

    PubMed

    Nannenga, Brent L; Iadanza, Matthew G; Vollmar, Breanna S; Gonen, Tamir

    2013-01-01

    Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.

  6. Protein crystal growth with a two-liquid system and stirring solution.

    PubMed

    Adachi, Hiroaki; Takano, Kazufumi; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo

    2004-01-01

    We developed two novel methods for growing large, high-quality protein crystals. A two-liquid system enables the convenient extraction of protein crystals without causing mechanical damage due to growth at the interface between two liquids. Since this system does not require limitations on solution volume, it is also suitable for the seed technique, and for growing large crystals. Another new concept is the mild stirring of the solution using the Floating And Stirring Technique (FAST) and the Micro-stirring technique. When compared to conventional techniques, both techniques result in a reduced number of crystals, as well as the growth of large crystals.

  7. Automated documentation generator for advanced protein crystal growth

    NASA Astrophysics Data System (ADS)

    Maddux, Gary A.; Provancha, Anna; Chattam, David; Ford, Ronald

    1993-01-01

    The System Management and Production Laboratory at the Research Institute, the University of Alabama in Huntsville (UAH), was tasked by the Microgravity Experiment Projects (MEP) Office of the Payload Projects Office (PPO) at Marshall Space Flight Center (MSFC) to conduct research in the current methods of written documentation control and retrieval. The goals of this research were to determine the logical interrelationships within selected NASA documentation, and to expand on a previously developed prototype system to deliver a distributable, electronic knowledge-based system. This computer application would then be used to provide a paperless interface between the appropriate parties for the required NASA document.

  8. Automated documentation generator for advanced protein crystal growth

    NASA Technical Reports Server (NTRS)

    Maddux, Gary A.; Provancha, Anna; Chattam, David

    1994-01-01

    To achieve an environment less dependent on the flow of paper, automated techniques of data storage and retrieval must be utilized. This software system, 'Automated Payload Experiment Tool,' seeks to provide a knowledge-based, hypertext environment for the development of NASA documentation. Once developed, the final system should be able to guide a Principal Investigator through the documentation process in a more timely and efficient manner, while supplying more accurate information to the NASA payload developer. The current system is designed for the development of the Science Requirements Document (SRD), the Experiment Requirements Document (ERD), the Project Plan, and the Safety Requirements Document.

  9. Extracellular matrix protein in calcified endoskeleton: a potential additive for crystal growth and design

    NASA Astrophysics Data System (ADS)

    Azizur Rahman, M.; Fujimura, Hiroyuki; Shinjo, Ryuichi; Oomori, Tamotsu

    2011-06-01

    In this study, we demonstrate a key function of extracellular matrix proteins (ECMPs) on seed crystals, which are isolated from calcified endoskeletons of soft coral and contain only CaCO 3 without any living cells. This is the first report that an ECMP protein extracted from a marine organism could potentially influence in modifying the surface of a substrate for designing materials via crystallization. We previously studied with the ECMPs from a different type of soft coral ( Sinularia polydactyla) without introducing any seed crystals in the process , which showed different results. Thus, crystallization on the seed in the presence of ECMPs of present species is an important first step toward linking function to individual proteins from soft coral. For understanding this interesting phenomenon, in vitro crystallization was initiated in a supersaturated solution on seed particles of calcite (1 0 4) with and without ECMPs. No change in the crystal growth shape occurred without ECMPs present during the crystallization process. However, with ECMPs, the morphology and phase of the crystals in the crystallization process changed dramatically. Upon completion of crystallization with ECMPs, an attractive crystal morphology was found. Scanning electron microscopy (SEM) was utilized to observe the crystal morphologies on the seeds surface. The mineral phases of crystals nucleated by ECMPs on the seeds surface were examined by Raman spectroscopy. Although 50 mM Mg 2+ is influential in making aragonite in the crystallization process, the ECMPs significantly made calcite crystals even when 50 mM Mg 2+ was present in the process. Crystallization with the ECMP additive seems to be a technically attractive strategy to generate assembled micro crystals that could be used in crystals growth and design in the Pharmaceutical and biotechnology industries.

  10. In situ measurement and dynamic control of the evaporation rate in vapor diffusion crystallization of proteins

    NASA Astrophysics Data System (ADS)

    Shu, Zhan-Yong; Gong, Hai-Yun; Bi, Ru-Chang

    1998-08-01

    A special device with a weight-sensitive facility was designed for monitoring and controlling the water evaporation in vapor diffusion protein crystallization. The device made it possible to measure the weight of the drop in real time while the crystallization experiment was going on normally. The precise water equilibration curves under different crystallization conditions could be obtained automatically. By monitoring and controlling the evaporation rate, the crystallization of hen egg-white lysozyme and trichosanthin, a plant protein from Chinese herb, was optimized by regulating the reservoir solution dynamically. The experimental results of these two proteins indicate both the feasibility of the device and the usefulness of dynamic control technique. Compared with traditional crystallization experiments, dynamically controlled crystallization can reduce the number of nuclei, increase the crystal size and save experimental time effectively.

  11. Self-interaction chromatography as a tool for optimizing conditions for membrane protein crystallization.

    PubMed

    Gabrielsen, Mads; Nagy, Lisa A; DeLucas, Lawrence J; Cogdell, Richard J

    2010-01-01

    The second virial coefficient, or B value, is a measurement of how well a protein interacts with itself in solution. These interactions can lead to protein crystallization or precipitation, depending on their strength, with a narrow range of B values (the 'crystallization slot') being known to promote crystallization. A convenient method of determining the B value is by self-interaction chromatography. This paper describes how the light-harvesting complex 1-reaction centre core complex from Allochromatium vinosum yielded single straight-edged crystals after iterative cycles of self-interaction chromatography and crystallization. This process allowed the rapid screening of small molecules and detergents as crystallization additives. Here, a description is given of how self-interaction chromatography has been utilized to improve the crystallization conditions of a membrane protein.

  12. Investigation of Advanced Processed Single-Crystal Turbine Blade Alloys

    NASA Technical Reports Server (NTRS)

    Peters, B. J.; Biondo, C. M.; DeLuca, D. P.

    1995-01-01

    This investigation studied the influence of thermal processing and microstructure on the mechanical properties of the single-crystal, nickel-based superalloys PWA 1482 and PWA 1484. The objective of the program was to develop an improved single-crystal turbine blade alloy that is specifically tailored for use in hydrogen fueled rocket engine turbopumps. High-gradient casting, hot isostatic pressing (HIP), and alternate heat treatment (HT) processing parameters were developed to produce pore-free, eutectic-free microstructures with different (gamma)' precipitate morphologies. Test materials were cast in high thermal gradient solidification (greater than 30 C/cm (137 F/in.)) casting furnaces for reduced dendrite arm spacing, improved chemical homogeneity, and reduced interdendritic pore size. The HIP processing was conducted in 40 cm (15.7 in.) diameter production furnaces using a set of parameters selected from a trial matrix study. Metallography was conducted on test samples taken from each respective trial run to characterize the as-HIP microstructure. Post-HIP alternate HT processes were developed for each of the two alloys. The goal of the alternate HT processing was to fully solution the eutectic gamma/(gamma)' phase islands and to develop a series of modified (gamma)' morphologies for subsequent characterization testing. This was accomplished by slow cooling through the (gamma)' solvus at controlled rates to precipitate volume fractions of large (gamma)'. Post-solution alternate HT parameters were established for each alloy providing additional volume fractions of finer precipitates. Screening tests included tensile, high-cycle fatigue (HCF), smooth and notched low-cycle fatigue (LCF), creep, and fatigue crack growth evaluations performed in air and high pressure (34.5 MPa (5 ksi)) hydrogen at room and elevated temperature. Under the most severe embrittling conditions (HCF and smooth and notched LCF in 34.5 MPa (5 ksi) hydrogen at 20 C (68 F), screening test

  13. System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

    DOEpatents

    Craig, George D.; Glass, Robert; Rupp, Bernhard

    1997-01-01

    A method for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10.sup.6 V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved.

  14. System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

    DOEpatents

    Craig, G.D.; Glass, R.; Rupp, B.

    1997-01-28

    A method is disclosed for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10{sup 6}V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved. 2 figs.

  15. Understanding the Physical Properties that Control Protein Crystallization by Analysis of Large-Scale Experimental Data

    SciTech Connect

    Price, W.; Chen, Y; Handelman, S; Neely, H; Manor, P; Karlin, R; Nair, R; Montelione, G; Hunt, J; et. al.

    2008-01-01

    Crystallization is the most serious bottleneck in high-throughput protein-structure determination by diffraction methods. We have used data mining of the large-scale experimental results of the Northeast Structural Genomics Consortium and experimental folding studies to characterize the biophysical properties that control protein crystallization. This analysis leads to the conclusion that crystallization propensity depends primarily on the prevalence of well-ordered surface epitopes capable of mediating interprotein interactions and is not strongly influenced by overall thermodynamic stability. We identify specific sequence features that correlate with crystallization propensity and that can be used to estimate the crystallization probability of a given construct. Analyses of entire predicted proteomes demonstrate substantial differences in the amino acid-sequence properties of human versus eubacterial proteins, which likely reflect differences in biophysical properties, including crystallization propensity. Our thermodynamic measurements do not generally support previous claims regarding correlations between sequence properties and protein stability.

  16. Real-time investigation of dynamic protein crystallization in living cellsa)

    PubMed Central

    Schönherr, R.; Klinge, M.; Rudolph, J. M.; Fita, K.; Rehders, D.; Lübber, F.; Schneegans, S.; Majoul, I. V.; Duszenko, M.; Betzel, C.; Brandariz-Nuñez, A.; Martinez-Costas, J.; Duden, R.; Redecke, L.

    2015-01-01

    X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus μNS by live-cell imaging, showing that dimensions of living cells did not limit crystal size. The crystallization process is highly dynamic and occurs in different cellular compartments. In vivo protein crystallization offers exciting new possibilities for proteins that do not form crystals in vitro. PMID:26798811

  17. Graphene as a protein crystal mounting material to reduce background scatter

    PubMed Central

    Wierman, Jennifer L.; Alden, Jonathan S.; Kim, Chae Un; McEuen, Paul L.; Gruner, Sol M.

    2013-01-01

    The overall signal-to-noise ratio per unit dose for X-ray diffraction data from protein crystals can be improved by reducing the mass and density of all material surrounding the crystals. This article demonstrates a path towards the practical ultimate in background reduction by use of atomically thin graphene sheets as a crystal mounting platform for protein crystals. The results show the potential for graphene in protein crystallography and other cases where X-ray scatter from the mounting material must be reduced and specimen dehydration prevented, such as in coherent X-ray diffraction imaging of microscopic objects. PMID:24068843

  18. Crystal structure of advanced lithium titanate with lithium oxide additives

    NASA Astrophysics Data System (ADS)

    Hoshino, Tsuyoshi; Sasaki, Kazuya; Tsuchiya, Kunihiko; Hayashi, Kimio; Suzuki, Akihiro; Hashimoto, Takuya; Terai, Takayuki

    2009-04-01

    Li 2TiO 3 is one of the most promising candidates among solid breeder materials proposed for fusion reactors. However, the mass of Li 2TiO 3 was found to decrease with time in the sweep gas mixed with hydrogen. This mass change indicates that the oxygen content of the sample decreased, suggesting the change from Ti 4+ to Ti 3+. In the present paper, the crystal structure and the non-stoichiometry of Li 2TiO 3 added with Li 2O have been extensively investigated by means of X-ray diffraction (XRD) and thermogravimetry. In the case of the Li 2TiO 3 samples used in the present study, LiO-C 2H 5 or LiO-i-C 3H 7 and Ti(O-i-C 3H 7) 4 were mixed in the proportion corresponding to the molar ratio Li 2O/TiO 2 of either 2.00 or 1.00. In thermogravimetry, the mass of this sample decreased with time due to lithium deficiency, where no presence of oxygen deficiency was indicated.

  19. Advances in Polymer and Polymeric Nanostructures for Protein Conjugation

    PubMed Central

    González-Toro, Daniella C.; Thayumanavan, S.

    2013-01-01

    Linear polymers have been considered the best molecular structures for the formation of efficient protein conjugates due to their biological advantages, synthetic convenience and ease of functionalization. In recent years, much attention has been dedicated to develop synthetic strategies that produce the most control over protein conjugation utilizing linear polymers as scaffolds. As a result, different conjugate models, such as semitelechelic, homotelechelic, heterotelechelic and branched or star polymer conjugates, have been obtained that take advantage of these well-controlled synthetic strategies. Development of protein conjugates using nanostructures and the formation of said nanostructures from protein-polymer bioconjugates are other areas in the protein bioconjugation field. Although several polymer-protein technologies have been developed from these discoveries, few review articles have focused on the design and function of these polymers and nanostructures. This review will highlight some recent advances in protein-linear polymer technologies that employ protein covalent conjugation and successful protein-nanostructure bioconjugates (covalent conjugation as well) that have shown great potential for biological applications. PMID:24058205

  20. Automation of Vapor-Diffusion Growth of Protein Crystals

    NASA Technical Reports Server (NTRS)

    Hamrick, David T.; Bray, Terry L.

    2005-01-01

    Some improvements have been made in a system of laboratory equipment developed previously for studying the crystallization of proteins from solution by use of dynamically controlled flows of dry gas. The improvements involve mainly (1) automation of dispensing of liquids for starting experiments, (2) automatic control of drying of protein solutions during the experiments, and (3) provision for automated acquisition of video images for monitoring experiments in progress and for post-experiment analysis. The automation of dispensing of liquids was effected by adding an automated liquid-handling robot that can aspirate source solutions and dispense them in either a hanging-drop or a sitting-drop configuration, whichever is specified, in each of 48 experiment chambers. A video camera of approximately the size and shape of a lipstick dispenser was added to a mobile stage that is part of the robot, in order to enable automated acquisition of images in each experiment chamber. The experiment chambers were redesigned to enable the use of sitting drops, enable backlighting of each specimen, and facilitate automation.

  1. Repartitioning of NaCl and Protein Impurities in Lysozyme Crystallization

    NASA Technical Reports Server (NTRS)

    Vekilov, Peter G.; Monaco, Lisa A.; Thomas, Bill R.; Stojanoff, Vivian; Rosenberger, Franz

    1996-01-01

    Nonuniform precipitant and impurity incorporation in protein crystals can cause lattice strain and, thus, possibly decrease the X-ray diffraction resolution. To address this issue, a series of crystallization experiments were carried out, in which initial supersaturation, NaCl concentration, protein purity level and crystallized fraction were varied. Lysozyme and protein impurities, as well as sodium and chloride were independently determined in the initial solution, supernatant and crystals. The segregation coefficients for Na(+) and Cl(-) were found to be independent of supersaturation and NaCl concentration, and decreased with crystallized fraction/crystal size. Numerical evaluation of the extensive body of data, based on a nucleation-growth- repartitioning model, suggests a core of approx. 40 microns in which salt is incorporated in much greater concentrations than during later growth. Small crystals containing higher amounts of incorporated NaCl also had higher protein impurity contents. This suggests that the excess salt is associated with the protein impurities in the core. X-ray topography revealed strain fields in the center of the crystals comparable in size to the inferred core. The growth rates of crystals smaller than 30-40 pm in size were consistently 1.5-2 times lower than those of larger crystals, presumably due to higher chemical potentials in the core.

  2. Green Fluorescent Protein as a Model for Protein Crystal Growth Studies

    NASA Technical Reports Server (NTRS)

    Agena, Sabine; Smith, Lori; Karr, Laurel; Pusey, Marc

    1998-01-01

    Green fluorescent protein (GFP) from jellyfish Aequorea Victoria has become a popular marker for e.g. mutagenesis work. Its fluorescent property, which originates from a chromophore located in the center of the molecule, makes it widely applicable as a research too]. GFP clones have been produced with a variety of spectral properties, such as blue and yellow emitting species. The protein is a single chain of molecular weight 27 kDa and its structure has been determined at 1.9 Angstrom resolution. The combination of GFP's fluorescent property, the knowledge of its several crystallization conditions, and its increasing use in biophysical and biochemical studies, all led us to consider it as a model material for macromolecular crystal growth studies. Initial preparations of GFP were from E.coli with yields of approximately 5 mg/L of culture media. Current yields are now in the 50 - 120 mg/L range, and we hope to further increase this by expression of the GFP gene in the Pichia system. The results of these efforts and of preliminary crystal growth studies will be presented.

  3. Mechanisms of protein and virus crystal growth: An atomic force microscopy study of canavalin and STMV crystallization

    SciTech Connect

    Land, T.A.; De Yoreo, J.J.; Malkin, A.J.; Kutznesov, Yu.G.; McPherson, A.

    1996-05-01

    The evolution of surface morphology and step dynamics during growth of rhombohedral crystals of the protein canavalin and crystals of the cubic satellite tobacco mosaic virus (STMV) have been investigated for the first time by in situ atomic force microscopy. These two crystals were observed to grow by very different mechanisms. Growth of canavalin occurs on complex vicinal hillocks formed by multiple, independently acting screw dislocations. Small clusters were observed on the terraces. STMV on the other hand, was observed to grow by 2D nucleation of islands. No dislocations were found on the crystal. The results are used to determine the growth mechanisms and estimate the fundamental materials parameters. The images also illustrate the important mechanism of defect incorporation and provide insight to the processes that limit the growth rate and uniformity of these crystals.

  4. Advanced Fluorescence Protein-Based Synapse-Detectors

    PubMed Central

    Lee, Hojin; Oh, Won Chan; Seong, Jihye; Kim, Jinhyun

    2016-01-01

    The complex information-processing capabilities of the central nervous system emerge from intricate patterns of synaptic input-output relationships among various neuronal circuit components. Understanding these capabilities thus requires a precise description of the individual synapses that comprise neural networks. Recent advances in fluorescent protein engineering, along with developments in light-favoring tissue clearing and optical imaging techniques, have rendered light microscopy (LM) a potent candidate for large-scale analyses of synapses, their properties, and their connectivity. Optically imaging newly engineered fluorescent proteins (FPs) tagged to synaptic proteins or microstructures enables the efficient, fine-resolution illumination of synaptic anatomy and function in large neural circuits. Here we review the latest progress in fluorescent protein-based molecular tools for imaging individual synapses and synaptic connectivity. We also identify associated technologies in gene delivery, tissue processing, and computational image analysis that will play a crucial role in bridging the gap between synapse- and system-level neuroscience. PMID:27445785

  5. Recent advances in the photochemical control of protein function

    PubMed Central

    Riggsbee, Chad W.; Deiters, Alexander

    2010-01-01

    Biological processes are regulated with a high level of spatial and temporal resolution. In order to understand and manipulate these processes, scientists need to be able to regulate them with Nature’s level of precision. In this context, light is a unique regulatory element because it can be precisely controlled in location, timing and amplitude. Moreover, most biological laboratories have a wide range of light sources as standard equipment. This review article summarizes the most recent advances in light-mediated regulation of protein function and the application in a cellular context. Specifically, the photocaging of small molecule modulators of protein function and of select amino acid residues in proteins will be discussed. In addition, examples of the photochemical control of protein function through the application of natural light-receptors are presented. PMID:20667607

  6. Advanced Fluorescence Protein-Based Synapse-Detectors.

    PubMed

    Lee, Hojin; Oh, Won Chan; Seong, Jihye; Kim, Jinhyun

    2016-01-01

    The complex information-processing capabilities of the central nervous system emerge from intricate patterns of synaptic input-output relationships among various neuronal circuit components. Understanding these capabilities thus requires a precise description of the individual synapses that comprise neural networks. Recent advances in fluorescent protein engineering, along with developments in light-favoring tissue clearing and optical imaging techniques, have rendered light microscopy (LM) a potent candidate for large-scale analyses of synapses, their properties, and their connectivity. Optically imaging newly engineered fluorescent proteins (FPs) tagged to synaptic proteins or microstructures enables the efficient, fine-resolution illumination of synaptic anatomy and function in large neural circuits. Here we review the latest progress in fluorescent protein-based molecular tools for imaging individual synapses and synaptic connectivity. We also identify associated technologies in gene delivery, tissue processing, and computational image analysis that will play a crucial role in bridging the gap between synapse- and system-level neuroscience. PMID:27445785

  7. Crystal Structure of the Protein Kinase Domain of Yeast AMP-Activated Protein Kinase Snf1

    SciTech Connect

    Rudolph,M.; Amodeo, G.; Bai, Y.; Tong, L.

    2005-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator, and is an important target for drug development against diabetes, obesity, and other diseases. AMPK is a hetero-trimeric enzyme, with a catalytic ({alpha}) subunit, and two regulatory ({beta} and {gamma}) subunits. Here we report the crystal structure at 2.2 Angstrom resolution of the protein kinase domain (KD) of the catalytic subunit of yeast AMPK (commonly known as SNF1). The Snf1-KD structure shares strong similarity to other protein kinases, with a small N-terminal lobe and a large C-terminal lobe. Two negative surface patches in the structure may be important for the recognition of the substrates of this kinase.

  8. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments

    PubMed Central

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C.; Johnson, Jennifer L.; Entzminger, Kevin; Jain, Avni; Heaner, David P.; Morales, Ivan A.; Truskett, Thomas M.; Maynard, Jennifer A.; Lieberman, Raquel L.

    2014-01-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although non-complementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts. PMID:24615866

  9. Watching Proteins Direct Crystal Growth One Step at a Time

    SciTech Connect

    2009-01-01

    Researchers at Berkeley Labs Molecular Foundry use an atomic force microscope to record this movie of a peptide being adsorbed to a crystal surface while two successive crystal steps interact, then progress beyond the peptide. The peptide temporarily slows the step before transferring up to the next atomic layer. The lattice pattern on the surface corresponds to the molecular structure of the underlying crystal.

  10. High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density.

    PubMed

    Teplitsky, Ella; Joshi, Karan; Ericson, Daniel L; Scalia, Alexander; Mullen, Jeffrey D; Sweet, Robert M; Soares, Alexei S

    2015-07-01

    We describe a high throughput method for screening up to 1728 distinct chemicals with protein crystals on a single microplate. Acoustic droplet ejection (ADE) was used to co-position 2.5nL of protein, precipitant, and chemicals on a MiTeGen in situ-1 crystallization plate™ for screening by co-crystallization or soaking. ADE-transferred droplets follow a precise trajectory which allows all components to be transferred through small apertures in the microplate lid. The apertures were large enough for 2.5nL droplets to pass through them, but small enough so that they did not disrupt the internal environment created by the mother liquor. Using this system, thermolysin and trypsin crystals were efficiently screened for binding to a heavy-metal mini-library. Fluorescence and X-ray diffraction were used to confirm that each chemical in the heavy-metal library was correctly paired with the intended protein crystal. A fragment mini-library was screened to observe two known lysozyme ligands using both co-crystallization and soaking. A similar approach was used to identify multiple, novel thaumatin binding sites for ascorbic acid. This technology pushes towards a faster, automated, and more flexible strategy for high throughput screening of chemical libraries (such as fragment libraries) using as little as 2.5nL of each component.

  11. Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction

    SciTech Connect

    Emamzadah, Soheila; Petty, Tom J.; De Almeida, Victor; Nishimura, Taisuke; Joly, Jacques; Ferrer, Jean-Luc; Halazonetis, Thanos D.

    2009-09-01

    A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction. Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus’ molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 Å resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts.

  12. High-pressure hydrogen testing of single crystal superalloys for advanced rocket engine turbopump turbine blades

    NASA Technical Reports Server (NTRS)

    Alter, W. S.; Parr, R. A.; Johnston, M. H.; Strizak, J. P.

    1984-01-01

    A screening program to determine the effects of high pressure hydrogen on selected candidate materials for advanced single crystal turbine blade applications is examined. The alloys chosen for the investigation are CM SX-2, CM SX-4C, Rene N-4, and PWA1480. Testing is carried out in hydrogen and helium at 34 MPa and room temperature, with both notched and unnotched single crystal specimens. Results show a significant variation in susceptibility to Hydrogen Environment Embrittlement (HEE) among the four alloys and a marked difference in fracture topography between hydrogen and helium environment specimens.

  13. High-pressure hydrogen testing of single crystal superalloys for advanced rocket engine turbopump turbine blades

    NASA Technical Reports Server (NTRS)

    Parr, R. A.; Alter, W. S.; Johnston, M. H.; Strizak, J. P.

    1985-01-01

    A screening program to determine the effects of high pressure hydrogen on selected candidate materials for advanced single crystal turbine blade applications is examined. The alloys chosen for the investigation are CM SX-2, CM SX-4C, Rene N-4, and PWA1480. Testing is carried out in hydrogen and helium at 34 MPa and room temperature, with both notched and unnotched single crystal specimens. Results show a significant variation in susceptibility to Hydrogen Environment Embrittlement (HEE) among the four alloys and a marked difference in fracture topography between hydrogen and helium environment specimens.

  14. Recent advances in designing substrate-competitive protein kinase inhibitors.

    PubMed

    Han, Ki-Cheol; Kim, So Yeon; Yang, Eun Gyeong

    2012-01-01

    Protein kinases play central roles in cellular signaling pathways and their abnormal phosphorylation activity is inseparably linked with various human diseases. Therefore, modulation of kinase activity using potent inhibitors is an attractive strategy for the treatment of human disease. While most protein kinase inhibitors in clinical development are mainly targeted to the highly conserved ATP-binding sites and thus likely promiscuously inhibit multiple kinases including kinases unrelated to diseases, protein substrate-competitive inhibitors are more selective and expected to be promising therapeutic agents. Most substrate-competitive inhibitors mimic peptides derived from substrate proteins, or from inhibitory domains within kinases or inhibitor proteins. In addition, bisubstrate inhibitors are generated by conjugating substrate-competitive peptide inhibitors to ATP-competitive inhibitors to improve affinity and selectivity. Although structural information on protein kinases provides invaluable guidance in designing substrate-competitive inhibitors, other strategies including bioinformatics, computational modeling, and high-throughput screening are often employed for developing specific substrate-competitive kinase inhibitors. This review focuses on recent advances in the design and discovery of substrate-competitive inhibitors of protein kinases.

  15. A historical perspective on protein crystallization from 1840 to the present day.

    PubMed

    Giegé, Richard

    2013-12-01

    Protein crystallization has been known since 1840 and can prove to be straightforward but, in most cases, it constitutes a real bottleneck. This stimulated the birth of the biocrystallogenesis field with both 'practical' and 'basic' science aims. In the early years of biochemistry, crystallization was a tool for the preparation of biological substances. Today, biocrystallogenesis aims to provide efficient methods for crystal fabrication and a means to optimize crystal quality for X-ray crystallography. The historical development of crystallization methods for structural biology occurred first in conjunction with that of biochemical and genetic methods for macromolecule production, then with the development of structure determination methodologies and, recently, with routine access to synchrotron X-ray sources. Previously, the identification of conditions that sustain crystal growth occurred mostly empirically but, in recent decades, this has moved progressively towards more rationality as a result of a deeper understanding of the physical chemistry of protein crystal growth and the use of idea-driven screening and high-throughput procedures. Protein and nucleic acid engineering procedures to facilitate crystallization, as well as crystallization methods in gelled-media or by counter-diffusion, represent recent important achievements, although the underlying concepts are old. The new nanotechnologies have brought a significant improvement in the practice of protein crystallization. Today, the increasing number of crystal structures deposited in the Protein Data Bank could mean that crystallization is no longer a bottleneck. This is not the case, however, because structural biology projects always become more challenging and thereby require adapted methods to enable the growth of the appropriate crystals, notably macromolecular assemblages.

  16. Crystal Structure of the Human Astrovirus Capsid Protein

    PubMed Central

    Toh, Yukimatsu; Harper, Justin; Dryden, Kelly A.; Yeager, Mark; Méndez, Ernesto

    2016-01-01

    ABSTRACT Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a nonenveloped virus with a T=3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90-kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of HAstV requires proteolytic processing of the astrovirus CP both inside and outside the host cell, resulting in the removal of the C-terminal domain and the breakdown of the rest of the CP into three predominant protein species with molecular masses of ∼34, 27/29, and 25/26 kDa, respectively. We have now solved the crystal structure of VP9071–415 (amino acids [aa] 71 to 415 of VP90) of human astrovirus serotype 8 at a 2.15-Å resolution. VP9071–415 encompasses the conserved N-terminal domain of VP90 but lacks the hypervariable domain, which forms the capsid surface spikes. The structure of VP9071–415 is comprised of two domains: an S domain, which adopts the typical jelly-roll β-barrel fold, and a P1 domain, which forms a squashed β-barrel consisting of six antiparallel β-strands similar to what was observed in the hepatitis E virus (HEV) capsid structure. Fitting of the VP9071–415 structure into the cryo-electron microscopy (EM) maps of HAstV produced an atomic model for a continuous, T=3 icosahedral capsid shell. Our pseudoatomic model of the human HAstV capsid shell provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation needed for virus infectivity. Such information has potential applications in the development of a virus-like particle (VLP) vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation. IMPORTANCE Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. As a nonenveloped virus

  17. An approach to crystallizing proteins by metal-mediated synthetic symmetrization

    SciTech Connect

    Laganowsky, Arthur; Zhao, Minglei; Soriaga, Angela B.; Sawaya, Michael R.; Cascio, Duilio; Yeates, Todd O.

    2011-12-09

    Combining the concepts of synthetic symmetrization with the approach of engineering metal-binding sites, we have developed a new crystallization methodology termed metal-mediated synthetic symmetrization. In this method, pairs of histidine or cysteine mutations are introduced on the surface of target proteins, generating crystal lattice contacts or oligomeric assemblies upon coordination with metal. Metal-mediated synthetic symmetrization greatly expands the packing and oligomeric assembly possibilities of target proteins, thereby increasing the chances of growing diffraction-quality crystals. To demonstrate this method, we designed various T4 lysozyme (T4L) and maltose-binding protein (MBP) mutants and cocrystallized them with one of three metal ions: copper (Cu2+), nickel (Ni2+), or zinc (Zn2+). The approach resulted in 16 new crystal structures-eight for T4L and eight for MBP-displaying a variety of oligomeric assemblies and packing modes, representing in total 13 new and distinct crystal forms for these proteins. We discuss the potential utility of the method for crystallizing target proteins of unknown structure by engineering in pairs of histidine or cysteine residues. As an alternate strategy, we propose that the varied crystallization-prone forms of T4L or MBP engineered in this work could be used as crystallization chaperones, by fusing them genetically to target proteins of interest.

  18. Nuclear resonance vibrational spectroscopy (NRVS) of rubredoxin and MoFe protein crystals

    PubMed Central

    Guo, Yisong; Brecht, Eric; Aznavour, Kristen; Nix, Jay C.; Xiao, Yuming; Wang, Hongxin; George, Simon J.; Bau, Robert; Keable, Stephen; Peters, John W.; Adams, Michael W.W.; Jenney, Francis; Sturhahn, Wolfgang; Alp, Ercan E.; Zhao, Jiyong; Yoda, Yoshitaka; Cramer, Stephen P.

    2014-01-01

    We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Fe-S protein crystals, including oxidized wild type rubredoxin crystals from Pyrococcus furiosus, and the MoFe protein of nitrogenase from Azotobacter vinelandii. Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type rubredoxin crystals from Pyrococcus furiosus. The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the X-ray crystal structure. PMID:26052177

  19. The role of flexibility and molecular shape in the crystallization of proteins by surface mutagenesis.

    PubMed

    Devedjiev, Yancho D

    2015-02-01

    Proteins are dynamic systems and interact with their environment. The analysis of crystal contacts in the most accurately determined protein structures (d < 1.5 Å) reveals that in contrast to current views, static disorder and high side-chain entropy are common in the crystal contact area. These observations challenge the validity of the theory that presumes that the occurrence of well ordered patches of side chains at the surface is an essential prerequisite for a successful crystallization event. The present paper provides evidence in support of the approach for understanding protein crystallization as a process dependent on multiple factors, each with its relative contribution, rather than a phenomenon driven by a few dominant physicochemical characteristics. The role of the molecular shape as a factor in the crystallization of proteins by surface mutagenesis is discussed.

  20. Nuclear resonance vibrational spectroscopy (NRVS) of rubredoxin and MoFe protein crystals

    NASA Astrophysics Data System (ADS)

    Guo, Yisong; Brecht, Eric; Aznavour, Kristen; Nix, Jay C.; Xiao, Yuming; Wang, Hongxin; George, Simon J.; Bau, Robert; Keable, Stephen; Peters, John W.; Adams, Michael W. W.; , Francis E. Jenney, Jr.; Sturhahn, Wolfgang; Alp, Ercan E.; Zhao, Jiyong; Yoda, Yoshitaka; Cramer, Stephen P.

    2013-12-01

    We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Iron-sulfur protein crystals, including oxidized wild type rubredoxin crystals from Pyrococcus furiosus, and the MoFe protein of nitrogenase from Azotobacter vinelandii. Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type rubredoxin crystals from Pyrococcus furiosus. The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the crystal structure.

  1. Femtosecond X-ray diffraction from two-dimensional protein crystals.

    PubMed

    Frank, Matthias; Carlson, David B; Hunter, Mark S; Williams, Garth J; Messerschmidt, Marc; Zatsepin, Nadia A; Barty, Anton; Benner, W Henry; Chu, Kaiqin; Graf, Alexander T; Hau-Riege, Stefan P; Kirian, Richard A; Padeste, Celestino; Pardini, Tommaso; Pedrini, Bill; Segelke, Brent; Seibert, M Marvin; Spence, John C H; Tsai, Ching-Ju; Lane, Stephen M; Li, Xiao-Dan; Schertler, Gebhard; Boutet, Sebastien; Coleman, Matthew; Evans, James E

    2014-03-01

    X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

  2. Femtosecond X-ray Diffraction From Two-Dimensional Protein Crystals

    SciTech Connect

    Frank, Matthias; Carlson, David B.; Hunter, Mark; Williams, Garth J.; Messerschmidt, Marc; Zatsepin, Nadia A.; Barty, Anton; Benner, Henry; Chu, Kaiqin; Graf, Alexander; Hau-Riege, Stefan; Kirian, Rick; Padeste, Celestino; Pardini, Tommaso; Pedrini, Bill; Segelke, Brent; Seibert, M. M.; Spence, John C.; Tsai, Ching-Ju; Lane, Steve M.; Li, Xiao-Dan; Schertler, Gebhard; Boutet, Sebastien; Coleman, Matthew A.; Evans, James E.

    2014-02-28

    Here we present femtosecond x-ray diffraction patterns from two-dimensional (2-D) protein crystals using an x-ray free electron laser (XFEL). To date it has not been possible to acquire x-ray diffraction from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permits a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy methodology at the Linac Coherent Light Source, we observed Bragg diffraction to better than 8.5 Å resolution for two different 2-D protein crystal samples that were maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

  3. Monitoring the stability of crosslinked protein crystals biotemplates: a feasibility study.

    PubMed

    Cohen-Hadar, Noa; Wine, Yariv; Nachliel, Esther; Huppert, Danny; Gutman, Menahem; Frolow, Felix; Freeman, Amihay

    2006-08-01

    Protein crystals, routinely prepared for the elucidation of protein 3D structures by X-ray crystallography, present an ordered and highly accurate 3D array of protein molecules. Inherent to the 3D arrangement of the protein molecules in the crystal is a complementary 3D array of voids made of interconnected cavities and exhibiting highly ordered porosity. The permeability of the porosity of chemically crosslinked enzyme protein crystals to low molecular weight solutes, was used for enzyme mediated organic synthesis and size exclusion chromatography. This permeability might be extended to explore new potential applications for protein crystals, for example, their use as bio-templates for the fabrication of novel, nano-structured composite materials. The quality of composites obtained from "filling" of the ordered voids in protein crystals and their potential applications will be strongly dependent upon an accurate preservation of the order in the original protein crystal 3D array during the "filling" process. Here we propose and demonstrate the feasibility of monitoring the changes in 3D order of the protein array by a step-by-step molecular level monitoring of a model system for hydrogel bio-templating by glutaraldehyde crosslinked lysozyme crystals. This monitoring is based on step-by-step comparative analysis of data obtained from (i) X-ray crystallography: resolution, unit cell dimensions and B-factor values and (ii) fluorescence decay kinetics of ultra-fast laser activated dye, impregnated within these crystals. Our results demonstrated feasibility of the proposed monitoring approach and confirmed that the stabilized protein crystal template retained its 3D structure throughout the process.

  4. Recent advances in production of recombinant spider silk proteins.

    PubMed

    Chung, Hannah; Kim, Tae Yong; Lee, Sang Yup

    2012-12-01

    Spider silk has been drawing much attention as a great biomaterial having many applications in biotechnology and biomedicine owing to its several desired material characteristics such as outstanding strength, toughness, and elasticity as well as biodegradability and biocompatibility. With various applications foreseeable in industry, there has been much effort to produce recombinant spider silk protein in large amounts. However, owing to the difficulties in its production using spiders, alternative host systems and engineering methods have been investigated to develop suitable production systems that can efficiently produce spider silk protein. Here, we review recent advances in production of spider silk proteins in various heterologous host systems with focus given on the development of metabolic and cellular engineering strategies. PMID:22521455

  5. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    PubMed Central

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

    2015-01-01

    Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s−1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening. PMID:25615864

  6. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    SciTech Connect

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

    2014-06-01

    Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s-1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

  7. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    DOE PAGES

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; et al

    2014-06-01

    Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s-1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from themore » inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.« less

  8. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening.

    PubMed

    Cuttitta, Christina M; Ericson, Daniel L; Scalia, Alexander; Roessler, Christian G; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M; Sweet, Robert M; Soares, Alexei S

    2015-01-01

    Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s(-1)) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

  9. Automated harvesting and processing of protein crystals through laser photoablation.

    PubMed

    Zander, Ulrich; Hoffmann, Guillaume; Cornaciu, Irina; Marquette, Jean-Pierre; Papp, Gergely; Landret, Christophe; Seroul, Gaël; Sinoir, Jérémy; Röwer, Martin; Felisaz, Frank; Rodriguez-Puente, Sonia; Mariaule, Vincent; Murphy, Peter; Mathieu, Magali; Cipriani, Florent; Márquez, José Antonio

    2016-04-01

    Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities. PMID:27050125

  10. Automated harvesting and processing of protein crystals through laser photoablation

    PubMed Central

    Zander, Ulrich; Hoffmann, Guillaume; Cornaciu, Irina; Marquette, Jean-Pierre; Papp, Gergely; Landret, Christophe; Seroul, Gaël; Sinoir, Jérémy; Röwer, Martin; Felisaz, Frank; Rodriguez-Puente, Sonia; Mariaule, Vincent; Murphy, Peter; Mathieu, Magali; Cipriani, Florent; Márquez, José Antonio

    2016-01-01

    Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities. PMID:27050125

  11. Intracrystalline Proteins Promote Dissolution of Urinary Calcium Oxalate Crystals in Cultured Renal Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Grover, Phulwinder K.; Thurgood, Lauren A.; Fleming, David E.; van Bronswijk, Wilhelm; Ryall, Rosemary L.

    2007-04-01

    We have proposed that internalized calcium oxalate (CaOx) crystals containing intracrystalline proteins would be vulnerable to intracellular dissolution. The aims of this study were (1) to measure non-uniform strain and crystallite size in CaOx monohydrate (COM) crystals containing increasing amounts of intracrystalline crystal matrix extract (CME) and (2) to compare the rates of crystal dissolution in Madin-Darby canine kidney (MDCKII) cells. CME was isolated by demineralization of COM crystals generated from human urine. Cold and 14C-oxalate-labelled COM crystals were precipitated from ultrafiltered urine containing CME at final concentrations of 0-5mg/L. Non-uniform strain and crystallite size were determined using synchrotron X-ray diffraction with Rietveld whole-pattern peak fitting and profile analysis, and the protein content of the crystals was analyzed using SDS-PAGE and Western blotting for prothrombin fragment 1. Radiolabeled crystals were added to MDCKII cells and dissolution was expressed as radioactive label released into the medium relative to that in the crystals at zero time. Non-uniform strain increased and crystallite size decreased proportionally with rising CME concentration, reaching saturation between approximately 1 and 5 mg/L, and demonstrating unequivocally the inclusion of increasing quantities of proteins in the crystals. This was confirmed by SDS-PAGE and Western blotting. Crystal dissolution also followed saturation kinetics. These findings were confirmed by field emission scanning electron microscopy (FESEM), which showed that the degree of crystal degradation increased relative to CME concentration. We conclude that intracrystalline proteins enhance intracellular dissolution of CaOx crystals and thus may provide a natural defense against stone pathogenesis.

  12. Towards protein-crystal centering using second-harmonic generation (SHG) microscopy

    PubMed Central

    Kissick, David J.; Dettmar, Christopher M.; Becker, Michael; Mulichak, Anne M.; Cherezov, Vadim; Ginell, Stephan L.; Battaile, Kevin P.; Keefe, Lisa J.; Fischetti, Robert F.; Simpson, Garth J.

    2013-01-01

    The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-­ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-­ray minibeam. SHG imaging was found to provide about 2 µm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed. PMID:23633594

  13. Crystallization and evaluation of hen egg-white lysozyme crystals for protein pH titration in the crystalline state.

    PubMed

    Iwai, Wakari; Yagi, Daichi; Ishikawa, Takuya; Ohnishi, Yuki; Tanaka, Ichiro; Niimura, Nobuo

    2008-05-01

    To observe the ionized status of the amino acid residues in proteins at different pH (protein pH titration in the crystalline state) by neutron diffraction, hen egg-white lysozyme was crystallized over a wide pH range (2.5-8.0). Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined. At pH < 4.5 the border between the metastable region and the nucleation region shifted to the left (lower precipitant concentration) in the phase diagram, and at pH > 4.5 the border shifted to the right (higher precipitant concentration). The qualities of these crystals were characterized using the Wilson plot method. The qualities of all crystals at different pH were more or less equivalent (B-factor values within 25-40). It is expected that neutron diffraction analysis of these crystals of different pH provides equivalent data in quality for discussions of protein pH titration in the crystalline state of hen egg-white lysozyme.

  14. Advances in the development of piezoelectric quartz-crystal oscillators, hydrogen masers, and superconducting frequency standards

    NASA Technical Reports Server (NTRS)

    Suter, Joseph J.

    1988-01-01

    This paper describes recent research advances made in the development of radiation-hardened piezoelectric quartz oscillators, hydrogen masers, and superconducting oscillators, with emphasis placed on the principles involved in the operation of these oscillators and the factors affecting the operation. Particular attention is given to the radiation-susceptibility studies of quartz-crystal resonators, the hydrogen-maser relaxation process and noise sources, and low-phase-noise superconducting oscillators. Diagrams of these devices and performance graphs are included.

  15. Membrane protein crystallization: observations and use of short chain phospholipids as amphiphiles

    NASA Astrophysics Data System (ADS)

    Eiselé, Jean-Luc; Keller, Thomas A.; König, Nicola; Stauffer, Kathrin A.; Rosenbusch, Jürg P.; Low, Philip S.

    1991-03-01

    An integral membrane protein, porin OmpF located in E. coli outer membrane, has been crystallized in the sole presence of short chain phospholipids. Although the amphiphilic characteristics of these molecules are comparable to those of conventional detergents, they appear to be unique probes for the study of the phospholipid-protein interactions by crystallographic methods. Here we also discuss the influence of the detergent head group on the crystal packing, and the role of additives. The stabilization of crystals after termination of growth in protein-free buffer is described.

  16. Life in the fast lane for protein crystallization and X-ray crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

    2005-01-01

    The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high-rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today's high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from

  17. Life in the Fast Lane for Protein Crystallization and X-Ray Crystallography

    NASA Technical Reports Server (NTRS)

    Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

    2004-01-01

    The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today s high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from

  18. Life in the fast lane for protein crystallization and X-ray crystallography

    SciTech Connect

    Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

    2010-07-20

    The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high-rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain 'low-hanging fruit' protein structures. We review the practical aspects of today's high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from

  19. Can radiation damage to protein crystals be reduced using small-molecule compounds?

    SciTech Connect

    Kmetko, Jan; Warkentin, Matthew; Englich, Ulrich; Thorne, Robert E.

    2011-10-01

    Free-radical scavengers that are known to be effective protectors of proteins in solution are found to increase global radiation damage to protein crystals. Protective mechanisms may become deleterious in the protein-dense environment of a crystal. Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.

  20. Systematic analysis of protein-detergent complexes applying dynamic light scattering to optimize solutions for crystallization trials.

    PubMed

    Meyer, Arne; Dierks, Karsten; Hussein, Rana; Brillet, Karl; Brognaro, Hevila; Betzel, Christian

    2015-01-01

    Detergents are widely used for the isolation and solubilization of membrane proteins to support crystallization and structure determination. Detergents are amphiphilic molecules that form micelles once the characteristic critical micelle concentration (CMC) is achieved and can solubilize membrane proteins by the formation of micelles around them. The results are presented of a study of micelle formation observed by in situ dynamic light-scattering (DLS) analyses performed on selected detergent solutions using a newly designed advanced hardware device. DLS was initially applied in situ to detergent samples with a total volume of approximately 2 µl. When measured with DLS, pure detergents show a monodisperse radial distribution in water at concentrations exceeding the CMC. A series of all-trans n-alkyl-β-D-maltopyranosides, from n-hexyl to n-tetradecyl, were used in the investigations. The results obtained verify that the application of DLS in situ is capable of distinguishing differences in the hydrodynamic radii of micelles formed by detergents differing in length by only a single CH2 group in their aliphatic tails. Subsequently, DLS was applied to investigate the distribution of hydrodynamic radii of membrane proteins and selected water-insoluble proteins in presence of detergent micelles. The results confirm that stable protein-detergent complexes were prepared for (i) bacteriorhodopsin and (ii) FetA in complex with a ligand as examples of transmembrane proteins. A fusion of maltose-binding protein and the Duck hepatitis B virus X protein was added to this investigation as an example of a non-membrane-associated protein with low water solubility. The increased solubility of this protein in the presence of detergent could be monitored, as well as the progress of proteolytic cleavage to separate the fusion partners. This study demonstrates the potential of in situ DLS to optimize solutions of protein-detergent complexes for crystallization applications.

  1. Preparation of 2D crystals of membrane proteins for high-resolution electron crystallography data collection.

    PubMed

    Abeyrathne, Priyanka D; Chami, Mohamed; Pantelic, Radosav S; Goldie, Kenneth N; Stahlberg, Henning

    2010-01-01

    Electron crystallography is a powerful technique for the structure determination of membrane proteins as well as soluble proteins. Sample preparation for 2D membrane protein crystals is a crucial step, as proteins have to be prepared for electron microscopy at close to native conditions. In this review, we discuss the factors of sample preparation that are key to elucidating the atomic structure of membrane proteins using electron crystallography.

  2. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of macrophage growth locus A (MglA) protein from Francisella tularensis

    SciTech Connect

    Subburaman, P.; Austin, B.P.; Shaw, G.X.; Waugh, D.S.; Ji, X.

    2010-11-03

    Francisella tularensis, a potential bioweapon, causes a rare infectious disease called tularemia in humans and animals. The macrophage growth locus A (MglA) protein from F. tularensis associates with RNA polymerase to positively regulate the expression of multiple virulence factors that are required for its survival and replication within macrophages. The MglA protein was overproduced in Escherichia coli, purified and crystallized. The crystals diffracted to 7.5 {angstrom} resolution at the Advanced Photon Source, Argonne National Laboratory and belonged to the hexagonal space group P6{sub 1} or P6{sub 5}, with unit-cell parameters a = b = 125, c = 54 {angstrom}.

  3. Protein crystal growth aboard the U.S. space shuttle flights STS-31 and STS-32.

    PubMed

    DeLucas, L J; Smith, C D; Carter, D C; Twigg, P; He, X M; Snyder, R S; Weber, P C; Schloss, J V; Einspahr, H M; Clancy, L L; McPherson, A; Koszelak, S; Vandonselaar, M M; Prasad, L; Quail, J W; Delbaere, L T; Bugg, C E

    1992-01-01

    The first microgravity protein crystal growth experiments were performed on Spacelab I by Littke and John. These experiments indicated that the space grown crystals, which were obtained using a liquid-liquid diffusion system, were larger than crystals obtained by the same experimental system on earth. Subsequent experiments were performed by other investigators on a series of space shuttle missions from 1985 through 1990. The results from two of these shuttle flights (STS-26 and STS-29) have been described previously. The results from these missions indicated that the microgravity grown crystals for a number of different proteins were larger, displayed more uniform morphologies, and yielded diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth. This paper presents the results obtained from shuttle flight STS-32 (flown in January, 1990) and preliminary results from the most recent shuttle flight, STS-31 (flown in April, 1990).

  4. PCAM: a multi-user facility-based protein crystallization apparatus for microgravity

    NASA Astrophysics Data System (ADS)

    Carter, Daniel C.; Wright, Brenda; Miller, Teresa; Chapman, Jenny; Twigg, Pam; Keeling, Kim; Moody, Kerry; White, Melissa; Click, James; Ruble, John R.; Ho, Joseph X.; Adcock-Downey, Lawana; Dowling, Tim; Chang, Chong-Hwan; Ala, Paul; Rose, John; Wang, B. C.; Declercq, Jean-Paul; Evrard, Christine; Rosenberg, John; Wery, Jean-Pierre; Clawson, David; Wardell, Mark; Stallings, W.; Stevens, A.

    1999-01-01

    A facility-based protein crystallization apparatus for microgravity (PCAM) has been constructed and flown on a series of Space Shuttle Missions. The hardware development was undertaken largely because of the many important examples of quality improvements gained from crystal growth in the diffusion-limited environment in space. The concept was based on the adaptation for microgravity of a commonly available crystallization tray to increase sample density, to facilitate co-investigator participation and to improve flight logistics and handling. A co-investigator group representing scientists from industry, academia, and government laboratories has been established. Microgravity applications of the hardware have produced improvements in a number of structure-based crystallographic studies and include examples of enabling research. Additionally, the facility has been used to support fundamental research in protein crystal growth which has delineated factors contributing to the effect of microgravity on the growth and quality of protein crystals.

  5. Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth

    PubMed Central

    Celik, Yeliz; Drori, Ran; Pertaya-Braun, Natalya; Altan, Aysun; Barton, Tyler; Bar-Dolev, Maya; Groisman, Alex; Davies, Peter L.; Braslavsky, Ido

    2013-01-01

    Antifreeze proteins (AFPs) are a subset of ice-binding proteins that control ice crystal growth. They have potential for the cryopreservation of cells, tissues, and organs, as well as for production and storage of food and protection of crops from frost. However, the detailed mechanism of action of AFPs is still unclear. Specifically, there is controversy regarding reversibility of binding of AFPs to crystal surfaces. The experimentally observed dependence of activity of AFPs on their concentration in solution appears to indicate that the binding is reversible. Here, by a series of experiments in temperature-controlled microfluidic devices, where the medium surrounding ice crystals can be exchanged, we show that the binding of hyperactive Tenebrio molitor AFP to ice crystals is practically irreversible and that surface-bound AFPs are sufficient to inhibit ice crystal growth even in solutions depleted of AFPs. These findings rule out theories of AFP activity relying on the presence of unbound protein molecules. PMID:23300286

  6. A synergistic approach to protein crystallization: Combination of a fixed-arm carrier with surface entropy reduction

    PubMed Central

    Moon, Andrea F; Mueller, Geoffrey A; Zhong, Xuejun; Pedersen, Lars C

    2010-01-01

    Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail. PMID:20196072

  7. Roles of Electrostatics and Conformation in Protein-Crystal Interactions

    PubMed Central

    Azzopardi, Paul V.; O'Young, Jason; Lajoie, Gilles; Karttunen, Mikko; Goldberg, Harvey A.; Hunter, Graeme K.

    2010-01-01

    In vitro studies have shown that the phosphoprotein osteopontin (OPN) inhibits the nucleation and growth of hydroxyapatite (HA) and other biominerals. In vivo, OPN is believed to prevent the calcification of soft tissues. However, the nature of the interaction between OPN and HA is not understood. In the computational part of the present study, we used molecular dynamics simulations to predict the adsorption of 19 peptides, each 16 amino acids long and collectively covering the entire sequence of OPN, to the {100} face of HA. This analysis showed that there is an inverse relationship between predicted strength of adsorption and peptide isoelectric point (P<0.0001). Analysis of the OPN sequence by PONDR (Predictor of Naturally Disordered Regions) indicated that OPN sequences predicted to adsorb well to HA are highly disordered. In the experimental part of the study, we synthesized phosphorylated and non-phosphorylated peptides corresponding to OPN sequences 65–80 (pSHDHMDDDDDDDDDGD) and 220–235 (pSHEpSTEQSDAIDpSAEK). In agreement with the PONDR analysis, these were shown by circular dichroism spectroscopy to be largely disordered. A constant-composition/seeded growth assay was used to assess the HA-inhibiting potencies of the synthetic peptides. The phosphorylated versions of OPN65-80 (IC50 = 1.93 µg/ml) and OPN220-235 (IC50 = 1.48 µg/ml) are potent inhibitors of HA growth, as is the nonphosphorylated version of OPN65-80 (IC50 = 2.97 µg/ml); the nonphosphorylated version of OPN220-235 has no measurable inhibitory activity. These findings suggest that the adsorption of acidic proteins to Ca2+-rich crystal faces of biominerals is governed by electrostatics and is facilitated by conformational flexibility of the polypeptide chain. PMID:20174473

  8. How proteins pack into crystals: nuclei achieve translation symmetries by growing.

    PubMed

    Feng, Dan; Zeng, Zong-Hao

    2004-07-01

    How protein molecules pack into a crystal remains problematic. Packing units are direct materials for packing into crystals. The group generator method is introduced for automatically identifying the packing unit. By introducing deviations into the nucleation stage of crystallization, we proved that these deviations diminish in further packing. This process illustrates how translation symmetries are achieved by the growing of nuclei. Two effects, the size effect and the close up effect, are found to behave differently in this process.

  9. Towards protein-crystal centering using second-harmonic generation (SHG) microscopy

    SciTech Connect

    Kissick, David J.; Dettmar, Christopher M.; Becker, Michael; Mulichak, Anne M.; Cherezov, Vadim; Ginell, Stephan L.; Battaile, Kevin P.; Keefe, Lisa J.; Fischetti, Robert F.; Simpson, Garth J.

    2013-05-01

    The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals has been explored. The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β{sub 2} adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam. SHG imaging was found to provide about 2 µm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed.

  10. Enhancement of crystal homogeneity of protein crystals under application of an external alternating current electric field

    SciTech Connect

    Koizumi, H.; Uda, S.; Fujiwara, K.; Nozawa, J.; Tachibana, M.; Kojima, K.

    2014-10-06

    X-ray diffraction rocking-curve measurements were performed on tetragonal hen egg white (HEW) lysozyme crystals grown with and without the application of an external alternating current (AC) electric field. The crystal quality was assessed by the full width at half maximum (FWHM) value for each rocking curve. For two-dimensional maps of the FWHMs measured on the 440 and the 12 12 0 reflection, the crystal homogeneity was improved under application of an external electric field at 1 MHz, compared with that without. In particular, the significant improvement of the crystal homogeneity was observed for the 12 12 0 reflection.

  11. Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization.

    SciTech Connect

    Wallace, E.; Dranow, D.; Laible, P. D.; Christensen, J.; Nollert, P.

    2011-01-01

    The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization

  12. Utilisation of adsorption and desorption for simultaneously improving protein crystallisation success rate and crystal quality

    PubMed Central

    Guo, Yun-Zhu; Sun, Li-Hua; Oberthuer, Dominik; Zhang, Chen-Yan; Shi, Jian-Yu; Di, Jiang-Lei; Zhang, Bao-Liang; Cao, Hui-Ling; Liu, Yong-Ming; Li, Jian; Wang, Qian; Huang, Huan-Huan; Liu, Jun; Schulz, Jan-Mirco; Zhang, Qiu-Yu; Betzel, Christian; He, Jian-Hua; Yin, Da-Chuan

    2014-01-01

    High-quality protein crystals of suitable size are an important prerequisite for applying X-ray crystallography to determine the 3-dimensional structure of proteins. However, it is often difficult to obtain protein crystals of appropriate size and quality because nucleation and growth processes can be unsuccessful. Here, we show that by adsorbing proteins onto porous polystyrene-divinylbenzene microspheres (SDB) floating on the surface of the crystallisation solution, a localised high supersaturation region at the surface of the microspheres and a low supersaturation region below the microspheres can coexist in a single solution. The crystals will easily nucleate in the region of high supersaturation, but when they grow to a certain size, they will sediment to the region of low supersaturation and continue to grow. In this way, the probability of crystallisation and crystal quality can be simultaneously increased in a single solution without changing other crystallisation parameters. PMID:25471817

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

    NASA Technical Reports Server (NTRS)

    Vekilov, Peter G.; Witherow, W. (Technical Monitor)

    2003-01-01

    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.

  14. Protein crystal quality in diffusive environments and its evaluation

    NASA Astrophysics Data System (ADS)

    Lopez-Jaramillo, F. J.; Otálora, F.; Gavira, J. A.

    2003-01-01

    We have analyzed the crystal quality along a capillary by a precise protocol that comprises the study of tetragonal lysozyme cylindrical crystals that fill the capillary diameter (i.e. rods), the careful definition of the diffraction parameters and the use of a single software for the data reduction in order to avoid any bias in the comparison of the quality of different data sets. Our results cannot be explained on the basis of the different redundancy of the data sets and they demonstrate that the gel acupuncture method promotes a gradient of supersaturation along the capillary that yields in the same experiment crystals of increasing quality as a function of the position. However, despite being single crystals, rods have regions that show different crystal quality because they grew at different supersaturations. Our data are in agreement with the existence of a relation between length of the c-axis and crystal quality reported by other groups, but a deeper analysis of the cell parameters reveals the existence of a significant linear relation ( R=0.87) with the c/ a-axis ratio. This result points to the hypothesis of an ideal unit cell that yields the best crystals in terms of I/ σ( I).

  15. Advances in the growth of alkaline-earth halide single crystals for scintillator detectors

    SciTech Connect

    Boatner, Lynn A; Ramey, Joanne Oxendine; Kolopus, James A; Neal, John S; Cherepy, Nerine; Payne, Stephen A.; Beck, P; Burger, Arnold; Rowe, E; Bhattacharya, P.

    2014-01-01

    Alkaline-earth scintillators such as strontium iodide and other alkaline-earth halides activated with divalent europium represent some of the most efficient and highest energy resolution scintillators for use as gamma-ray detectors in a wide range of applications. These applications include the areas of nuclear nonproliferation, homeland security, the detection of undeclared nuclear material, nuclear physics and materials science, medical diagnostics, space physics, high energy physics, and radiation monitoring systems for first responders, police, and fire/rescue personnel. Recent advances in the growth of large single crystals of these scintillator materials hold the promise of higher crystal yields and significantly lower detector production costs. In the present work, we describe new processing protocols that, when combined with our molten salt filtration methods, have led to advances in achieving a significant reduction of cracking effects during the growth of single crystals of SrI2:Eu2+. In particular, we have found that extended pumping on the molten crystal-growth charge under vacuum for time periods extending up to 48 hours is generally beneficial in compensating for variations in the alkaline-earth halide purity and stoichiometry of the materials as initially supplied by commercial sources. These melt-pumping and processing techniques are now being applied to the purification of CaI2:Eu2+ and some mixed-anion europium-doped alkaline-earth halides prior to single-crystal growth by means of the vertical Bridgman technique. The results of initial studies of the effects of aliovalent doping of SrI2:Eu2+ on the scintillation characteristics of this material are also described.

  16. Crystallization Process of Protein Rv0731c from Mycobacterium Tuberculosis for a Successful Atomic Resolution Crystal Structure at 1.2 Angstrom

    SciTech Connect

    Zhu, Liang Cong

    2009-06-08

    Proteins are bio-macromolecules consisting of basic 20 amino acids and have distinct three-dimensional folds. They are essential parts of organisms and participate in every process within cells. Proteins are crucial for human life, and each protein within the body has a specific function, such as antibodies, contractile proteins, enzymes, hormonal proteins, structural proteins, storage proteins and transport proteins. Determining three-dimensional structure of a protein can help researchers discover the remarkable protein folding, binding site, conformation and etc, in order to understand well of protein interaction and aid for possible drug design. The research on protein structure by X-ray protein crystallography carried by Li-Wei Hung's research group in the Physical Bioscience Division at Lawrence Berkeley National Laboratory (LBNL) is focusing on protein crystallography. The research in this lab is in the process of from crystallizing the proteins to determining the three dimensional crystal structures of proteins. Most protein targets are selected from Mycobacterium Tuberculosis. TB (Tuberculosis) is a possible fatal infectious disease. By studying TB target protein can help discover antituberculer drugs, and find treatment for TB. The high-throughput mode of crystallization, crystal harvesting, crystal screening and data collection are applied to the research pipeline (Figure 1). The X-ray diffraction data by protein crystals can be processed and analyzed to result in a three dimensional representation of electron density, producing a detailed model of protein structure. Rv0731c is a conserved hypothetical protein with unknown function from Mycobacterium Tuberculosis. This paper is going to report the crystallization process and brief structure information of Rv0731c.

  17. Study of Fluid Flow Control in Protein Crystallization using Strong Magnetic Fields

    NASA Technical Reports Server (NTRS)

    Ramachandran, Narayanan; Leslie, Fred; Ciszak, Ewa

    2002-01-01

    An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in "microgravity", researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately

  18. Evaporative microdialysis: an effective improvement in an established method of protein crystallization.

    PubMed

    Bunick, C; North, A C; Stubbs, G

    2000-11-01

    Evaporative dialysis is a simple variant of conventional microdialysis in which the reservoir solution is allowed to evaporate slowly. The slow increase in precipitant concentration allows crystals to grow without increasing nucleation. The method is useful for proteins that have a very narrow metastable zone (the range of solution conditions under which crystals grow but nuclei do not form at an appreciable rate). The method is demonstrated with the coat protein of potato virus X.

  19. Crystallization and preliminary X-ray diffraction analysis of Val57 mutants of the amyloidogenic protein human cystatin C

    SciTech Connect

    Orlikowska, Marta; Jankowska, Elzbieta; Borek, Dominika; Otwinowski, Zbyszek; Skowron, Piotr; Szymanska, Aneta

    2012-03-15

    Human cystatin C (hCC) is a low-molecular-mass protein (120 amino-acid residues, 13 343 Da) found in all nucleated cells. Its main physiological role is regulation of the activity of cysteine proteases. Biologically active hCC is a monomeric protein, but all crystallization efforts have resulted in a dimeric domain-swapped structure. Recently, two monomeric structures were reported for cystatin C variants. In one of them stabilization was achieved by abolishing the possibility of domain swapping by the introduction of an additional disulfide bridge connecting the two protein domains (Cys47-Cys69). In the second structure, reported by this group, the monomeric hCC fold was preserved by stabilization of the conformationally constrained loop (L1) by a single-amino-acid substitution (V57N). To further assess the influence of changes in the sequence and properties of loop L1 on the dimerization propensity of cystatin C, two additional hCC mutants were obtained: one with a residue favoured in {beta}-turns (V57D) and another with proline (V57P), a residue that is known to be a structural element that can rigidify but also broaden turns. Here, the expression, purification and crystallization of V57D and V57P variants of recombinant human cystatin C are described. Crystals were grown by the vapour-diffusion method. Several diffraction data sets were collected using a synchrotron source at the Advanced Photon Source, Argonne National Laboratory, Chicago, USA.

  20. Development of high-performance X-ray transparent crystallization plates for in situ protein crystal screening and analysis

    PubMed Central

    Soliman, Ahmed S. M.; Warkentin, Matthew; Apker, Benjamin; Thorne, Robert E.

    2011-01-01

    X-ray transparent crystallization plates based upon a novel drop-pinning technology provide a flexible, simple and inexpensive approach to protein crystallization and screening. The plates consist of open cells sealed top and bottom by thin optically, UV and X-ray transparent films. The plates do not need wells or depressions to contain liquids. Instead, protein drops and reservoir solution are held in place by rings with micrometre dimensions that are patterned onto the bottom film. These rings strongly pin the liquid contact lines, thereby improving drop shape and position uniformity, and thus crystallization reproducibility, and simplifying automated image analysis of drop contents. The same rings effectively pin solutions containing salts, proteins, cryoprotectants, oils, alcohols and detergents. Strong pinning by rings allows the plates to be rotated without liquid mixing to 90° for X-ray data collection or to be inverted for hanging-drop crystallization. The plates have the standard SBS format and are compatible with standard liquid-handling robots. PMID:21697603

  1. Crystallization of proteins by dynamic control of evaporation

    NASA Astrophysics Data System (ADS)

    Wilson, L. G.; Bray, T. L.; Suddath, F. L.

    1991-03-01

    It is expected that the kinetics of supersaturation, which is directly related to the evaporation of solvent from a crystallization solution, will greatly affect both nucleation and crystal growth processes. Therefore, a novel device has been developed which allows computer regulation of the flow of N 2(g) over a hanging drop to dynamically control the evaporation of solvent. A thermal conductivity detector is used to monitor the amount of water vapor transferred from the drop to the gas stream and provides closed loop control of the evaporation process. Data acquisition and control are accomplished using a custom program written with LabVIEW software (National Instruments) on a Macintosh II microcomputer. Quantitation of several evaporation protocols has been accomplished using both the thermal conductivity detector and a novel conductance cell that allows continuous measurement of solution analyte concentrations. Crystals of hen egg white lysozyme have been grown at different evaporation rates and analyzed according to size and number of single crystals.

  2. Step and Kink Dynamics in Inorganic and Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, A. A.; Rashkovich, L. N.; Vekilov, P. G.; DeYoreo, J. J.

    2004-01-01

    Behavior of low-kink-density steps in solution growth and consequences for general understanding of spiral crystal growth processes will be overviewed. Also, influence of turbulence on step bunching and possibility to diminish this bunching will be presented.

  3. Michelson interferometric studies of protein and virus crystal growth

    NASA Astrophysics Data System (ADS)

    Kuznetsov, Yu. G.; Malkin, A. J.; Greenwood, A.; McPherson, A.

    1996-09-01

    In situ laser Michelson interferometry was utilized to investigate the growth kinetics and surface morphology in canavalin, thaumatin, and turnip yellow mosaic virus (TYMV) crystallization. Interferometric patterns and kinetic measurements from growing macromolecular crystals as small as 20 μm were obtained. This study shows that for the crystallization of canavalin, dislocations are the sources of growth steps on the surfaces of growing crystals. Supersaturation dependencies of the normal growth rates, tangential growth step velocities, and the slopes of the dislocation hillocks were determined. The kinetic coefficient β was estimated for canavalin grown from two different precipitant systems to be 3.2 × 10 -4 and 5.3 × 10 -4 cm s -1, respectively. The change in activities of dislocation sources under different growth conditions was analyzed.

  4. Crystal structure of prokaryotic ribosomal protein L9: a bi-lobed RNA-binding protein.

    PubMed Central

    Hoffman, D W; Davies, C; Gerchman, S E; Kycia, J H; Porter, S J; White, S W; Ramakrishnan, V

    1994-01-01

    The crystal structure of protein L9 from the Bacillus stearothermophilus ribosome has been determined at 2.8 A resolution using X-ray diffraction methods. This primary RNA-binding protein has a highly elongated and unusual structure consisting of two separated domains joined by a long exposed alpha-helix. Conserved, positively charged and aromatic amino acids on the surfaces of both domains probably represent the sites of specific interactions with 23S rRNA. Comparisons with other prokaryotic L9 sequences show that while the length of the connecting alpha-helix is invariant, the sequence within the exposed central region is not conserved. This suggests that the alpha-helix has an architectural role and serves to fix the relative separation and orientation of the N- and C-terminal domains within the ribosome. The N-terminal domain has structural homology to the smaller ribosomal proteins L7/L12 and L30, and the eukaryotic RNA recognition motif (RRM). Images PMID:8306963

  5. Large scale crystallization of protein pharmaceuticals in microgravity via temperature change

    NASA Technical Reports Server (NTRS)

    Long, Marianna M.

    1992-01-01

    The major objective of this research effort is the temperature driven growth of protein crystals in large batches in the microgravity environment of space. Pharmaceutical houses are developing protein products for patient care, for example, human insulin, human growth hormone, interferons, and tissue plasminogen activator or TPA, the clot buster for heart attack victims. Except for insulin, these are very high value products; they are extremely potent in small quantities and have a great value per gram of material. It is feasible that microgravity crystallization can be a cost recoverable, economically sound final processing step in their manufacture. Large scale protein crystal growth in microgravity has significant advantages from the basic science and the applied science standpoints. Crystal growth can proceed unhindered due to lack of surface effects. Dynamic control is possible and relatively easy. The method has the potential to yield large quantities of pure crystalline product. Crystallization is a time honored procedure for purifying organic materials and microgravity crystallization could be the final step to remove trace impurities from high value protein pharmaceuticals. In addition, microgravity grown crystals could be the final formulation for those medicines that need to be administered in a timed release fashion. Long lasting insulin, insulin lente, is such a product. Also crystalline protein pharmaceuticals are more stable for long-term storage. Temperature, as the initiation step, has certain advantages. Again, dynamic control of the crystallization process is possible and easy. A temperature step is non-invasive and is the most subtle way to control protein solubility and therefore crystallization. Seeding is not necessary. Changes in protein and precipitant concentrations and pH are not necessary. Finally, this method represents a new way to crystallize proteins in space that takes advantage of the unique microgravity environment. The results

  6. Design of a confined environment using protein cages and crystals for the development of biohybrid materials.

    PubMed

    Abe, Satoshi; Maity, Basudev; Ueno, Takafumi

    2016-05-01

    There is growing interest in the design of protein assemblies for use in materials science and bionanotechnology. Protein assemblies, such as cages and crystalline protein structures, provide confined chemical environments that allow immobilization of metal complexes, nanomaterials, and proteins by metal coordination, assembly/disassembly reactions, genetic manipulation and crystallization methods. Protein assembly composites can be used to prepare hybrid materials with catalytic, magnetic and optical properties for cellular applications due to their high stability, solubility and biocompatibility. In this feature article, we focus on the recent development of ferritin as the most promising molecular template protein cage and in vivo and in vitro engineering of protein crystals as solid protein materials with functional properties.

  7. The good, the bad and the twisted: a survey of ligand geometry in protein crystal structures.

    PubMed

    Liebeschuetz, John; Hennemann, Jana; Olsson, Tjelvar; Groom, Colin R

    2012-02-01

    The protein databank now contains the structures of over 11,000 ligands bound to proteins. These structures are invaluable in applied areas such as structure-based drug design, but are also the substrate for understanding the energetics of intermolecular interactions with proteins. Despite their obvious importance, the careful analysis of ligands bound to protein structures lags behind the analysis of the protein structures themselves. We present an analysis of the geometry of ligands bound to proteins and highlight the role of small molecule crystal structures in enabling molecular modellers to critically evaluate a ligand model's quality and investigate protein-induced strain. PMID:22246295

  8. Protein crystal growth aboard the U.S. Space Shuttle flights STS-31 and STS-32

    NASA Technical Reports Server (NTRS)

    Delucas, Lawrence J.; Smith, Craig D.; Carter, Daniel C.; Twigg, Pam; He, Xiao-Min; Snyder, Robert S.; Weber, Patricia C.; Schloss, J. V.; Einspahr, H. M.; Clancy, L. L.

    1992-01-01

    Results obtained from the Shuttle flight STS-32 flown in January 1990, and preliminary results from the most recent Shuttle flight, STS-31, flown in April 1990, are presented. Crystals grown in microgravity environment include Canavalin, isocitrate lyase, human serum albumin, and Anti-HPr Fab. It is concluded that about 20 percent of proteins flown exhibit better morphologies or better quality data than their earth-grown counterparts. About 40 percent do not yield crystals at all and the remaining 40 percent yield crystals that are either too small for X-ray analysis or produce data of poorer quality than the best earth-grown crystals.

  9. Protein crystallization aboard the Space Shuttle and the Mir space station

    NASA Technical Reports Server (NTRS)

    Delbaere, Louis T. J.; Vandonselaar, Margaret; Prasad, Lata; Quail, J. W.; Birnbaum, George I.; Delucas, Lawrence J.; Moore, Karen; Bugg, Charles E.

    1993-01-01

    Two different protein crystallizations, namely ,the free Fab fragment of the Je142 monoclonal antibody and the complex of Fab fragment/HPr with antigen, were performed aboard the Discovery Space Shuttle flights and the Mir space station, respectively. Medium sized crystals of the Je142 Fab fragment were obtained. The Je142 Fab fragment/Hpr complex produced two medium-sized crystals after two months aboard the Mir space station. Microgravity was found to eliminate the tendency of these crystals to form clusters.

  10. Development of a technology for automation and miniaturization of protein crystallization.

    PubMed

    Mueller, U; Nyarsik, L; Horn, M; Rauth, H; Przewieslik, T; Saenger, W; Lehrach, H; Eickhoff, H

    2001-01-23

    The usage of standard 96 well microplates for the screening of crystallization conditions of recombinant proteins offers several advantages when compared to commonly used crystallization plate formats. The adoption of robotic technology for plate and glass slide preparation within a "hanging drop" vapour diffusion crystallization experiment enables to work with an increased throughput at reduced costs. In addition to commercial pipetting devices with a 96-channel aspirator/dispenser, solenoid ink-jet technology was applied to form 250 nl droplets with a diameter of approximately 1 mm. This allows miniaturization of crystallization screening set-ups with an estimated ten-fold cost reduction when compared to commonly used 24 well plates.

  11. Liposomes on a streptavidin crystal: a system to study membrane proteins by cryo-EM

    PubMed Central

    Wang, Liguo; Sigworth, Fred J.

    2013-01-01

    In this chapter we describe the preparation of cryo-EM specimens for random spherically-constrained (RSC) single-particle reconstruction of membrane proteins. The specimen consists of liposomes into which the purified membrane protein is reconstituted at low density. The substrate is a 2D streptavidin crystal, which serves as an affinity surface that tethers the liposomes, which are doped with biotinylated lipids; the crystal also can serve as an image-quality and image-calibration reference. After subtraction of the crystal and lipid membrane contributions to the image, the remaining particle images can be used for 3D reconstruction. PMID:20887857

  12. Purification, crystallization and preliminary X-ray diffraction analysis of the plant Rho protein ROP5

    SciTech Connect

    Thomas, Christoph Berken, Antje

    2007-12-01

    Crystals of the plant Rho protein ROP5 from A. thaliana have been obtained that diffract to 1.53 Å resolution. The small G protein ROP5 from the model plant Arabidopsis thaliana was purified and crystallized using the hanging-drop vapour-diffusion method. ROP5 crystals were obtained using PEG 3000 as precipitant and belong to space group P2{sub 1}. A data set was collected to 1.53 Å resolution using synchrotron radiation at 100 K. A clear molecular-replacement solution was found using ROP4–GDP of the ROP4–GDP–PRONE8 complex as the search model.

  13. Crystallization of DIR1, a LTP2-like resistance signalling protein from Arabidopsis thaliana

    SciTech Connect

    Lascombe, Marie-Bernard; Buhot, Nathalie; Bakan, Bénédicte; Marion, Didier; Blein, Jean Pierre; Lamb, Chris J.; Prangé, Thierry

    2006-07-01

    DIR1, a putative LTP2 protein from Arabidopsis thaliana implicated in systemic acquired resistance in planta, has been crystallized in space group P2{sub 1}2{sub 1}2{sub 1} with one molecule per asymmetric unit. DIR1, a putative LTP2 protein from Arabidopsis thaliana implicated in systemic acquired resistance in planta, has been crystallized in space group P2{sub 1}2{sub 1}2{sub 1} with one molecule per asymmetric unit. The crystals diffract to a resolution of 1.6 Å.

  14. Study of Fluid Flow Control In Protein Crystallization Using Strong Magnetic Fields

    NASA Technical Reports Server (NTRS)

    Ramachandran, N.; Leslie, F.; Ciszak, E.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in 'microgravity', researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately

  15. Approach for growth of high-quality and large protein crystals.

    PubMed

    Matsumura, Hiroyoshi; Sugiyama, Shigeru; Hirose, Mika; Kakinouchi, Keisuke; Maruyama, Mihoko; Murai, Ryota; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Mori, Yusuke; Inoue, Tsuyoshi

    2011-01-01

    Three crystallization methods for growing large high-quality protein crystals, i.e. crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study the effectiveness of crystallization in the presence of a semi-solid agarose gel has been further evaluated by crystallizing additional proteins in the presence of 2.0% (w/v) agarose gel, resulting in complete gelification with high mechanical strength. In TSSG the seed crystals are hung by a seed holder protruding from the top of the growth vessel to prevent polycrystallization. In the large-scale hanging-drop method, a cut pipette tip was used to maintain large-scale droplets consisting of protein-precipitant solution. Here a novel crystallization method that combines TSSG and the large-scale hanging-drop method is reported. A large and single crystal of lysozyme was obtained by this method.

  16. Stability of Magnetically-Suppressed Solutal Convection In Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    Leslie, F. W.; Ramachandran, N.

    2005-01-01

    The effect of convection during the crystallization of proteins is not very well understood. In a gravitational field, convection is caused by crystal sedimentation and by solutal buoyancy induced flow and these can lead to crystal imperfections. While crystallization in microgravity can approach diffusion limited growth conditions (no convection), terrestrially strong magnetic fields can be used to control fluid flow and sedimentation effects. In this work, a theory is presented on the stability of solutal convection of a magnetized fluid in the presence of a magnetic field. The requirements for stability are developed and compared to experiments performed within the bore of a superconducting magnet. The theoretical predictions are in good agreement with the experiments and show solutal convection can be stabilized if the surrounding fluid has larger magnetic susceptibility and the magnetic field has a specific structure. Discussion on the application of the technique to protein crystallization is also provided.

  17. Protein crystallization in a 100 nl solution with new stirring equipment

    PubMed Central

    Maki, S.; Murai, R.; Yoshikawa, H. Y.; Kitatani, T.; Nakata, S.; Kawahara, H.; Hasenaka, H.; Kobayashi, A.; Okada, S.; Sugiyama, S.; Adachi, H.; Matsumura, H.; Takano, K.; Murakami, S.; Inoue, T.; Sasaki, T.; Mori, Y.

    2008-01-01

    To investigate quantitatively the effects of stirring on protein crystallization, a new stirring system which can agitate a protein solution, ∼100 nl, by providing Hagen–Poiseuille flow has been successfully developed. In addition, this new stirring system provides flow with a well defined pattern and velocity. Using this system, hen egg-white lysozyme was crystallized in 100–200 nl solutions while being stirred. The optimum stirring conditions for lysozyme crystals have been explored by evaluating the Reynolds (Re) number and the crystals obtained. Intermittent flow, as well as a low Re number, was found to contribute significantly to the growth of a smaller number of larger crystals. PMID:18421156

  18. Intercalating dyes for enhanced contrast in second-harmonic generation imaging of protein crystals.

    PubMed

    Newman, Justin A; Scarborough, Nicole M; Pogranichniy, Nicholas R; Shrestha, Rashmi K; Closser, Richard G; Das, Chittaranjan; Simpson, Garth J

    2015-07-01

    The second-harmonic generation (SHG) activity of protein crystals was found to be enhanced by up to ∼1000-fold by the intercalation of SHG phores within the crystal lattice. Unlike the intercalation of fluorophores, the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates. The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice. In addition, the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response. Kinetics studies indicate that intercalation arises over a timeframe of several minutes in lysozyme, with detectable enhancements within seconds. These results provide a potential means to increase the overall diversity of protein crystals and crystal sizes amenable to characterization by SHG microscopy. PMID:26143918

  19. Intercalating dyes for enhanced contrast in second-harmonic generation imaging of protein crystals

    PubMed Central

    Newman, Justin A.; Scarborough, Nicole M.; Pogranichniy, Nicholas R.; Shrestha, Rashmi K.; Closser, Richard G.; Das, Chittaranjan; Simpson, Garth J.

    2015-01-01

    The second-harmonic generation (SHG) activity of protein crystals was found to be enhanced by up to ∼1000-fold by the intercalation of SHG phores within the crystal lattice. Unlike the intercalation of fluorophores, the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates. The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice. In addition, the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response. Kinetics studies indicate that intercalation arises over a timeframe of several minutes in lysozyme, with detectable enhancements within seconds. These results provide a potential means to increase the overall diversity of protein crystals and crystal sizes amenable to characterization by SHG microscopy. PMID:26143918

  20. In Vitro Calcite Crystal Morphology Is Modulated by Otoconial Proteins Otolin-1 and Otoconin-90

    PubMed Central

    Moreland, K. Trent; Hong, Mina; Lu, Wenfu; Rowley, Christopher W.; Ornitz, David M.; De Yoreo, James J.; Thalmann, Ruediger

    2014-01-01

    Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia. PMID:24748133

  1. Crystallization of Ranasmurfin, a blue-coloured protein from Polypedates leucomystax

    SciTech Connect

    McMahon, Stephen A.; Walsh, Martin A.; Ching, Rosalind Tan Yan; Carter, Lester G.; Dorward, Mark; Johnson, Kenneth A.; Liu, Huanting; Oke, Muse; Bloch, Carlos Jr; Kennedy, Malcolm W.; Latiff, Aishah A.; Cooper, Alan; Taylor, Garry L.; White, Malcolm F.; Naismith, James H.

    2006-11-01

    A novel blue protein from frog nests has been crystallized. Ranasmurfin, a previously uncharacterized ∼13 kDa blue protein found in the nests of the frog Polypedates leucomystax, has been purified and crystallized. The crystals are an intense blue colour and diffract to 1.51 Å with P2{sub 1} symmetry and unit-cell parameters a = 40.9, b = 59.9, c = 45.0 Å, β = 93.3°. Self-rotation function analysis indicates the presence of a dimer in the asymmetric unit. Biochemical data suggest that the blue colour of the protein is related to dimer formation. Sequence data for the protein are incomplete, but thus far have identified no model for molecular replacement. A fluorescence scan shows a peak at 9.676 keV, indicating that the protein binds zinc and suggesting a route for structure solution.

  2. The Effect of Ionic Liquids on Protein Crystallization and X-ray Diffraction Resolution

    SciTech Connect

    Judge, Russell A.; Takahashi, Sumiko; Longenecker, Kenton L.; Fry, Elizabeth H.; Abad-Zapatero, Cele; Chiu, Mark L.

    2009-09-08

    Ionic liquids exhibit a variety of properties that make them attractive solvents for biomaterials. Given the potential for productive interaction between ionic liquids and biological macromolecules, we investigated the use of ionic liquids as precipitating agents and additives for protein crystallization for six model proteins (lysozyme, catalase, myoglobin, trypsin, glucose isomerase, and xylanase). The ionic liquids produced changes in crystal morphology and mediated significant increases in crystal size in some cases. Crystals grown using ionic liquids as precipitating agents or as additives provided X-ray diffraction resolution similar to or better than that obtained without ionic liquids. Based upon the experiments performed with model proteins, the ionic liquids were used as additives for the crystallization of the poorly diffracting monoclonal antibody 106.3 Fab in complex with the B-type natriuretic peptide (5-13). The ionic liquids improved the crystallization behavior and provided improved diffraction resulting in the determination of the structure. Ionic liquids should be considered as useful additives for the crystallization of other proteins.

  3. Flow-induced 2D protein crystallization: characterization of the coupled interfacial and bulk flows.

    PubMed

    Young, James E; Posada, David; Lopez, Juan M; Hirsa, Amir H

    2015-05-14

    Two-dimensional crystallization of the protein streptavidin, crystallizing below a biotinylated lipid film spread on a quiescent air-water interface is a well studied phenomenon. More recently, 2D crystallization induced by a shearing interfacial flow has been observed at film surface pressures significantly lower than those required in a quiescent system. Here, we quantify the interfacial and bulk flow associated with 2D protein crystallization through numerical modeling of the flow along with a Newtonian surface model. Experiments were conducted over a wide range of conditions resulting in a state diagram delineating the flow strength required to induce crystals for various surface pressures. Through measurements of the velocity profile at the air-water interface, we found that even in the cases where crystals are formed, the macroscopic flow at the interface is well described by the Newtonian model. However, the results show that even in the absence of any protein in the system, the viscous response of the biotinylated lipid film is complicated and strongly dependent on the strength of the flow. This observation suggests that the insoluble lipid film plays a key role in flow-induced 2D protein crystallization.

  4. Design rules for the self-assembly of a protein crystal

    NASA Astrophysics Data System (ADS)

    Whitelam, Stephen; Haxton, Thomas

    2014-03-01

    Theories and models of protein crystallization based on spheres that form close-packed crystals suggest that protein crystallization can be enhanced by metastable liquid-liquid criticality or demixing, and can be predicted by the osmotic second virial coefficient. However, most protein crystals are open structures, stabilized by anisotropic interactions. I will use analytic theory and computer simulations to argue that the self-assembly of open crystal lattices should not in general be best near the metastable liquid-liquid critical point or binodal (although assembly can certainly happen there), and to argue that the second virial coefficient cannot be a fully predictive measure of assembly propensity (although it is a useful starting point). Instead, the conditions that lead to best self-assembly of one particular computer model of a porous protein crystal are closer to the conditions that lead to best self-assembly of certain model viral capsids than they are to the conditions that optimize assembly of close-packed crystals. Work done at the Molecular Foundry at Lawrence Berkeley National Lab, supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

  5. Recent Advances in High-Growth Rate Single-Crystal CVD Diamond

    SciTech Connect

    Liang, Q.; Yan, C; Meng, Y; Lai, J; Krasnicki, S; Mao, H; Hemley, R

    2009-01-01

    There have been important advances in microwave plasma chemical vapor deposition (MPCVD) of large single-crystal CVD diamond at high growth rates and applications of this diamond. The types of gas chemistry and growth conditions, including microwave power, pressure, and substrate surface temperatures, have been varied to optimize diamond quality and growth rates. The diamond has been characterized by a variety of spectroscopic and diffraction techniques. We have grown single-crystal CVD diamond over ten carats and above 1 cm in thickness at growth rates of 50-100 {micro}m/h. Colorless and near colorless single crystals up to two carats have been produced by further optimizing the process. The nominal Vickers fracture toughness of this high-growth rate diamond can be tuned to exceed 20 MPa m{sup 1/2} in comparison to 5-10 MPa m{sup 1/2} for conventional natural and CVD diamond. Post-growth high-pressure/high-temperature (HPHT) and low-pressure/high-temperature (LPHT) annealing have been carried out to alter the optical, mechanical, and electronic properties. Most recently, single-crystal CVD diamond has been successfully annealed by LPHT methods without graphitization up to 2200 C and < 300 Torr for periods of time ranging from a fraction of minute to a few hours. Significant changes observed in UV, visible, infrared, and photoluminescence spectra are attributed to changes in various vacancy centers and extended defects.

  6. Interaction of Tenebrio Molitor Antifreeze Protein with Ice Crystal: Insights from Molecular Dynamics Simulations.

    PubMed

    Ramya, L; Ramakrishnan, Vigneshwar

    2016-07-01

    Antifreeze proteins (AFP) observed in cold-adapting organisms bind to ice crystals and prevent further ice growth. However, the molecular mechanism of AFP-ice binding and AFP-inhibited ice growth remains unclear. Here we report the interaction of the insect antifreeze protein (Tenebrio molitor, TmAFP) with ice crystal by molecular dynamics simulation studies. Two sets of simulations were carried out at 263 K by placing the protein near the primary prism plane (PP) and basal plane (BL) of the ice crystal. To delineate the effect of temperatures, both the PP and BL simulations were carried out at 253 K as well. The analyses revealed that the protein interacts strongly with the ice crystal in BL simulation than in PP simulation both at 263 K and 253 K. Further, it was observed that the interactions are primarily mediated through the interface waters. We also observed that as the temperature decreases, the interaction between the protein and the ice increases which can be attributed to the decreased flexibility and the increased structuring of the protein at low temperature. In essence, our study has shed light on the interaction mechanism between the TmAFP antifreeze protein and the ice crystal. PMID:27492241

  7. Modification of an apparatus for tumor-suppressor protein crystal growth in the International Space Station

    NASA Astrophysics Data System (ADS)

    de Morais Mendonca Teles, Antonio

    Some human diseases as tumors are being studied continuously for the development of vaccines against them. And a way of doing that is by means of proteins research. There are some kinds of proteins, like the p53 and p73 proteins, which are tumor suppressors. There are other diseases such as A.I.D.S., hansenosis, the Parkinson's and Chagas' diseases which are protein-related. The determination of how proteins geometrically order themselves, during its biological functions is very necessary to understand how a protein's structure affects its function, to design vaccines that intercede in tumor-protein activities and in other proteins related to those other diseases. The protein crystal growth in microgravity environment produces purer crystallization than on the ground, and it is a powerful tool to produce better vaccines. Several data have already been acquired using ground-based research and in spaceflight experiments aboard the Spacelab and Space Shuttle missions, and in the MIR and in the International Space Station (ISS). Here in this paper, I propose to be performed in the ISS Biological Research Facility (which is being developed), multiple crystal growth of proteins related to cancer (as tumors suppressors and oncoproteins), A.I.D.S., hansenosis, the Parkinson's and Chagas' diseases, for the future obtaining of possible vaccines against them. I also propose a simple and practical equipment, a modification of the crystallization plates (which use a vapor diffusion technique) inside each cylinder of the Protein Crystallization Apparatus in Microgravity (PCAM), with multiple chambers with different sizes. Instead of using some chambers with the same size it is better to use several chambers with different sizes. Why is that? The answer is: the energy associated with the surface tension of the liquid in the chamber is directly related to the circle area of it. So, to minimize the total energy of the surface tension of a proteins liquid -making it more stable

  8. Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals.

    PubMed

    Rigaud, J-L

    2002-07-01

    Reconstitution of membrane proteins into lipid bilayers is a powerful tool to analyze functional as well as structural areas of membrane protein research. First, the proper incorporation of a purified membrane protein into closed lipid vesicles, to produce proteoliposomes, allows the investigation of transport and/or catalytic properties of any membrane protein without interference by other membrane components. Second, the incorporation of a large amount of membrane proteins into lipid bilayers to grow crystals confined to two dimensions has recently opened a new way to solve their structure at high resolution using electron crystallography. However, reconstitution of membrane proteins into functional proteoliposomes or 2-D crystallization has been an empirical domain, which has been viewed for a long time more like "black magic" than science. Nevertheless, in the last ten years, important progress has been made in acquiring knowledge of lipid-protein-detergent interactions and has permitted to build upon a set of basic principles that has limited the empirical approach of reconstitution experiments. Reconstitution strategies have been improved and new strategies have been developed, facilitating the success rate of proteoliposome formation and 2-D crystallization. This review deals with the various strategies available to obtain proteoliposomes and 2-D crystals from detergent-solubilized proteins. It gives an overview of the methods that have been applied, which may be of help for reconstituting more proteins into lipid bilayers in a form suitable for functional studies at the molecular level and for high-resolution structural analysis.

  9. Crystallization and preliminary crystallographic analysis of recombinant immunoglobulin G-binding protein from Streptococcus suis

    SciTech Connect

    Khan, Abdul Hamid; Chu, Fuliang; Feng, Youjun; Zhang, Qinagmin; Qi, Jianxun; Gao, George Fu

    2008-08-01

    Crystallization of recombinant IgG-binding protein expressed in Escherichia coli using the hanging-drop vapour-diffusion method is described. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å. Streptococcus suis, an important zoonotic pathogen, expresses immunoglobulin G-binding protein, which is thought to be helpful to the organism in eluding the host defence system. Recombinant IgG-binding protein expressed in Escherichia coli has been crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å and one molecule in the asymmetric unit. Diffraction data were collected to 2.60 Å resolution.

  10. A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion

    PubMed Central

    Hansen, Carl L.; Skordalakes, Emmanuel; Berger, James M.; Quake, Stephen R.

    2002-01-01

    Producing robust and scalable fluid metering in a microfluidic device is a challenging problem. We developed a scheme for metering fluids on the picoliter scale that is scalable to highly integrated parallel architectures and is independent of the properties of the working fluid. We demonstrated the power of this method by fabricating and testing a microfluidic chip for rapid screening of protein crystallization conditions, a major hurdle in structural biology efforts. The chip has 480 active valves and performs 144 parallel reactions, each of which uses only 10 nl of protein sample. The properties of microfluidic mixing allow an efficient kinetic trajectory for crystallization, and the microfluidic device outperforms conventional techniques by detecting more crystallization conditions while using 2 orders of magnitude less protein sample. We demonstrate that diffraction-quality crystals may be grown and harvested from such nanoliter-volume reactions. PMID:12486223

  11. Expression, purification and crystallization of human 5-lipoxygenase-activating protein with leukotriene-biosynthesis inhibitors

    SciTech Connect

    Xu, Shihua; McKeever, Brian M.; Wisniewski, Douglas; Miller, Douglas K.; Spencer, Robert H.; Chu, Lin; Ujjainwalla, Feroze; Yamin, Ting-Ting; Evans, Jilly F.; Becker, Joseph W.; Ferguson, Andrew D.

    2007-12-01

    The expression, purification and crystallization of human 5-lipoxygenase-activating protein in complex with two leukotriene-biosynthesis inhibitors is decribed. The processes that were used to generate diffraction quality crystals are presented in detail. The nuclear membrane protein 5-lipoxygenase-activating protein (FLAP) plays an essential role in leukotriene synthesis. Recombinant full-length human FLAP with a C-terminal hexahistidine tag has been expressed and purified from the cytoplasmic membrane of Escherichia coli. Diffraction-quality crystals of FLAP in complex with leukotriene-synthesis inhibitor MK-591 and with an iodinated analogue of MK-591 have been grown using the sitting-drop vapor-diffusion method. The crystals exhibit tetragonal symmetry (P42{sub 1}2) and diffracted to a resolution limit of 4 Å.

  12. In situ observation of containerless protein crystallization by magnetically levitating crystal growth

    NASA Astrophysics Data System (ADS)

    Maki, Syou; Tanimoto, Yoshifumi; Udagawa, Chikako; Morimoto, Shotaro; Hagiwara, Masayuki

    2016-03-01

    We report on the results of the crystal growth of hen-egg lysozyme by magnetically levitating crystals in a small amount of buffer solution. The concentrations of lysozyme and the precipitating agent (gadolinium chloride) were 6.53 wt % and 0.362 mol/kg, respectively. Gadolinium chloride, which induces the magneto-Archimedes effect, was utilized to levitate the crystals with Bz · (dBz/dz) = 22.46 T2/m, where Bz is the vertical (z) component of the magnetic flux density vector. Although the collected crystals were small, we succeeded in maintaining the levitation of the crystals into a specific place in the liquid phase from the beginning of nucleation. In situ observation revealed that a state of pseudo-weightlessness was generated in the vicinity of the magnet bore edge, and small crystals were concentrated inside the domain moving along an hourglass-shaped surface. We found by numerical computations that the formation of the hourglass-shaped domain is attributable to the radial component of the magnetic force.

  13. (PCG) Protein Crystal Growth Canavalin Crystals grown on Earth and Space

    NASA Technical Reports Server (NTRS)

    1993-01-01

    The major storage protein of leguminous plants and a major source of dietary protein for humans and domestic animals. It is studied in efforts to enhance nutritional value of proteins through protein engineerings. It is isolated from Jack Bean because of it's potential as a nutritional substance. Principal Investigator was Alexander McPherson.

  14. Recent advances in covalent, site-specific protein immobilization

    PubMed Central

    Meldal, Morten; Schoffelen, Sanne

    2016-01-01

    The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. Covalent, site-specific immobilization strategies are robust and can provide the level of control that is desired in this kind of application. Recent advances include the use of enzymes, such as sortase A, to couple proteins in a site-specific manner to materials such as microbeads, glass, and hydrogels. Also, self-labeling tags such as the SNAP-tag can be employed. Last but not least, chemical approaches based on bioorthogonal reactions, like the azide–alkyne cycloaddition, have proven to be powerful tools. The lack of comparative studies and quantitative analysis of these immobilization methods hampers the selection process of the optimal strategy for a given application. However, besides immobilization efficiency, the freedom in selecting the site of conjugation and the size of the conjugation tag and the researcher’s expertise regarding molecular biology and/or chemical techniques will be determining factors in this regard. PMID:27785356

  15. Advances in the growth of alkaline-Earth halide single crystals for scintillator detectors

    NASA Astrophysics Data System (ADS)

    Boatner, L. A.; Ramey, J. O.; Kolopus, J. A.; Neal, J. S.; Cherepy, N. J.; Beck, P. R.; Payne, S. A.; Burger, A.; Rowe, E.; Bhattacharya, P.

    2014-09-01

    Alkaline-earth scintillators such as strontium iodide and other alkaline-earth halides activated with divalent europium represent some of the most efficient and highest energy resolution scintillators for use as gamma-ray detectors in a wide range of applications. These applications include the areas of nuclear nonproliferation, homeland security, the detection of undeclared nuclear material, nuclear physics and materials science, medical diagnostics, space physics, high energy physics, and radiation monitoring systems for first responders, police, and fire/rescue personnel. Recent advances in the growth of large single crystals of these scintillator materials hold the promise of higher crystal yields and significantly lower detector production costs. In the present work, we describe new processing protocols that, when combined with our molten salt filtration methods, have led to advances in achieving a significant reduction of cracking effects during the growth of single crystals of SrI2:Eu2+. In particular, we have found that extended pumping on the molten crystalgrowth charge under vacuum for time periods extending up to 48 hours is generally beneficial in compensating for variations in the alkaline-earth halide purity and stoichiometry of the materials as initially supplied by commercial sources. These melt-pumping and processing techniques are now being applied to the purification of CaI2:Eu2+ and some mixed-anion europium-doped alkaline-earth halides prior to single-crystal growth by means of the vertical Bridgman technique. The results of initial studies of the effects of aliovalent doping of SrI2:Eu2+ on the scintillation characteristics of this material are also described.

  16. Large-scale identification of membrane proteins with properties favorable for crystallization.

    PubMed

    Kim, Jared; Kagawa, Allison; Kurasaki, Kellie; Ataie, Niloufar; Cho, Il Kyu; Li, Qing X; Ng, Ho Leung

    2015-11-01

    Membrane protein crystallography is notoriously difficult due to challenges in protein expression and issues of degradation and structural stability. We have developed a novel method for large-scale screening of native sources for integral membrane proteins that have intrinsic biochemical properties favorable for crystallization. Highly expressed membrane proteins that are thermally stable and nonaggregating in detergent solutions were identified by mass spectrometry from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebrum. Many of the membrane proteins identified had been crystallized previously, supporting the promise of the approach. Most identified proteins have known functions and include high-value targets such as transporters and ATPases. To validate the method, we recombinantly expressed and purified the yeast protein, Yop1, which is responsible for endoplasmic reticulum curvature. We demonstrate that Yop1 can be purified with the detergent dodecylmaltoside without aggregating.

  17. Crystal structure of ribosomal protein L1 from the bacterium Aquifex aeolicus

    NASA Astrophysics Data System (ADS)

    Nikonova, E. Yu.; Tishchenko, S. V.; Gabdulkhakov, A. G.; Shklyaeva, A. A.; Garber, M. B.; Nikonov, S. V.; Nevskaya, N. A.

    2011-07-01

    The crystal structure of ribosomal protein L1 from the bacterium Aquifex aeolicus was solved by the molecular-replacement method and refined to R cryst = 19.4% and R free = 25.1% at 2.1 Å protein consists of two domains linked together by a flexible hinge region. In the structure under consideration, the domains are in close proximity and adopt a closed conformation. Earlier, this conformation has been found in the structure of protein L1 from the bacterium Thermus thermophilus, whereas the structures of archaeal L1 proteins and the structures of all L1 proteins in the RNA-bound form have an open conformation. The fact that a closed conformation was found in the structures of two L1 proteins which crystallize in different space groups and belong to different bacteria suggests that this conformation is a characteristic feature of L1 bacterial proteins in the free form.

  18. The crystal structure of the thiocyanate-forming protein from Thlaspi arvense, a kelch protein involved in glucosinolate breakdown.

    PubMed

    Gumz, Frauke; Krausze, Joern; Eisenschmidt, Daniela; Backenköhler, Anita; Barleben, Leif; Brandt, Wolfgang; Wittstock, Ute

    2015-09-01

    Kelch repeat-containing proteins are involved in diverse cellular processes, but only a small subset of plant kelch proteins has been functionally characterized. Thiocyanate-forming protein (TFP) from field-penny cress, Thlaspi arvense (Brassicaceae), is a representative of specifier proteins, a group of kelch proteins involved in plant specialized metabolism. As components of the glucosinolate-myrosinase system of the Brassicaceae, specifier proteins determine the profile of bioactive products formed when plant tissue is disrupted and glucosinolates are hydrolyzed by myrosinases. Here, we describe the crystal structure of TaTFP at a resolution of 1.4 Å. TaTFP crystallized as homodimer. Each monomer forms a six-blade β-propeller with a wide "top" and a narrower "bottom" openi