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Sample records for carrier protein crystal

  1. 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.

  2. 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

  3. Crystallization and preliminary X-ray analysis of enoyl-acyl carrier protein reductase (FabK) from Streptococcus pneumoniae

    SciTech Connect

    Saito, Jun Yamada, Mototsugu; Watanabe, Takashi; Kitagawa, Hideo; Takeuchi, Yasuo

    2006-06-01

    Enoyl-acyl carrier protein (ACP) reductases are responsible for bacterial type II fatty-acid biosynthesis and are attractive targets for developing novel antibiotics. The S. pneumoniae enoyl-ACP reductase (FabK) was crystallized and selenomethionine MAD data were collected to 2 Å resolution. The enoyl-acyl carrier protein (ACP) reductase from Streptococcus pneumoniae (FabK; EC 1.3.1.9) is responsible for catalyzing the final step in each elongation cycle of fatty-acid biosynthesis. Selenomethionine-substituted FabK was purified and crystallized by the hanging-drop vapour-diffusion method at 277 K. The crystal belongs to space group P2{sub 1}, with unit-cell parameters a = 50.26, b = 126.70, c = 53.63 Å, β = 112.46°. Diffraction data were collected to 2.00 Å resolution using synchrotron beamline BL32B2 at SPring-8. Two molecules were estimated to be present in the asymmetric unit, with a solvent content of 45.1%.

  4. The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins

    PubMed Central

    Lohman, Jeremy R.; Ma, Ming; Cuff, Marianne E.; Bigelow, Lance; Bearden, Jessica; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

    2014-01-01

    Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl-carrier proteins (PCPs) or acyl-carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs. PMID:25050442

  5. Crystal Structure of a Sulfur Carrier Protein Complex Found in the Cysteine Biosynthetic Pathway of Mycobacterium tuberculosis

    SciTech Connect

    Jurgenson, Christopher T.; Burns, Kristin E.; Begley, Tadhg P.; Ealick, Steven E.

    2008-10-02

    The structure of the protein complex CysM-CysO from a new cysteine biosynthetic pathway found in the H37Rv strain of Mycobacterium tuberculosis has been determined at 1.53 {angstrom} resolution. CysM (Rv1336) is a PLP-containing {beta}-replacement enzyme and CysO (Rv1335) is a sulfur carrier protein with a ubiquitin-like fold. CysM catalyzes the replacement of the acetyl group of O-acetylserine by CysO thiocarboxylate to generate a protein-bound cysteine that is released in a subsequent proteolysis reaction. The protein complex in the crystal structure is asymmetric with one CysO protomer binding to one end of a CysM dimer. Additionally, the structures of CysM and CysO were determined individually at 2.8 and 2.7 {angstrom} resolution, respectively. Sequence alignments with homologues and structural comparisons with CysK, a cysteine synthase that does not utilize a sulfur carrier protein, revealed high conservation of active site residues; however, residues in CysM responsible for CysO binding are not conserved. Comparison of the CysM-CysO binding interface with other sulfur carrier protein complexes revealed a similarity in secondary structural elements that contribute to complex formation in the ThiF-ThiS and MoeB-MoaD systems, despite major differences in overall folds. Comparison of CysM with and without bound CysO revealed conformational changes associated with CysO binding.

  6. 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.

  7. Crystal structures and kinetic properties of enoyl-acyl carrier protein reductase I from Candidatus Liberibacter asiaticus

    PubMed Central

    Jiang, Ling; Gao, Zengqiang; Li, Yanhua; Wang, Shennan; Dong, Yuhui

    2014-01-01

    Huanglongbing (HLB) is a destructive citrus disease. The leading cause of HLB is Candidatus Liberibacter asiaticus. Fatty acid biosynthesis is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterial agents. Enoyl−acyl carrier protein reductase (also called ENR or FabI and a product of the fabI gene) is an enzyme required in a critical step of bacterial fatty acid biosynthesis and has attracted attention as a target of novel antimicrobial agents. We determined the crystal structures of FabI from Ca. L. asiaticus in its apoform as well as in complex with b-nicotinamide adenine dinucleotide (NAD) at 1.7 and 2.7 Å resolution, respectively, to facilitate the design and screening of small molecule inhibitors of FabI. The monomeric ClFabI is highly similar to other known FabI structures as expected; however, unlike the typical tetramer, ClFabI exists as a hexamer in crystal, whereas as dimer in solution, on the other hand, the substrate binding loop which always disordered in apoform FabI structures is ordered in apo-ClFabI. Interestingly, the structure of ClFabI undergoes remarkable conformational change in the substrate-binding loop in the presence of NAD. We conclude that the signature sequence motif of FabI can be considered as Gly-(Xaa)5-Ser-(Xaa)n-Val-Tyr-(Xaa)6-Lys-(Xaa)n-Thr instead of Tyr-(Xaa)6-Lys. We have further identified isoniazid as a competitive inhibitor with NADH. PMID:24407918

  8. Crystal structures and kinetic properties of enoyl-acyl carrier protein reductase I from Candidatus Liberibacter asiaticus.

    PubMed

    Jiang, Ling; Gao, Zengqiang; Li, Yanhua; Wang, Shennan; Dong, Yuhui

    2014-04-01

    Huanglongbing (HLB) is a destructive citrus disease. The leading cause of HLB is Candidatus Liberibacter asiaticus. Fatty acid biosynthesis is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterial agents. Enoyl-acyl carrier protein reductase (also called ENR or FabI and a product of the fabI gene) is an enzyme required in a critical step of bacterial fatty acid biosynthesis and has attracted attention as a target of novel antimicrobial agents. We determined the crystal structures of FabI from Ca. L. asiaticus in its apoform as well as in complex with b-nicotinamide adenine dinucleotide (NAD) at 1.7 and 2.7 Å resolution, respectively, to facilitate the design and screening of small molecule inhibitors of FabI. The monomeric ClFabI is highly similar to other known FabI structures as expected; however, unlike the typical tetramer, ClFabI exists as a hexamer in crystal, whereas as dimer in solution, on the other hand, the substrate binding loop which always disordered in apoform FabI structures is ordered in apo-ClFabI. Interestingly, the structure of ClFabI undergoes remarkable conformational change in the substrate-binding loop in the presence of NAD. We conclude that the signature sequence motif of FabI can be considered as Gly-(Xaa)5-Ser-(Xaa)n-Val-Tyr-(Xaa)6-Lys-(Xaa)n-Thr instead of Tyr-(Xaa)6-Lys. We have further identified isoniazid as a competitive inhibitor with NADH. PMID:24407918

  9. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3.

    PubMed

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2007-04-01

    Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein-protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPDeltaN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 complexes as well as crystals of BPL*, BPL** and BCCPDeltaN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*-BCCPDeltaN76 and BPL**-BCCPDeltaN76 crystals were collected at 100 K to 2.7 and 2.0 A resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2(1), with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 A, beta = 95.9 degrees . Assuming two subunits of the complex per asymmetric unit gives a V(M) value of 2.45 A(3) Da(-1) and a solvent content of 50%. PMID:17401210

  10. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3

    PubMed Central

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2007-01-01

    Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein–protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPΔN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*–BCCPΔN76 and BPL**–BCCPΔN76 complexes as well as crystals of BPL*, BPL** and BCCPΔN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals were collected at 100 K to 2.7 and 2.0 Å resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P21, with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 Å, β = 95.9°. Assuming two subunits of the complex per asymmetric unit gives a V M value of 2.45 Å3 Da−1 and a solvent content of 50%. PMID:17401210

  11. Crystallization and preliminary X-ray crystallographic studies of the biotin carboxyl carrier protein and biotin protein ligase complex from Pyrococcus horikoshii OT3

    SciTech Connect

    Bagautdinov, Bagautdin; Matsuura, Yoshinori; Bagautdinova, Svetlana; Kunishima, Naoki

    2007-04-01

    A truncated form of biotin carboxyl carrier protein containing the C-terminal half fragment (BCCPΔN76) and the biotin protein ligase (BPL) with the mutation R48A (BPL*) or the double mutation R48A K111A (BPL**) were successfully cocrystallized in the presence of ATP and biotin. The BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals belong to space group P2{sub 1} and diffract X-rays to 2.7 and 2.0 Å resolution, respectively. Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. To elucidate the exact details of the protein–protein interactions in the biotinylation function, the C-terminal half fragment of BCCP (BCCPΔN76), the R48A mutant of BPL (BPL*) and the R48A K111A double mutant of BPL (BPL**), all of which are from Pyrococcus horikoshii OT3, have been expressed, purified and successfully cocrystallized. Cocrystals of the BPL*–BCCPΔN76 and BPL**–BCCPΔN76 complexes as well as crystals of BPL*, BPL** and BCCPΔN76 were obtained by the oil-microbatch method using PEG 20 000 as a precipitant at 295 K. Complete X-ray diffraction data sets for BPL*–BCCPΔN76 and BPL**–BCCPΔN76 crystals were collected at 100 K to 2.7 and 2.0 Å resolution, respectively, using synchrotron radiation. They belong to the monoclinic space group P2{sub 1}, with similar unit-cell parameters a = 69.85, b = 63.12, c = 75.64 Å, β = 95.9°. Assuming two subunits of the complex per asymmetric unit gives a V{sub M} value of 2.45 Å{sup 3} Da{sup −1} and a solvent content of 50%.

  12. X-Ray Crystal Structure of Mycobacterium Tuberculosis β-Ketoacyl Acyl Carrier Protein Synthase II (mtKasB)

    PubMed Central

    Sridharan, Sudharsan; Wang, Lei; Brown, Alistair K.; Dover, Lynn G.; Kremer, Laurent; Besra, Gurdyal S.; Sacchettini, James C.

    2007-01-01

    Summary Mycolic acids are long chain α-alkyl branched, β-hydroxy fatty acids that represent a characteristic component of the Mycobacterium tuberculosis cell wall. Through their covalent attachment to peptidoglycan via an arabinogalactan polysaccharide, they provide the basis for an essential outer envelope membrane. Mycobacteria possess two fatty acid synthases (FAS); FAS-I carries out de novo synthesis of fatty acids while FAS-II is considered to elongate medium chain length fatty acyl primers to provide long chain (C56) precursors of mycolic acids. Here we report the crystal structure of Mycobacterium tuberculosis β-ketoacyl acyl carrier protein synthase (ACP) II mtKasB, a mycobacterial elongation condensing enzyme involved in FAS-II. This enzyme, along with the M. tuberculosis β-ketoacyl ACP synthase I mtKasA, catalyzes the Claisen-type condensation reaction responsible for fatty acyl elongation in FAS-II and are potential targets for development of novel anti-tubercular drugs. The crystal structure refined to 2.4 Å resolution revealed that, like other KAS-II enzymes, mtKasB adopts a thiolase fold but contains unique structural features in the capping region that may be crucial to its preference for longer fatty acyl chains than its counterparts from other bacteria. Modeling of mtKasA using the mtKasB structure as a template predicts the overall structures to be almost identical, but a larger entrance to the active site tunnel is envisaged that might contribute to the greater sensitivity of mtKasA to the inhibitor thiolactomycin (TLM). Modeling of TLM binding in mtKasB shows that the drug fits the active site poorly and results of enzyme inhibition assays using TLM analogues are wholly consistent with our structural observations. Consequently, the structure described here further highlights the potential of TLM as an anti-tubercular lead compound and will aid further exploration of the TLM scaffold towards the design of novel compounds which inhibit

  13. Crystal Structure of Epiphyas Postvittana Takeout 1 With Bound Ubiquinone Supports a Role As Ligand Carriers for Takeout Proteins in Insects

    SciTech Connect

    Hamiaux, C.; Stanley, D.; Greenwood, D.R.; Baker, E.N.; Newcomb, R.D.

    2009-05-19

    Takeout (To) proteins are found exclusively in insects and have been proposed to have important roles in various aspects of their physiology and behavior. Limited sequence similarity with juvenile hormone-binding proteins (JHBPs), which specifically bind and transport juvenile hormones in Lepidoptera, suggested a role for To proteins in binding hydrophobic ligands. We present the first crystal structure of a To protein, EpTo1 from the light brown apple moth Epiphyas postvittana, solved in-house by the single-wavelength anomalous diffraction technique using sulfur anomalous dispersion, and refined to 1.3 {angstrom} resolution. EpTo1 adopts the unusual {alpha}/{beta}-wrap fold, seen only for JHBP and several mammalian lipid carrier proteins, a scaffold tailored for the binding and/or transport of hydrophobic ligands. EpTo1 has a 45 {angstrom} long, purely hydrophobic, internal tunnel that extends for the full length of the protein and accommodates a bound ligand. The latter was shown by mass spectrometry to be ubiquinone-8 and is probably derived from Escherichia coli. The structure provides the first direct experimental evidence that To proteins are ligand carriers; gives insights into the nature of endogenous ligand(s) of EpTo1; shows, by comparison with JHBP, a basis for different ligand specificities; and suggests a mechanism for the binding/release of ligands.

  14. Crystal Structure of the Human Fatty Acid Synthase Enoyl-Acyl Carrier Protein-Reductase Domain Complexed with Triclosan Reveals Allosteric Protein-Protein Interface Inhibition*

    PubMed Central

    Sippel, Katherine H.; Vyas, Nand K.; Zhang, Wei; Sankaran, Banumathi; Quiocho, Florante A.

    2014-01-01

    Human fatty acid synthase (FAS) is a large, multidomain protein that synthesizes long chain fatty acids. Because these fatty acids are primarily provided by diet, FAS is normally expressed at low levels; however, it is highly up-regulated in many cancers. Human enoyl-acyl carrier protein-reductase (hER) is one of the FAS catalytic domains, and its inhibition by drugs like triclosan (TCL) can increase cytotoxicity and decrease drug resistance in cancer cells. We have determined the structure of hER in the presence and absence of TCL. TCL was not bound in the active site, as predicted, but rather at the protein-protein interface (PPI). TCL binding induces a dimer orientation change that causes downstream structural rearrangement in critical active site residues. Kinetics studies indicate that TCL is capable of inhibiting the isolated hER domain with an IC50 of ∼55 μm. Given the hER-TCL structure and the inhibition observed in the hER domain, it seems likely that TCL is observed in the physiologically relevant binding site and that it acts as an allosteric PPI inhibitor. TCL may be a viable scaffold for the development of anti-cancer PPI FAS inhibitors. PMID:25301948

  15. 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.

  16. 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.

  17. Crystal structure of enoyl-acyl carrier protein reductase (FabK) from Streptococcus pneumoniae reveals the binding mode of an inhibitor.

    PubMed

    Saito, Jun; Yamada, Mototsugu; Watanabe, Takashi; Iida, Maiko; Kitagawa, Hideo; Takahata, Sho; Ozawa, Tomohiro; Takeuchi, Yasuo; Ohsawa, Fukuichi

    2008-04-01

    Enoyl-acyl carrier protein (ACP) reductases are critical for bacterial type II fatty acid biosynthesis and thus are attractive targets for developing novel antibiotics. We determined the crystal structure of enoyl-ACP reductase (FabK) from Streptococcus pneumoniae at 1.7 A resolution. There was one dimer per asymmetric unit. Each subunit formed a triose phosphate isomerase (TIM) barrel structure, and flavin mononucleotide (FMN) was bound as a cofactor in the active site. The overall structure was similar to the enoyl-ACP reductase (ER) of fungal fatty acid synthase and to 2-nitropropane dioxygenase (2-ND) from Pseudomonas aeruginosa, although there were some differences among these structures. We determined the crystal structure of FabK in complex with a phenylimidazole derivative inhibitor to envision the binding site interactions. The crystal structure reveals that the inhibitor binds to a hydrophobic pocket in the active site of FabK, and this is accompanied by induced-fit movements of two loop regions. The thiazole ring and part of the ureido moiety of the inhibitor are involved in a face-to-face pi-pi stacking interaction with the isoalloxazine ring of FMN. The side-chain conformation of the proposed catalytic residue, His144, changes upon complex formation. Lineweaver-Burk plots indicate that the inhibitor binds competitively with respect to NADH, and uncompetitively with respect to crotonoyl coenzyme A. We propose that the primary basis of the inhibitory activity is competition with NADH for binding to FabK, which is the first step of the two-step ping-pong catalytic mechanism. PMID:18305197

  18. 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.

  19. Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins.

    PubMed Central

    Lindqvist, Y; Huang, W; Schneider, G; Shanklin, J

    1996-01-01

    The three-dimensional structure of recombinant homodimeric delta9 stearoyl-acyl carrier protein desaturase, the archetype of the soluble plant fatty acid desaturases that convert saturated to unsaturated fatty acids, has been determined by protein crystallographic methods to a resolution of 2.4 angstroms. The structure was solved by a combination of single isomorphous replacement, anomalous contribution from the iron atoms to the native diffraction data and 6-fold non-crystallographic symmetry averaging. The 363 amino acid monomer consists of a single domain of 11 alpha-helices. Nine of these form an antiparallel helix bundle. The enzyme subunit contains a di-iron centre, with ligands from four of the alpha-helices in the helix bundle. The iron ions are bound in a highly symmetric environment, with one of the irons forming interactions with the side chains of E196 and H232 and the second iron with the side chains of E105 and H146. Two additional glutamic acid side chains, from E143 and E229, are within coordination distance to both iron ions. A water molecule is found within the second coordination sphere from the iron atoms. The lack of electron density corresponding to a mu-oxo bridge, and the long (4.2 angstroms) distance between the iron ions suggests that this probably represents the diferrous form of the enzyme. A deep channel which probably binds the fatty acid extends from the surface into the interior of the enzyme. Modelling of the substrate, stearic acid, into this channel places the delta9 carbon atom in the vicinity of one of the iron ions. Images PMID:8861937

  20. (PCG) Protein Crystal Growth on STS-26

    NASA Technical Reports Server (NTRS)

    1988-01-01

    Mission Specialist George (Pinky) D. Nelson uses a 35 mm camera to photograph a protein crystal grown during the STS-26 Protein Crystal Growth (PCG-II-01) experiment. The protein crystal growth (PCG) carrier is shown deployed from the PCG Refrigerator/Incubator Mocule (R/IM) located in the middeck forward locker. The R/IM contained three Vapor Diffusion Apparatus (VDS) trays (one of which is shown). A total of sixty protein crystal samples were processed during the STS-26 mission.

  1. 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.

  2. 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.

  3. 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.

  4. 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)

  5. 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.

  6. 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.

  7. 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)

  8. 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.

  9. Microgravity Crystallization of Alpha-Crustacyanin Onboard the Unmanned Carrier, EURECA

    NASA Technical Reports Server (NTRS)

    Boggon, T. J.; Snell, E. H.; Helliwell, J. R.; Chayen, N. E.; Zagalsky, P. F.

    1998-01-01

    alpha-Crustacyanin, the lobster carapace astaxanthin-protein, was crystallized using the European Space Agency's (ESA) automated Protein Crystallization Facility (PCF) which flew onboard the unmanned EUropean REtrievable CArrier (EURECA). A free interface linear, liquid - liquid diffusion, method was used. Crystals grew larger and thicker in the microgravity case compared to the biggest crystals grown on earth. Video observation on EURECA revealed variations in crystal sizes through-out the reactor neatly correlated with depletion of this coloured protein from the solution. The video observations most importantly revealed no visible movement of crystals over the initial 7 weeks of the experiment, although an obvious temperature induced jump occurred at that time in a mission spanning 11 months. An important observation from this mission, over the first 7 weeks, of completely stationary crystal growth contrasts with crystal motions viewed on manned microgravity missions, even using linear liquid - liquid geometries, and much shorter flights (eg. 12 to 16 days).

  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. 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

  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. 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. PMID:24419610

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  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. Protein crystals and their growth

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2003-01-01

    Recent results on the associations between protein molecules in crystal lattices, crystal-solution surface energy, elastic properties, strength, and spontaneous crystal cracking are reviewed and discussed. In addition, some basic approaches to understanding the solubility of proteins are followed by an overview of crystal nucleation and growth. It is argued that variability of mixing in batch crystallization may be a source of the variation in the number of crystals ultimately appearing in the sample. The frequency at which new molecules join a crystal lattice is measured by the kinetic coefficient and is related to the observed crystal growth rate. Numerical criteria used to discriminate diffusion- and kinetic-limited growth are discussed on this basis. Finally, the creation of defects is discussed with an emphasis on the role of impurities and convection on macromolecular crystal perfection.

  6. (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.

  7. Carrier doping and interlayer coupling in HTSC single crystals

    SciTech Connect

    Kishio, K.; Shimoyama, J.; Kimura, T.; Kotaka, Y.; Kitazawa, K.; Yamafuji, K.; Li, Q.; Suenaga, M.

    1994-09-01

    Experimental results of the effect of carrier doping on the irreversibility lines in (La,Sr){sub 2}CuO{sub 4{minus}{delta}} and Bi{sub 2}Sr{sub 2}CaCu{sub 2}O{sub 8 + {delta}} single crystals are summarized. As a function of Sr or oxygen contents, systematic and dramatic widening of the irreversible regions in the B {minus} T phase diagram was observed in both systems. The present study suggests the critical importance of carrier concentration which directly affects the interlayer coupling strength and dimensionality of the flux line lattice in all the layered HTSC compounds as a universal feature.

  8. Protein crystal growth tray assembly

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor); Miller, Teresa Y. (Inventor)

    1992-01-01

    A protein crystal growth tray assembly includes a tray that has a plurality of individual crystal growth chambers. Each chamber has a movable pedestal which carries a protein crystal growth compartment at an upper end. The several pedestals for each tray assembly are ganged together for concurrent movement so that the solutions in the various pedestal growth compartments can be separated from the solutions in the tray's growth chambers until the experiment is to be activated.

  9. Trapping of the Enoyl-Acyl Carrier Protein Reductase–Acyl Carrier Protein Interaction

    PubMed Central

    Tallorin, Lorillee; Finzel, Kara; Nguyen, Quynh G.; Beld, Joris; La Clair, James J.; Burkart, Michael D.

    2016-01-01

    An ideal target for metabolic engineering, fatty acid biosynthesis remains poorly understood on a molecular level. These carrier protein-dependent pathways require fundamental protein–protein interactions to guide reactivity and processivity, and their control has become one of the major hurdles in successfully adapting these biological machines. Our laboratory has developed methods to prepare acyl carrier proteins (ACPs) loaded with substrate mimetics and cross-linkers to visualize and trap interactions with partner enzymes, and we continue to expand the tools for studying these pathways. We now describe application of the slow-onset, tight-binding inhibitor triclosan to explore the interactions between the type II fatty acid ACP from Escherichia coli, AcpP, and its corresponding enoyl-ACP reductase, FabI. We show that the AcpP–triclosan complex demonstrates nM binding, inhibits in vitro activity, and can be used to isolate FabI in complex proteomes. PMID:26938266

  10. 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. PMID:25607090

  11. 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.

  12. 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.

  13. Containerless protein crystal growth method

    NASA Technical Reports Server (NTRS)

    Rhim, Won-Kyu; Chung, Sang K.

    1991-01-01

    A method of growing protein crystals from levitated drops is introduced and unique features of containerless approach in 1-g and micro-G laboratories are discussed. Electrostatic multidrop levitation system which is capable of simultaneous four drop levitation is described. A method of controlling protein saturation level in a programmed way is introduced and discussed. Finally, some of the unique features of containerless approach of protein crystal growth in space are discussed and summarized.

  14. Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

    SciTech Connect

    Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.; Grooms, Michael; Daines, Robert A.; Lonsdale, John T.; Khandekar, Sanjay S.

    2010-07-20

    {beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.

  15. 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.

  16. Relatedness of acyl carrier proteins shown by amino acid compositions.

    PubMed

    Walker, T A; Ernst-Fonberg, M L

    1982-01-01

    1. Relatedness among the following carrier proteins was assessed on the basis of amino acid compositions: eight acyl carrier proteins (ACP's) associated with fatty acid synthesis, ACP's associated with citrate lyase and citramalate lyase, a biotin carboxyl carrier protein and cytochrome 552. Two independent indices of amino acid composition were used. 2. The fatty acid synthesis-associated ACP's of many organisms and the lyase-associated ACP's show a high degree of relatedness among one another. 3. The ACP's show no relatedness to biotin carboxyl carrier protein or cytochrome 552. PMID:7128903

  17. 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.

  18. Characterization of the "Escherichia Coli" Acyl Carrier Protein Phosphodiesterase

    ERIC Educational Resources Information Center

    Thomas, Jacob

    2009-01-01

    Acyl carrier protein (ACP) is a small essential protein that functions as a carrier of the acyl intermediates of fatty acid synthesis. ACP requires the posttranslational attachment of a 4'phosphopantetheine functional group, derived from CoA, in order to perform its metabolic function. A Mn[superscript 2+] dependent enzymatic activity that removes…

  19. Nucleation precursors in protein crystallization

    PubMed Central

    Vekilov, Peter G.; Vorontsova, Maria A.

    2014-01-01

    Protein crystal nucleation is a central problem in biological crystallography and other areas of science, technology and medicine. Recent studies have demonstrated that protein crystal nuclei form within crucial precursors. Here, methods of detection and characterization of the precursors are reviewed: dynamic light scattering, atomic force microscopy and Brownian microscopy. Data for several proteins provided by these methods have demonstrated that the nucleation precursors are clusters consisting of protein-dense liquid, which are metastable with respect to the host protein solution. The clusters are several hundred nanometres in size, the cluster population occupies from 10−7 to 10−3 of the solution volume, and their properties in solutions supersaturated with respect to crystals are similar to those in homogeneous, i.e. undersaturated, solutions. The clusters exist owing to the conformation flexibility of the protein molecules, leading to exposure of hydrophobic surfaces and enhanced intermolecular binding. These results indicate that protein conformational flexibility might be the mechanism behind the metastable mesoscopic clusters and crystal nucleation. Investigations of the cluster properties are still in their infancy. Results on direct imaging of cluster behaviors and characterization of cluster mechanisms with a variety of proteins will soon lead to major breakthroughs in protein biophysics. PMID:24598910

  20. Charge carrier rearrangement in spinel crystals irradiated at low temperatures

    NASA Astrophysics Data System (ADS)

    Gritsyna, V. T.; Afanasyev-Charkin, I. V.; Kobyakov, V. A.; Voitsenya, T. I.; Sickafus, K. E.

    2000-05-01

    The results of an investigation of thermoluminescence (TL) in nominally pure MgAl2O4 spinel single crystals in the temperature range between 80-670 K are presented. For a heating rate of 0.21 K/s, TL spectra exhibit glow peaks in three distinct temperature ranges: 100-160, 270-370 and 470-670 K. The most prominent peaks are at 115, 140, 305, 335, 525, 570 and 605 K. The locations of the temperature maxima, as well as the intensity of the peaks, vary depending on the treatment of the crystals, the type of irradiation and the temperature of irradiation. Measurements of the glow peaks at different emission wavelengths and the use of partial bleaching and isothermal decay techniques for TL, allowed us to propose mechanisms for charge carrier rearrangement at lattice defects and impurity ions, during irradiation and subsequent heating.

  1. Carrier mobility and crystal perfection of tetracene thin film FET

    NASA Astrophysics Data System (ADS)

    Moriguchi, N.; Nishikawa, T.; Anezaki, T.; Unno, A.; Tachibana, M.; Kojima, K.

    2006-04-01

    It is well-known that the carrier mobility of an organic field effect semiconductor (FET) depended on the crystal quality and/or the crystal perfection of the organic thin films [T.W. Kelly, D.V. Muyres, P.F. Baude, T.P. Smith, T.D. Jones, Mater. Res. Soc. Symp. Proc. 771 (2003) L6.5.1; D.J. Gundlach, J.A. Nichols, L. Zhou, T.N. Jackson, Appl. Phys. Lett. 80 (2002) 2925; H.K. Lauk, M. Halik, U. Zschieschang, G. Schmid, W. Radlik, J. Appl. Phys. 92 (2002) 5259; M. Shtein, J. Mapel, J.B. Benziger, S.R. Forrest, Appl. Phys. Lett. 81 (2002) 268; D. Knipp, R.A. Street, A.R. Volkel, Appl. Phys. Lett. 82 (2003) 3907; R. Ruiz, A.C. Mayer, G.G. Malliaras, Appl. Phys. Lett. 85 (2004) 4926; R.W.I. de Boer, M.E. Gershenson, A.F. Morpurgo, V. Podzorov, Phys. Stat. Sol. A 201 (2004) 1031]. To improve the crystal quality of the thin film many efforts were made. One of the important improvements was the surface treatment of the substrate. The tetracene thin film FET (top contact structure) was fabricated using the substrate, which was coated by a spin-coating method with a 0.1% poly α-methylstyrene (AMS) solution. The crystal quality was improved by this treatment so that the carrier mobility was higher than that of non-treatment. The maximum mobility of the AMS-treated sample was obtained to be 0.12 cm 2/V s.

  2. 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.

  3. 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.

  4. Direct Acylation of Carrier Proteins with Functionalized β-Lactones

    PubMed Central

    Amoroso, Jon W.; Borketey, Lawrence S.; Prasad, Gitanjeli

    2014-01-01

    As the key component of many biosynthetic assemblies, acyl-carrier proteins offer a robust entry point for introduction of small molecule probes and pathway intermediates. Current labeling strategies primarily rely on modifications to the phosphopantetheine cofactor or its biosynthetic precursors followed by attachment to the apo form of a given carrier protein. As a greatly simplified alternative, direct and selective acylation of holo-acyl-carrier proteins using readily accessible β-lactones as electrophilic partners for the phosphopantetheine-thiol has been demonstrated. PMID:20433156

  5. Preclinical studies on new proteins as carrier for glycoconjugate vaccines.

    PubMed

    Tontini, M; Romano, M R; Proietti, D; Balducci, E; Micoli, F; Balocchi, C; Santini, L; Masignani, V; Berti, F; Costantino, P

    2016-07-29

    Glycoconjugate vaccines are made of carbohydrate antigens covalently bound to a carrier protein to enhance their immunogenicity. Among the different carrier proteins tested in preclinical and clinical studies, five have been used so far for licensed vaccines: Diphtheria and Tetanus toxoids, the non-toxic mutant of diphtheria toxin CRM197, the outer membrane protein complex of Neisseria meningitidis serogroup B and the Protein D derived from non-typeable Haemophilus influenzae. Availability of novel carriers might help to overcome immune interference in multi-valent vaccines containing several polysaccharide-conjugate antigens, and also to develop vaccines which target both protein as well saccharide epitopes of the same pathogen. Accordingly we have conducted a study to identify new potential carrier proteins. Twenty-eight proteins, derived from different bacteria, were conjugated to the model polysaccharide Laminarin and tested in mice for their ability in inducing antibodies against the carbohydrate antigen and eight of them were subsequently tested as carrier for serogroup meningococcal C oligosaccharides. Four out of these eight were able to elicit in mice satisfactory anti meningococcal serogroup C titers. Based on immunological evaluation, the Streptococcus pneumoniae protein spr96/2021 was successfully evaluated as carrier for serogroups A, C, W, Y and X meningococcal capsular saccharides. PMID:27317455

  6. 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

  7. 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.

  8. 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

  9. Sterol Carrier Protein-2: Binding Protein for Endocannabinoids

    PubMed Central

    Liedhegner, Elizabeth Sabens; Vogt, Caleb D.; Sem, Daniel S.; Cunningham, Christopher W.

    2015-01-01

    The endocannabinoid (eCB) system, consisting of eCB ligands and the type 1 cannabinoid receptor (CB1R), subserves retrograde, activity-dependent synaptic plasticity in the brain. eCB signaling occurs “on-demand,” thus the processes regulating synthesis, mobilization and degradation of eCBs are also primary mechanisms for the regulation of CB1R activity. The eCBs, N-arachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are poorly soluble in water. We hypothesize that their aqueous solubility, and, therefore, their intracellular and transcellular distribution, are facilitated by protein binding. Using in silico docking studies, we have identified the nonspecific lipid binding protein, sterol carrier protein 2 (SCP-2), as a potential AEA binding protein. The docking studies predict that AEA and AM404 associate with SCP-2 at a putative cholesterol binding pocket with ΔG values of −3.6 and −4.6 kcal/mol, respectively. These values are considerably higher than cholesterol (−6.62 kcal/mol) but consistent with a favorable binding interaction. In support of the docking studies, SCP-2-mediated transfer of cholesterol in vitro is inhibited by micromolar concentrations of AEA; and heterologous expression of SCP-2 in HEK 293 cells increases time-related accumulation of AEA in a temperature-dependent fashion. These results suggest that SCP-2 facilitates cellular uptake of AEA. However, there is no effect of SCP-2 transfection on the cellular accumulation of AEA determined at equilibrium or the IC50 values for AEA, AM404 or 2-AG to inhibit steady state accumulation of radiolabelled AEA. We conclude that SCP-2 is a low affinity binding protein for AEA that can facilitate its cellular uptake but does not contribute significantly to intracellular sequestration of AEA. PMID:24510313

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Structural and bioinformatic characterization of an Acinetobacter baumannii type II carrier protein

    SciTech Connect

    Allen, C. Leigh; Gulick, Andrew M.

    2014-06-01

    The high-resolution crystal structure of a free-standing carrier protein from Acinetobacter baumannii that belongs to a larger NRPS-containing operon, encoded by the ABBFA-003406–ABBFA-003399 genes of A. baumannii strain AB307-0294, that has been implicated in A. baumannii motility, quorum sensing and biofilm formation, is presented. Microorganisms produce a variety of natural products via secondary metabolic biosynthetic pathways. Two of these types of synthetic systems, the nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), use large modular enzymes containing multiple catalytic domains in a single protein. These multidomain enzymes use an integrated carrier protein domain to transport the growing, covalently bound natural product to the neighboring catalytic domains for each step in the synthesis. Interestingly, some PKS and NRPS clusters contain free-standing domains that interact intermolecularly with other proteins. Being expressed outside the architecture of a multi-domain protein, these so-called type II proteins present challenges to understand the precise role they play. Additional structures of individual and multi-domain components of the NRPS enzymes will therefore provide a better understanding of the features that govern the domain interactions in these interesting enzyme systems. The high-resolution crystal structure of a free-standing carrier protein from Acinetobacter baumannii that belongs to a larger NRPS-containing operon, encoded by the ABBFA-003406–ABBFA-003399 genes of A. baumannii strain AB307-0294, that has been implicated in A. baumannii motility, quorum sensing and biofilm formation, is presented here. Comparison with the closest structural homologs of other carrier proteins identifies the requirements for a conserved glycine residue and additional important sequence and structural requirements within the regions that interact with partner proteins.

  16. (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.

  17. Laser Irradiated Growth of Protein Crystal

    NASA Astrophysics Data System (ADS)

    Adachi, Hiroaki; Takano, Kazufumi; Hosokawa, Youichiroh; Inoue, Tsuyoshi; Mori, Yusuke; Matsumura, Hiroyoshi; Yoshimura, Masashi; Tsunaka, Yasuo; Morikawa, Masaaki; Kanaya, Shigenori; Masuhara, Hiroshi; Kai, Yasushi; Sasaki, Takatomo

    2003-07-01

    We succeeded in the first ever generation of protein crystals by laser irradiation. We call this process Laser Irradiated Growth Technique (LIGHT). Effective crystallization was confirmed by applying an intense femtosecond laser. The crystallization period was dramatically shortened by LIGHT. In addition, protein crystals were obtained by LIGHT from normally uncrystallized conditions. These results indicate that intense femtosecond laser irradiation generates crystal nuclei; protein crystals can then be grown from the nuclei that act as seeds in a supersaturated solution. The nuclei formation is possible primarily due to nonlinear nucleation processes of an intense femtosecond laser with a peak intensity of over a gigawatt (GW).

  18. The structure of the human sterol carrier protein X/sterol carrier protein 2 gene (SCP2)

    SciTech Connect

    Ohba, Takashi; Rennert, H.; Pfeifer, S.M.

    1994-11-15

    Sterol carrier protein X (SCPx) is a 58-kDa protein that is localized to peroxisomes. The amino acid sequence of the protein suggests that SCPx may function as a thiolase. The gene encoding SCPx also codes for a 15.3-kDa protein called sterol carrier protein 2 (SCP{sub 2}). Here the authors report the structure of this gene (SCP2), which spans approximately 80 kb and consists of 16 exons and 15 introns. Multiple transcription start sites were identified. The 5{prime} flanking region has characteristics of other peroxisomal protein promoters, which include the absence of a TATA box and G+C-enriched region containing several reverse GC boxes. 24 refs., 3 figs., 1 tab.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. Membrane Protein Crystallization Using Laser Irradiation

    NASA Astrophysics Data System (ADS)

    Adachi, Hiroaki; Murakami, Satoshi; Niino, Ai; Matsumura, Hiroyoshi; Takano, Kazufumi; Inoue, Tsuyoshi; Mori, Yusuke; Yamaguchi, Akihito; Sasaki, Takatomo

    2004-10-01

    We demonstrate the crystallization of a membrane protein using femtosecond laser irradiation. This method, which we call the laser irradiated growth technique (LIGHT), is useful for producing AcrB crystals in a solution of low supersaturation range. LIGHT is characterized by reduced nucleation times. This feature is important for crystallizing membrane proteins because of their labile properties when solubilized as protein-detergent micelles. Using LIGHT, high-quality crystals of a membrane transporter protein, AcrB, were obtained. The resulting crystals were found to be of sufficiently high resolution for X-ray diffraction. The results reported here indicate that LIGHT is a powerful tool for membrane protein crystallization, as well as for the growth of soluble proteins.

  6. Ultrafast carrier dynamics in single-crystal In2Se3 thin layers

    NASA Astrophysics Data System (ADS)

    Tao, Xin; Mafi, Elham; Gu, Yi

    2013-11-01

    Carrier dynamics in single-crystal In2Se3 thin layers with various thicknesses was studied by femtosecond optical pump-probe reflectivity and ultrafast photocurrent measurements. The results suggest that, in thinner (thicker) layers, the carrier recombination dynamics is dominated by three-carrier (bimolecular) Auger process. The Auger time constant was found to decrease with deceasing layer thickness. Surface states were suggested to be the origin of the transition between different Auger processes as the layer thickness varies.

  7. 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

  8. 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.

  9. Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection

    PubMed Central

    Dolezal, Pavel; Aili, Margareta; Tong, Janette; Jiang, Jhih-Hang; Marobbio, Carlo M.; Lee, Sau fung; Schuelein, Ralf; Belluzzo, Simon; Binova, Eva; Mousnier, Aurelie; Frankel, Gad; Giannuzzi, Giulia; Palmieri, Ferdinando; Gabriel, Kipros; Naderer, Thomas; Hartland, Elizabeth L.; Lithgow, Trevor

    2012-01-01

    The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms. PMID:22241989

  10. Protein carriers of conjugate vaccines: characteristics, development, and clinical trials.

    PubMed

    Pichichero, Michael E

    2013-12-01

    The immunogenicity of polysaccharides as human vaccines was enhanced by coupling to protein carriers. Conjugation transformed the T cell-independent polysaccharide vaccines of the past to T cell-dependent antigenic vaccines that were much more immunogenic and launched a renaissance in vaccinology. This review discusses the conjugate vaccines for prevention of infections caused by Hemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis. Specifically, the characteristics of the proteins used in the construction of the vaccines including CRM, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane complex, and Hemophilus influenzae protein D are discussed. The studies that established differences among and key features of conjugate vaccines including immunologic memory induction, reduction of nasopharyngeal colonization and herd immunity, and antibody avidity and avidity maturation are presented. Studies of dose, schedule, response to boosters, of single protein carriers with single and multiple polysaccharides, of multiple protein carriers with multiple polysaccharides and conjugate vaccines administered concurrently with other vaccines are discussed along with undesirable consequences of conjugate vaccines. The clear benefits of conjugate vaccines in improving the protective responses of the immature immune systems of young infants and the senescent immune systems of the elderly have been made clear and opened the way to development of additional vaccines using this technology for future vaccine products. PMID:23955057

  11. Method for controlling protein crystallization

    NASA Technical Reports Server (NTRS)

    Noever, David A. (Inventor)

    1993-01-01

    A method and apparatus for controlling the crystallization of protein by solvent evaporation including placing a drop of protein solution between and in contact with a pair of parallel plates and driving one of the plates toward and away from the other plate in a controlled manner to adjust the spacing between the plates is presented. The drop of solution forms a liquid cylinder having a height dependent upon the plate spacing thereby effecting the surface area available for solvent evaporation. When the spacing is close, evaporation is slow. Evaporation is increased by increasing the spacing between the plates until the breaking point of the liquid cylinder. One plate is mounted upon a fixed post while the other plate is carried by a receptacle movable relative to the post and driven by a belt driven screw drive. The temperature and humidity of the drop of protein solution are controlled by sealing the drop within the receptacle and mounting a heater and dessicant within the receptacle.

  12. 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

  13. 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

  14. 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.

  15. Peptide binding to a bacterial signal peptidase visualized by peptide tethering and carrier-driven crystallization

    PubMed Central

    Ting, Yi Tian; Harris, Paul W. R.; Batot, Gaelle; Brimble, Margaret A.; Baker, Edward N.; Young, Paul G.

    2016-01-01

    Bacterial type I signal peptidases (SPases) are membrane-anchored serine proteases that process the signal peptides of proteins exported via the Sec and Tat secretion systems. Despite their crucial importance for bacterial virulence and their attractiveness as drug targets, only one such enzyme, LepB from Escherichia coli, has been structurally characterized, and the transient nature of peptide binding has stymied attempts to directly visualize SPase–substrate complexes. Here, the crystal structure of SpsB, the type I signal peptidase from the Gram-positive pathogen Staphylococcus aureus, is reported, and a peptide-tethering strategy that exploits the use of carrier-driven crystallization is described. This enabled the determination of the crystal structures of three SpsB–peptide complexes, both with cleavable substrates and with an inhibitory peptide. SpsB–peptide interactions in these complexes are almost exclusively limited to the canonical signal-peptide motif Ala-X-Ala, for which clear specificity pockets are found. Minimal contacts are made outside this core, with the variable side chains of the peptides accommodated in shallow grooves or exposed faces. These results illustrate how high fidelity is retained despite broad sequence diversity, in a process that is vital for cell survival. PMID:26870377

  16. 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.

  17. 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

  18. 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

  19. The MORPHEUS II protein crystallization screen

    PubMed Central

    Gorrec, Fabrice

    2015-01-01

    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. PMID:26144227

  20. Carrier transport simulation of anomalous temperature dependence in nematic liquid crystals.

    PubMed

    Goto, Masanao; Takezoe, Hideo; Ishikawa, Ken

    2007-10-01

    We investigated the carrier transport phenomena in model liquid crystalline systems, which were constructed on the basis of the Gay-Berne potential and Monte Carlo calculation. The carrier transport was analyzed under the condition that the molecular arrangement in the system was fixed and thermally activated carriers were transported by hopping in the system. The carrier transport simulation was performed by Monte Carlo method using Miller-Abrahams hopping ratio. By these calculations, we reproduced the experimental results of the electronic conduction in nematic liquid crystals. PMID:17994925

  1. Carrier transport simulation of anomalous temperature dependence in nematic liquid crystals

    NASA Astrophysics Data System (ADS)

    Goto, Masanao; Takezoe, Hideo; Ishikawa, Ken

    2007-10-01

    We investigated the carrier transport phenomena in model liquid crystalline systems, which were constructed on the basis of the Gay-Berne potential and Monte Carlo calculation. The carrier transport was analyzed under the condition that the molecular arrangement in the system was fixed and thermally activated carriers were transported by hopping in the system. The carrier transport simulation was performed by Monte Carlo method using Miller-Abrahams hopping ratio. By these calculations, we reproduced the experimental results of the electronic conduction in nematic liquid crystals.

  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. Which strategy for a protein crystallization project?

    PubMed

    Kundrot, C E

    2004-03-01

    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. PMID:15004692

  4. 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.

  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. Crystal structures of MBP fusion proteins.

    PubMed

    Waugh, David S

    2016-03-01

    Although chaperone-assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose-binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter-domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP-mediated structural distortions are very rare. PMID:26682969

  7. Protein Crystals Grown in the hand-held Protein Crystallization Apparatus for Microgravity (PCAM)

    NASA Technical Reports Server (NTRS)

    1986-01-01

    Crystals grown in the hand-held Protein Crystallization Apparatus for Microgravity (PCAM) onboard STS-61C. The PCAM has a pedestal in the center of a circular chamber, the surrounding chamber holds an absorbent reservoir that contains a solution of the precipitant. Vapor pressure differences between the protein solution and the reservoir solution force water to move from the protein solution to the reservoir. As protein concentrations increase, protein crystals begin to nucleate and grow.

  8. Charge carrier dynamics in bulk MoS2 crystal studied by transient absorption microscopy

    NASA Astrophysics Data System (ADS)

    Kumar, Nardeep; He, Jiaqi; He, Dawei; Wang, Yongsheng; Zhao, Hui

    2013-04-01

    We report a transient absorption microscopy study of charge carrier dynamics in bulk MoS2 crystals at room temperature. Charge carriers are injected by interband absorption of a 555-nm pulse, and probed by measuring differential reflection of a time-delayed and spatially scanned 660-nm pulse. We find an intervalley transfer time of about 0.35 ps, an energy relaxation time of hot carriers on the order of 50 ps, and a carrier lifetime of 180 ± 20 ps. By monitoring the spatiotemporal dynamics of carriers, we obtained a diffusion coefficient of thermalized electrons of 4.2 ± 0.5 cm2/s, corresponding to a mobility of 170 ± 20 cm2/Vs. We also observed a time-varying diffusion coefficient of hot carriers.

  9. 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.

  10. Role of acyl carrier protein isoforms in plant lipid metabolism

    SciTech Connect

    Not Available

    1990-01-01

    Although acyl carrier protein (ACP) is the best studied protein in plant fatty acid biosynthesis, the in vivo forms of ACPs and their steady state pools have not been examined previously in either seed or leaf. Information about the relative pool sizes of free ACP and its acyl-ACP intermediates is essential for understanding regulation of de novo fatty acid biosynthesis in plants. In this study we utilized antibodies directed against spinach ACP as a sensitive assay to analyze the acyl groups while they were still covalently attached to ACPs. 4 refs., 4 figs.

  11. 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.

  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 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.

  14. 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.

  15. Thermal crystallization mechanism of silk fibroin protein

    NASA Astrophysics Data System (ADS)

    Hu, Xiao

    In this thesis, the thermal crystallization mechanism of silk fibroin protein from Bombyx mori silkworm, was treated as a model for the general study of protein based materials, combining theories from both biophysics and polymer physics fields. A systematic and scientific path way to model the dynamic beta-sheet crystallization process of silk fibroin protein was presented in the following sequence: (1) The crystallinity, fractions of secondary structures, and phase compositions in silk fibroin proteins at any transition stage were determined. Two experimental methods, Fourier transform infrared spectroscopy (FTIR) with Fourier self-deconvolution, and specific reversing heat capacity, were used together for the first time for modeling the static structures and phases in the silk fibroin proteins. The protein secondary structure fractions during the crystallization were quantitatively determined. The possibility of existence of a "rigid amorphous phase" in silk protein was also discussed. (2) The function of bound water during the crystallization process of silk fibroin was studied using heat capacity, and used to build a silk-water dynamic crystallization model. The fundamental concepts and thermal properties of silk fibroin with/without bound water were discussed. Results show that intermolecular bound water molecules, acting as a plasticizer, will cause silk to display a water-induced glass transition around 80°C. During heating, water is lost, and the change of the microenvironment in the silk fibroin chains induces a mesophase prior to thermal crystallization. Real time FTIR during heating and isothermal holding above Tg show the tyrosine side chain changes only during the former process, while beta sheet crystallization occurs only during the latter process. Analogy is made between the crystallization of synthetic polymers according to the four-state scheme of Strobl, and the crystallization process of silk fibroin, which includes an intermediate precursor

  16. Topological Predictions for Integral Membrane Channel and Carrier Proteins

    PubMed Central

    Abhinay, Reddy; Jaehoon, Cho; Sam, Ling; Vamsee, Reddy; Maksim, Shlykov; Milton, Saier

    2014-01-01

    We evaluated topological predictions for nine different programs, HMMTOP, TMHMM, SVMTOP, DAS, SOSUI, TOPCONS, PHOBIUS, MEMSAT-SVM (hereinafter referred to as MEMSAT), and SPOCTOPUS. These programs were first evaluated using four large topologically well-defined families of secondary transporters, and the three best programs were further evaluated using topologically more diverse families of channels and carriers. In the initial studies, the order of accuracy was: SPOCTOPUS>MEMSAT>HMMTOP>TOPCONS>PHOBIUS>TMHMM>SVMTOP>DAS>S OSUI. Some families, such as the Sugar Porter family (2.A.1.1) of the Major Facilitator Superfamily (MFS; TC# 2.A.1) and the Amino acid/Polyamine/Organocation (APC) Family (TC# 2.A.3), were correctly predicted with high accuracy while others, such as the Mitochondrial Carrier (MC) (TC# 2.A.29) and the K+ transporter (Trk) families (TC# 2.A.38), were predicted with much lower accuracy. For small, topologically homogeneous families, SPOCTOPUS and MEMSAT were generally most reliable, while with large, more diverse superfamilies, HMMTOP often proved to have the greatest prediction accuracy. We next developed a novel program, TM-STATS, that tabulates HMMTOP, SPOCTOPUS or MEMSAT-based topological predictions for any subdivision (class, subclass, superfamily, family, subfamily, or any combination of these) of the Transporter Classification Database (TCDB; www.tcdb.org) and examined the following subclasses: α-type channel proteins (TC subclasses 1.A and 1.E), secreted poreforming toxins (TC subclass 1.C) and secondary carriers (subclass 2.A). Histograms 3 were generated for each of these subclasses, and the results were analyzed according to subclass, family and protein. The results provide an update of topological predictions for integral membrane transport proteins as well as guides for the development of more reliable topological prediction programs, taking family-specific characteristics into account. PMID:24992992

  17. Dependence of charge transfer reorganization energy on carrier localisation in organic molecular crystals.

    PubMed

    Bromley, Stefan T; Illas, Francesc; Mas-Torrent, Marta

    2008-01-01

    Taking the organic molecular material dithiophene-tetrathiafulvalene (DT-TTF) as an example of a high mobility organic molecular material, we use density functional calculations to calculate the dependency of the reorganization energy associated with charge carrier transport on: (i) the geometric and electronic responsiveness of the local molecular crystal environment, and, (ii) the local spatial extent of the charge carrier. We find that in our most realistic extended models the charge transfer reorganization energy is strongly dependent on carrier localization. In particular, whereas highly localized carriers are found to be highly susceptible to their charge transfer efficiency being affected by changes in the local crystal environment, more delocalized carriers are better able to maintain their low reorganization energies. Considering that maintaining a relatively small charge transfer reorganization energy magnitude is an important factor in achieving high carrier mobilities, we suggest that those materials better able to sustain carriers with short-range thermally resistant intermolecular delocalisation should be sought for device applications. PMID:18075690

  18. 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

  19. Spectroscopy of Charge Carriers and Traps in Field-Doped Single Crystal Organic Semiconductors

    SciTech Connect

    Zhu, Xiaoyang

    2014-12-10

    The proposed research aims to achieve quantitative, molecular level understanding of charge carriers and traps in field-doped crystalline organic semiconductors via in situ linear and nonlinear optical spectroscopy, in conjunction with transport measurements and molecular/crystal engineering. Organic semiconductors are emerging as viable materials for low-cost electronics and optoelectronics, such as organic photovoltaics (OPV), organic field effect transistors (OFETs), and organic light emitting diodes (OLEDs). Despite extensive studies spanning many decades, a clear understanding of the nature of charge carriers in organic semiconductors is still lacking. It is generally appreciated that polaron formation and charge carrier trapping are two hallmarks associated with electrical transport in organic semiconductors; the former results from the low dielectric constants and weak intermolecular electronic overlap while the latter can be attributed to the prevalence of structural disorder. These properties have lead to the common observation of low charge carrier mobilities, e.g., in the range of 10-5 - 10-3 cm2/Vs, particularly at low carrier concentrations. However, there is also growing evidence that charge carrier mobility approaching those of inorganic semiconductors and metals can exist in some crystalline organic semiconductors, such as pentacene, tetracene and rubrene. A particularly striking example is single crystal rubrene (Figure 1), in which hole mobilities well above 10 cm2/Vs have been observed in OFETs operating at room temperature. Temperature dependent transport and spectroscopic measurements both revealed evidence of free carriers in rubrene. Outstanding questions are: what are the structural features and physical properties that make rubrene so unique? How do we establish fundamental design principles for the development of other organic semiconductors of high mobility? These questions are critically important but not comprehensive, as the nature of

  20. (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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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).

  6. Induction of protein crystallization by platinum nanoparticles

    NASA Astrophysics Data System (ADS)

    Takeda, Yoshihiro; Mafuné, Fumitaka

    2016-03-01

    We have investigated effects of platinum nanoparticles (PtNPs) on protein crystal nucleation. The presence of PtNPs increased the number of crystals in a crystallization solution, indicating that the PtNPs have the ability to promote the crystal nucleation. Dynamic light scattering measurements revealed that the PtNP gathers more than 10 lysozyme molecules around it to form an embryonic complex of PtNP and lysozyme. Zeta potential measurements revealed that the charges of the lysozyme molecules were reduced by delocalization of their charges in the complex. As a result, the energy barrier of association between the complexes is reduced, followed by the nucleation.

  7. Droplet hydrodynamics during lysozyme protein crystallization.

    PubMed

    Pradhan, T; Asfer, M; Panigrahi, P K

    2012-11-01

    Various experimental studies in zero gravity have been reported in the literature for generation of superior quality crystals due to the importance of convective transport on protein crystal quality. However, limited experimental and numerical studies are available in the literature for a complete characterization of transport phenomena during the protein crystal growth process. The present investigation reports experimental results on convective motion inside the droplet during protein crystallization by the vapor diffusion method. Lysozyme crystals are grown using a sitting drop method and micro-particle image velocimetry is used for investigating the detailed hydrodynamics inside the droplet. Dynamic evolution of the flow field for the complete crystal growth process, i.e., during the prenucleation, nucleation, and postnucleation stage, is reported. Various flow transitions are observed during the complete cycle of the protein crystal growth process. Significant Marangoni convection is observed during the prenucleation period followed by buoyancy-driven convection during the postnucleation period. The Marangoni convection flow patterns observed during the prenucleation stage match those of evaporating droplets. The postnucleation convection patterns are similar to those of ethanol-water mixture evaporation with high ethanol concentration. PMID:23214788

  8. 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.

  9. 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

  10. Charge Carrier Lifetimes Exceeding 15 μs in Methylammonium Lead Iodide Single Crystals.

    PubMed

    Bi, Yu; Hutter, Eline M; Fang, Yanjun; Dong, Qingfeng; Huang, Jinsong; Savenije, Tom J

    2016-03-01

    The charge carrier lifetime in organic-inorganic perovskites is one of the most important parameters for modeling and design of solar cells and other types of devices. In this work, we use CH3NH3PbI3 single crystal as a model system to study optical absorption, charge carrier generation, and recombination lifetimes. We show that commonly applied photoluminescence lifetime measurements may dramatically underestimate the intrinsic carrier lifetime in CH3NH3PbI3, which could be due to severe charge recombination at the crystal surface and/or fast electron-hole recombination close to the surface. By using the time-resolved microwave conductivity technique, we investigated the lifetime of free mobile charges inside the crystals. Most importantly, we find that for homogeneous excitation throughout the crystal, the charge carrier lifetime exceeds 15 μs. This means that the diffusion length in CH3NH3PbI3 can be as large as 50 μm if it is no longer limited by the dimensions of the crystallites. PMID:26901658

  11. 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.

  12. 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.

  13. 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.

  14. Protein Crystallization for X-ray Crystallography

    PubMed Central

    Dessau, Moshe A.; Modis, Yorgo

    2011-01-01

    Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography. To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999). Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin

  15. 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

  16. Protein Crystallization: Specific Phenomena and General Insights on Crystallization Kinetics

    NASA Technical Reports Server (NTRS)

    Rosenberger, F.

    1998-01-01

    Experimental and simulation studies of the nucleation and growth kinetics of proteins have revealed phenomena that are specific for macromolecular crystallization, and others that provide a more detailed understanding of solution crystallization in general. The more specific phenomena, which include metastable liquid-liquid phase separations and gelation prior to solid nucleation, are due to the small ratio of the intermolecular interaction-range to the size of molecules involved. The apparently more generally applicable mechanisms include the cascade-like formation of macrosteps, as an intrinsic morphological instability that roots in the coupled bulk transport and nonlinear interface kinetics in systems with mixed growth rate control. Analyses of this nonlinear response provide (a) criteria for the choice of bulk transport conditions to minimize structural defect formation, and (b) indications that the "slow" protein crystallization kinetics stems from the mutual retardation of growth steps.

  17. Quantum effect on the nucleation of plastic deformation carriers and destruction in crystals

    SciTech Connect

    Khon, Yury A. Kaminskii, Petr P.

    2015-10-27

    New concepts on the irreversible crystal deformation as a structure transformation caused by a change in interatomic interactions at fluctuations of the electron density under loading are described. The change in interatomic interactions lead to the excitation of dynamical displacements of atoms. A model and a theory of a deformable pristine crystal taking into account the excitation of thermally activated and dynamical displacements of atoms are suggested. New mechanisms of the nucleation of plastic deformation carriers and destruction in pristine crystals at the real value of the deforming stress are studied.

  18. 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

  19. Datamining protein structure databanks for crystallization patterns of proteins.

    PubMed

    Valafar, Homayoun; Prestegard, James H; Valafar, Faramarz

    2002-12-01

    A study of 345 protein structures selected among 1,500 structures determined by nuclear magnetic resonance (NMR) methods, revealed useful correlations between crystallization properties and several parameters for the studied proteins. NMR methods of structure determination do not require the growth of protein crystals, and hence allow comparison of properties of proteins that have or have not been the subject of crystallographic approaches. One- and two-dimensional statistical analyses of the data confirmed a hypothesized relation between the size of the molecule and its crystallization potential. Furthermore, two-dimensional Bayesian analysis revealed a significant relationship between relative ratio of different secondary structures and the likelihood of success for crystallization trials. The most immediate result is an apparent correlation of crystallization potential with protein size. Further analysis of the data revealed a relationship between the unstructured fraction of proteins and the success of its crystallization. Utilization of Bayesian analysis on the latter correlation resulted in a prediction performance of about 64%, whereas a two-dimensional Bayesian analysis succeeded with a performance of about 75%. PMID:12594078

  20. 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.

  1. Carriers

    MedlinePlus

    ... for those known to be at risk for genetic diseases. Reproductive Choices For couples who are carriers, reproductive decisions can be sensitive. A number of options are available, such as adoption, prenatal testing, and pre-implantation genetic diagnosis (PGD). PGD screens ...

  2. 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

  3. 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

  4. 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.

  5. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8

    SciTech Connect

    Bagautdinov, Bagautdin Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-05-01

    The crystal structure of 3-oxoacyl-(acyl-carrier protein) synthase II from T. thermophilus HB8 has been determined at 2.0 Å resolution and compared with the structures of β-keto-ACP synthases from other sources. The β-ketoacyl-(acyl carrier protein) synthases (β-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 Å resolution. The crystal is orthorhombic, space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 Å, and contains one homodimer in the asymmetric unit. The subunits adopt the well known α-β-α-β-α thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ‘open’ conformation of the Phe396 side chain.

  6. 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

  7. Protein crystallization - is it rocket science?

    PubMed

    DeLucas, L J.

    2001-07-01

    Fueled by initial space shuttle results, the National Aeronautics and Space Administration (NASA) has been supporting fundamental studies of macromolecular crystal growth since 1985. The majority of this research is directed at understanding the relationship between experimental variables and important crystal characteristics. The program has resulted in new methods and technology that will benefit the crystallography community's effort to meet the ever-increasing demand for protein structural information. Microgravity crystallization results indicate a potential impact on structural biology's more challenging problems, as soon as long-duration experiments can be performed on the International Space Station. PMID:11445465

  8. 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

  9. 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.

  10. 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.

  11. Structural Insight into Amino Group-carrier Protein-mediated Lysine Biosynthesis

    PubMed Central

    Yoshida, Ayako; Tomita, Takeo; Fujimura, Tsutomu; Nishiyama, Chiharu; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2015-01-01

    In the biosynthesis of lysine by Thermus thermophilus, the metabolite α-ketoglutarate is converted to the intermediate α-aminoadipate (AAA), which is protected by the 54-amino acid acidic protein LysW. In this study, we determined the crystal structure of LysZ from T. thermophilus (TtLysZ), an amino acid kinase that catalyzes the second step in the AAA to lysine conversion, which was in a complex with LysW at a resolution of 1.85 Å. A crystal analysis coupled with isothermal titration calorimetry of the TtLysZ mutants for TtLysW revealed tight interactions between LysZ and the globular and C-terminal extension domains of the LysW protein, which were mainly attributed to electrostatic forces. These results provided structural evidence for LysW acting as a protecting molecule for the α-amino group of AAA and also as a carrier protein to guarantee better recognition by biosynthetic enzymes for the efficient biosynthesis of lysine. PMID:25392000

  12. 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.

  13. 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.

  14. 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.

  15. (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.

  16. 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).

  17. 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.

  18. 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

  19. (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.

  20. (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.

  1. Convective instability in protein crystal growth

    NASA Astrophysics Data System (ADS)

    Lima, D.; de Wit, A.

    2004-08-01

    The conditions for the onset of convection during protein crystalization from a solution are studied theoretically on the basis of diffusion-convection evolution equations for the concentrations coupled to the Navier-Stokes equation describing the flow velocity. We consider that the density of the solution depends on the concentration of two species, namely, a protein and a precipitating agent, a salt. While the protein is crystallized at the crystal/solution interface, the salt is rejected, and these mechanisms are described by means of boundary conditions for the interface. We find the base profiles for both protein and salt concentrations and perform a linear stability analysis of this basic state with regard to buoyancy induced perturbations. This gives information on the critical diameter of capillaries above which convection may be observed, as well as on the influence of the speed of growth V of the crystal interface on the stability of the system. Numerical integration of the model shows good agreement with the predictions of the linear stability analysis.

  2. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    PubMed Central

    Baum, Bernhard; Lecker, Laura S. M.; Zoltner, Martin; Jaenicke, Elmar; Schnell, Robert; Hunter, William N.; Brenk, Ruth

    2015-01-01

    Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials. PMID:26249693

  3. Structures of Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) and a C164Q mutant provide templates for antibacterial drug discovery and identify a buried potassium ion and a ligand-binding site that is an artefact of the crystal form

    SciTech Connect

    Baum, Bernhard; Lecker, Laura S. M.; Zoltner, Martin; Jaenicke, Elmar; Schnell, Robert; Hunter, William N.; Brenk, Ruth

    2015-07-28

    Three crystal structures of recombinant P. aeruginosa FabF are reported: the apoenzyme, an active-site mutant and a complex with a fragment of a natural product inhibitor. The characterization provides reagents and new information to support antibacterial drug discovery. Bacterial infections remain a serious health concern, in particular causing life-threatening infections of hospitalized and immunocompromised patients. The situation is exacerbated by the rise in antibacterial drug resistance, and new treatments are urgently sought. In this endeavour, accurate structures of molecular targets can support early-stage drug discovery. Here, crystal structures, in three distinct forms, of recombinant Pseudomonas aeruginosa β-ketoacyl-(acyl-carrier-protein) synthase II (FabF) are presented. This enzyme, which is involved in fatty-acid biosynthesis, has been validated by genetic and chemical means as an antibiotic target in Gram-positive bacteria and represents a potential target in Gram-negative bacteria. The structures of apo FabF, of a C164Q mutant in which the binding site is altered to resemble the substrate-bound state and of a complex with 3-(benzoylamino)-2-hydroxybenzoic acid are reported. This compound mimics aspects of a known natural product inhibitor, platensimycin, and surprisingly was observed binding outside the active site, interacting with a symmetry-related molecule. An unusual feature is a completely buried potassium-binding site that was identified in all three structures. Comparisons suggest that this may represent a conserved structural feature of FabF relevant to fold stability. The new structures provide templates for structure-based ligand design and, together with the protocols and reagents, may underpin a target-based drug-discovery project for urgently needed antibacterials.

  4. Inhibition of the Mycobacterium tuberculosis enoyl acyl carrier protein reductase InhA by arylamides

    PubMed Central

    He, Xin; Alian, Akram; Ortiz de Montellano, Paul R.

    2007-01-01

    InhA, the enoyl acyl carrier protein reductase (ENR) from Mycobacterium tuberculosis, is one of the key enzymes involved in the type II fatty acid biosynthesis pathway of M. tuberculosis. We report here the discovery, through high-throughput screening, of a series of arylamides as a novel class of potent InhA inhibitors. These direct InhA inhibitors require no mycobacterial enzymatic activation and thus circumvent the resistance mechanism to antitubercular prodrugs such as INH and ETA that is most commonly observed in drug-resistant clinical isolates. The crystal structure of InhA complexed with one representative inhibitor reveals the binding mode of the inhibitor within the InhA active site. Further optimization through a microtiter synthesis strategy followed by in situ activity screening led to the discovery of a potent InhA inhibitor with in vitro IC50 = 90 nM, representing a 34-fold potency improvement over the lead compound. PMID:17723305

  5. 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

  6. Mixing it up for Protein Crystallization

    NASA Astrophysics Data System (ADS)

    Hansen, Carl; Sommer, Morten; Berger, James; Quake, Stephen

    2005-03-01

    In the post-genomic era, X-ray crystallography has emerged as the workhorse of large-scale structural biology initiatives that seek to understand protein function and interaction at the atomic scale. Despite impressive technological advances in X-ray sources, phasing techniques, and computing power, the determination of protein structure continues to be severely hampered by the difficulties in obtaining high-quality protein crystals. Emergent technologies utilizing microfluidics now have the potential to solve these problems on several levels. We will present two microfluidic devices that have been shown to dramatically improve protein crystallization. The first is a formulation device which allows for the rapid combinatorial mixing of reagents to systematically explore protein solubility behavior. A priori solubility mapping allows for the rational design of optimal crystallization screens that are tailored to a specific target. A second screening device allows for massively parallel sample processing while exploiting the properties of mass transport manifest at the micron scale to ensure slow and efficient mixing kinetics that are difficult to achieve in macroscopic reactors.

  7. 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.

  8. Mycobacterium tuberculosis acyl carrier protein synthase adopts two different pH-dependent structural conformations

    SciTech Connect

    Gokulan, Kuppan; Aggarwal, Anup; Shipman, Lance; Besra, Gurdyal S.; Sacchettini, James C.

    2011-07-01

    Bacterial acyl carrier protein synthase plays an essential role in the synthesis of fatty acids, nonribosomal peptides and polyketides. In Mycobacterium tuberculosis, AcpS or group I phosphopentatheine transferase exhibits two different structural conformations depending upon the pH. The crystal structures of acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) and Corynebacterium ammoniagenes determined at pH 5.3 and pH 6.5, respectively, are reported. Comparison of the Mtb apo-AcpS structure with the recently reported structure of the Mtb AcpS–ADP complex revealed that AcpS adopts two different conformations: the orthorhombic and trigonal space-group structures show structural differences in the α2 helix and in the conformation of the α3–α4 connecting loop, which is in a closed conformation. The apo-AcpS structure shows electron density for the entire model and was obtained at lower pH values (4.4–6.0). In contrast, at a higher pH value (6.5) AcpS undergoes significant conformational changes, resulting in disordered regions that show no electron density in the AcpS model. The solved structures also reveal that C. ammoniagenes AcpS undergoes structural rearrangement in two regions, similar to the recently reported Mtb AcpS–ADP complex structure. In vitro reconstitution experiments show that AcpS has a higher post-translational modification activity between pH 4.4 and 6.0 than at pH values above 6.5, where the activity drops owing to the change in conformation. The results show that apo-AcpS and AcpS–ADP adopt different conformations depending upon the pH conditions of the crystallization solution.

  9. 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.

  10. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1988-01-01

    The solubility and growth of the protein canavalin, and the application of the schlieren technique to study fluid flow in protein crystal growth systems were investigated. These studies have resulted in the proposal of a model to describe protein crystal growth and the preliminary plans for a long-term space flight experiment. Canavalin, which may be crystallized from a basic solution by the addition of hydrogen (H+) ions, was shown to have normal solubility characteristics over the range of temperatures (5 to 25 C) and pH (5 to 7.5) studies. The solubility data combined with growth rate data gathered from the seeded growth of canavalin crystals indicated that the growth rate limiting step is a screw dislocation mechanism. A schlieren apparatus was constructed and flow patterns were observed in Rochelle salt (sodium potassium tartrate), lysozyme, and canavalin. The critical parameters were identified as the change in density with concentration (dp/dc) and the change in index of refraction with concentration (dn/dc). Some of these values were measured for the materials listed. The data for lyrozyme showed non-linearities in plots of optical properties and density vs. concentration. In conjunction with with W. A. Tiller, a model based on colloid stability theory was proposed to describe protein crystallization. The model was used to explain observations made by ourselves and others. The results of this research has lead to the development for a preliminary design for a long-term, low-g experiment. The proposed apparatus is univeral and capable of operation under microprocessor control.

  11. The chain-flipping mechanism of ACP (acyl carrier protein)-dependent enzymes appears universal.

    PubMed

    Cronan, John E

    2014-06-01

    ACPs (acyl carrier proteins) play essential roles in the synthesis of fatty acids, polyketides and non-ribosomal polypeptides. ACP function requires the modification of the protein by attachment of 4'-phosphopantetheine to a conserved serine residue. The phosphopantetheine thiol acts to tether the starting materials and intermediates as their thioesters. ACPs are small highly soluble proteins composed of four α-helices. The helices form a bundle that acts as a hydrophobic sleeve that sequesters the acyl chains and activated thioesters from solvent. However, in the synthesis of fatty acids and complex lipids the enzymes of the pathway must access the thioester and the proximal carbon atoms in order to perform the needed chemistry. How such access is provided without exposure of the acyl chains to solvent has been a longstanding question due to the lack of acyl-ACP-enzyme complexes, a situation generally attributed to the brevity of the interactions of acyl-ACPs with their cognate enzymes. As discussed in the present review the access question has now been answered by four recent crystal structures, each of which shows that the entire acyl chain plus the 4'-phosphopantetheine prosthetic group partitions from the ACP hydrophobic sleeve into a hydrophobic pocket or groove of the enzyme protein, a process termed chain flipping. PMID:24825445

  12. 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

  13. 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. PMID:25950977

  14. An investigation into the role of polymeric carriers on crystal growth within amorphous solid dispersion systems.

    PubMed

    Tian, Yiwei; Jones, David S; Andrews, Gavin P

    2015-04-01

    Using phase diagrams derived from Flory-Huggins theory, we defined the thermodynamic state of amorphous felodipine within three different polymeric carriers. Variation in the solubility and miscibility of felodipine within different polymeric materials (using F-H theory) has been identified and used to select the most suitable polymeric carriers for the production of amorphous drug-polymer solid dispersions. With this information, amorphous felodipine solid dispersions were manufactured using three different polymeric materials (HPMCAS-HF, Soluplus, and PVPK15) at predefined drug loadings, and the crystal growth rates of felodipine from these solid dispersions were investigated. Crystallization of amorphous felodipine was studied using Raman spectral imaging and polarized light microscopy. Using this data, we examined the correlation among several characteristics of solid dispersions to the crystal growth rate of felodipine. An exponential relationship was found to exist between drug loading and crystal growth rate. Moreover, crystal growth within all selected amorphous drug-polymer solid dispersion systems were viscosity dependent (η(-ξ)). The exponent, ξ, was estimated to be 1.36 at a temperature of 80 °C. Values of ξ exceeding 1 may indicate strong viscosity dependent crystal growth in the amorphous drug-polymer solid dispersion systems. We argue that the elevated exponent value (ξ > 1) is a result of drug-polymer mixing which leads to a less fragile amorphous drug-polymer solid dispersion system. All systems investigated displayed an upper critical solution temperature, and the solid-liquid boundary was always higher than the spinodal decomposition curve. Furthermore, for PVP-FD amorphous dispersions at drug loadings exceeding 0.6 volume ratio, the mechanism of phase separation within the metastable zone was found to be driven by nucleation and growth rather than liquid-liquid separation. PMID:25692314

  15. Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria

    SciTech Connect

    Halavaty, Andrei S.; Kim, Youngchang; Minasov, George; Shuvalova, Ludmilla; Dubrovska, Ievgeniia; Winsor, James; Zhou, Min; Onopriyenko, Olena; Skarina, Tatiana; Papazisi, Leka; Kwon, Keehwan; Peterson, Scott N.; Joachimiak, Andrzej; Savchenko, Alexei; Anderson, Wayne F.

    2012-10-01

    The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpS{sub SA}), Vibrio cholerae (AcpS{sub VC}) and Bacillus anthracis (AcpS{sub BA}) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS{sub BA} is emphasized because of the two 3′, 5′-adenosine diphosphate (3′, 5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′, 5′-ADP is bound as the 3′, 5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′, 5′-ADP–AcpS binary complexes. The position of the second 3′, 5′-ADP has never been described before. It is in close proximity to the first 3′, 5′-ADP and the ACP-binding site. The coordination of two ADPs in AcpS{sub BA} may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.

  16. 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.

  17. 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.

  18. Protein crystal growth in low gravity

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.

    1987-01-01

    The solubility and growth mechanism of canavalin were studied, and the applicability of the Schlieren technique to protein crystal growth was investigated. Canavalin which may be crystallized from a basic solution by the addition of hydrogen (H+) ions was shown to have normal solubility characteristics over the range of temperatures (5 to 25 C) and pH (5 to 7.5) studied. The solubility data combined with growth rate data gathered from the seeded growth of canavalin crystals indicated that the growth mechanism at high supersaturation ratios (>1.28) is screw dislocation like. A Schlieren apparatus was constructed and flow patterns were observed in Rochelle salt (sodium potassium tartrate), lysozyme, and canavalin. The critical parameters were identified as the change in density with concentration (dp/dc) and the change in index of refraction with concentration (dn/dc). Some of these values were measured for the materials listed.

  19. 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

  20. A glass polyalkenoate cement carrier for bone morphogenetic proteins.

    PubMed

    Alhalawani, Adel M F; Rodriguez, Omar; Curran, Declan J; Co, Russell; Kieran, Sean; Arshad, Saad; Keenan, Timothy J; Wren, Anthony W; Crasto, Gazelle; Peel, Sean A F; Towler, Mark R

    2015-03-01

    This work considers a glass polyalkenoate cement (GPC)-based carrier for the effective delivery of bone morphogenetic proteins (BMPs) at an implantation site. A 0.12 CaO-0.04 SrO-0.36 ZnO-0.48 SiO2 based glass and poly(acrylic acid) (PAA, Mw 213,000) were employed for the fabrication of the GPC. The media used for the water source in the GPC reaction was altered to produce a series of GPCs. The GPC liquid media was either 100 % distilled water with additions of albumin at 0, 2, 5 and 8 wt% of the glass content, 100 % formulation buffer (IFB), and 100 % BMP (150 µg rhBMP-2/ml IFB). Rheological properties, compressive strength, ion release profiles and BMP release were evaluated. Working times (Tw) of the formulated GPCs significantly increased with the addition of 2 % albumin and remained constant with further increases in albumin content or IFB solutions. Setting time (Ts) experienced an increase with 2 and 5 % albumin content, but a decrease with 8 % albumin. Changing the liquid source to IFB containing 5 % albumin had no significant effect on Ts compared to the 8 % albumin-containing BT101. Replacing the albumin with IFB/BMP-2 did not significantly affect Tw. However, Ts increased for the BT101_BMP-2 containing GPCs, compared to all other samples. The compressive strength evaluated 1 day post cement mixing was not affected significantly by the incorporation of BMPs, but the ion release did increase from the cements, particularly for Zn and Sr. The GPCs released BMP after the first day, which decreased in content during the following 6 days. This study has proven that BMPs can be immobilized into GPCs and may result in novel materials for clinical applications. PMID:25773232

  1. Crystallization of G Protein-Coupled Receptors

    PubMed Central

    Salom, David; Padayatti, Pius S.; Palczewski, Krzysztof

    2015-01-01

    Oligomerization is one of several mechanisms that can regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallography and NMR are the only methods able to reveal the details of receptor–receptor interactions at an atomic level, and several GPCR homodimers already have been described from crystal structures. Two clusters of symmetric interfaces have been identified from these structures that concur with biochemical data, one involving helices I, II, and VIII and the other formed mainly by helices V and VI. In this chapter, we describe the protocols used in our laboratory for the crystallization of rhodopsin and the β2-adrenergic receptor (β2-AR). For bovine rhodopsin, we developed a new purification strategy including a (NH4)2SO4-induced phase separation that proved essential to obtain crystals of photoactivated rhodopsin containing parallel dimers. Crystallization of native bovine rhodopsin was achieved by the classic vapor-diffusion technique. For β2-AR, we developed a purification strategy based on previously published protocols employing a lipidic cubic phase to obtain diffracting crystals of a β2-AR/T4-lysozyme chimera bound to the antagonist carazolol. PMID:24143992

  2. (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.

  3. 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.

  4. 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

  5. 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.

  6. 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

    2008-11-01

    The Microcapillary Protein Crystallization System (MPCS) is a new protein-crystallization technology used to generate nanolitre-sized crystallization experiments for crystal screening and optimization. Using the MPCS, diffraction-ready crystals were grown in the plastic MPCS CrystalCard and were used to solve the structure of methionine-R-sulfoxide reductase. 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, ∼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.

  7. Antibody response by cultured spleen fragments from carrier-primed mice to hapten-protein conjugates.

    PubMed

    Hurme, M; Nakamura, I; Kaartinen, M; Mäkelä, O

    1975-01-01

    Hapten-protein conjugates stimulated very poor anti-hapten responses in mouse spleen fragment cultures from unimmunized mice, whereas hapten coupled to type III pneumococcal polysaccharide or polylysine induced good responses. When the donors of the fragments were primed with the carrier protein, hapten-protein conjugates induced a strong anti-hapten response. Both the true primary and the carrier-primed response in vitro consisted mainly of IgA antibodies of 9-13S. In carrier-primed responses also IgM was produced at the beginning and IgG at the end of those responses. PMID:1080285

  8. Spiro-OMeTAD single crystals: Remarkably enhanced charge-carrier transport via mesoscale ordering.

    PubMed

    Shi, Dong; Qin, Xiang; Li, Yuan; He, Yao; Zhong, Cheng; Pan, Jun; Dong, Huanli; Xu, Wei; Li, Tao; Hu, Wenping; Brédas, Jean-Luc; Bakr, Osman M

    2016-04-01

    We report the crystal structure and hole-transport mechanism in spiro-OMeTAD [2,2',7,7'-tetrakis(N,N-di-p-methoxyphenyl-amine)9,9'-spirobifluorene], the dominant hole-transporting material in perovskite and solid-state dye-sensitized solar cells. Despite spiro-OMeTAD's paramount role in such devices, its crystal structure was unknown because of highly disordered solution-processed films; the hole-transport pathways remained ill-defined and the charge carrier mobilities were low, posing a major bottleneck for advancing cell efficiencies. We devised an antisolvent crystallization strategy to grow single crystals of spiro-OMeTAD, which allowed us to experimentally elucidate its molecular packing and transport properties. Electronic structure calculations enabled us to map spiro-OMeTAD's intermolecular charge-hopping pathways. Promisingly, single-crystal mobilities were found to exceed their thin-film counterparts by three orders of magnitude. Our findings underscore mesoscale ordering as a key strategy to achieving breakthroughs in hole-transport material engineering of solar cells. PMID:27152342

  9. Spiro-OMeTAD single crystals: Remarkably enhanced charge-carrier transport via mesoscale ordering

    PubMed Central

    Shi, Dong; Qin, Xiang; Li, Yuan; He, Yao; Zhong, Cheng; Pan, Jun; Dong, Huanli; Xu, Wei; Li, Tao; Hu, Wenping; Brédas, Jean-Luc; Bakr, Osman M.

    2016-01-01

    We report the crystal structure and hole-transport mechanism in spiro-OMeTAD [2,2′,7,7′-tetrakis(N,N-di-p-methoxyphenyl-amine)9,9′-spirobifluorene], the dominant hole-transporting material in perovskite and solid-state dye-sensitized solar cells. Despite spiro-OMeTAD’s paramount role in such devices, its crystal structure was unknown because of highly disordered solution-processed films; the hole-transport pathways remained ill-defined and the charge carrier mobilities were low, posing a major bottleneck for advancing cell efficiencies. We devised an antisolvent crystallization strategy to grow single crystals of spiro-OMeTAD, which allowed us to experimentally elucidate its molecular packing and transport properties. Electronic structure calculations enabled us to map spiro-OMeTAD’s intermolecular charge-hopping pathways. Promisingly, single-crystal mobilities were found to exceed their thin-film counterparts by three orders of magnitude. Our findings underscore mesoscale ordering as a key strategy to achieving breakthroughs in hole-transport material engineering of solar cells. PMID:27152342

  10. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8

    PubMed Central

    Bagautdinov, Bagautdin; Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-01-01

    The β-ketoacyl-(acyl carrier protein) synthases (β-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 Å resolution. The crystal is orthorhombic, space group P21212, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 Å, and contains one homodimer in the asymmetric unit. The subunits adopt the well known α-β-α-β-α thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ‘open’ conformation of the Phe396 side chain. PMID:18453702

  11. Structure of 3-oxoacyl-(acyl-carrier protein) synthase II from Thermus thermophilus HB8.

    PubMed

    Bagautdinov, Bagautdin; Ukita, Yoko; Miyano, Masashi; Kunishima, Naoki

    2008-05-01

    The beta-ketoacyl-(acyl carrier protein) synthases (beta-keto-ACP synthases; KAS) catalyse the addition of two-carbon units to the growing acyl chain during the elongation phase of fatty-acid synthesis. As key regulators of bacterial fatty-acid synthesis, they are promising targets for the development of new antibacterial agents. The crystal structure of 3-oxoacyl-ACP synthase II from Thermus thermophilus HB8 (TtKAS II) has been solved by molecular replacement and refined at 2.0 A resolution. The crystal is orthorhombic, space group P2(1)2(1)2, with unit-cell parameters a = 72.07, b = 185.57, c = 62.52 A, and contains one homodimer in the asymmetric unit. The subunits adopt the well known alpha-beta-alpha-beta-alpha thiolase fold that is common to ACP synthases. The structural and sequence similarities of TtKAS II to KAS I and KAS II enzymes of known structure from other sources support the hypothesis of comparable enzymatic activity. The dimeric state of TtKAS II is important to create each fatty-acid-binding pocket. Closer examination of KAS structures reveals that compared with other KAS structures in the apo form, the active site of TtKAS II is more accessible because of the ;open' conformation of the Phe396 side chain. PMID:18453702

  12. Decarboxylation of malonyl-(acyl carrier protein) by 3-oxoacyl-(acyl carrier protein) synthases in plant fatty acid biosynthesis.

    PubMed Central

    Winter, E; Brummel, M; Schuch, R; Spener, F

    1997-01-01

    In order to identify regulatory steps in fatty acid biosynthesis, the influence of intermediate 3-oxoacyl-(acyl carrier proteins) (3-oxoacyl-ACPs) and end-product acyl-ACPs of the fatty acid synthase reaction on the condensation reaction was investigated in vitro, using total fatty acid synthase preparations and purified 3-oxoacyl-ACP synthases (KASs; EC 2.3.1.41) from Cuphea lanceolata seeds. KAS I and II in the fatty acid synthase preparations were assayed for the elongation of octanoyl- and hexadecanoyl-ACP respectively, and the accumulation of the corresponding condensation product 3-oxoacyl-ACP was studied by modulating the content of the reducing equivalentS NADH and NADPH. Complete omission of reducing equivalents resulted with either KAS in the abnormal synthesis of acetyl-ACP from malonyl-ACP by a decarboxylation reaction. Supplementation with NADPH or NADH, separately or in combination with recombinant 3-oxoacyl-ACP reductase (EC 1.1.1.100), led to a decrease in the amount of acetyl-ACP and a simultaneous increase in elongation products. This demonstrates that the accumulation of 3-oxoacyl-ACP inhibits the condensation reaction on the one hand, and induces the decarboxylation of malonyl-ACP on the other. By carrying out similar experiments with purified enzymes, this decarboxylation was attributed to the action of KAS. Our data point to a regulatory mechanism for the degradation of malonyl-ACP in plants which is activated by the accumulation of the fatty acid synthase intermediate 3-oxoacyl-ACP. PMID:9020860

  13. Characterization of the yellow fever mosquito sterol carrier protein-2 like 3 gene and ligand-bound protein structure

    SciTech Connect

    Dyer, David H.; Vyazunova, Irina; Lorch, Jeffery M.; Forest, Katrina T.; Lan, Que

    2009-06-12

    The sterol carrier protein-2 like 3 gene (AeSCP-2L3), a new member of the SCP-2 protein family, is identified from the yellow fever mosquito, Aedes aegypti. The predicted molecular weight of AeSCP-2L3 is 13.4 kDa with a calculated pI of 4.98. AeSCP-2L3 transcription occurs in the larval feeding stages and the mRNA levels decrease in pupae and adults. The highest levels of AeSCP-2L3 gene expression are found in the body wall, and possibly originated in the fat body. This is the first report of a mosquito SCP-2-like protein with prominent expression in tissue other than the midgut. The X-ray protein crystal structure of AeSCP-2L3 reveals a bound C16 fatty acid whose acyl tail penetrates deeply into a hydrophobic cavity. Interestingly, the ligand-binding cavity is slightly larger than previously described for AeSCP-2 (Dyer et al. J Biol Chem 278:39085-39091, 2003) and AeSCP-2L2 (Dyer et al. J Lipid Res M700460-JLR200, 2007). There are also an additional 10 amino acids in SCP-2L3 that are not present in other characterized mosquito SCP-2s forming an extended loop between {beta}3 and {beta}4. Otherwise, the protein backbone is exceedingly similar to other SCP-2 and SCP-2-like proteins. In contrast to this observed high structural homology of members in the mosquito SCP2 family, the amino acid sequence identity between the members is less than 30%. The results from structural analysis imply that there have been evolutionary constraints that favor the SCP-2 C{alpha} backbone fold while the specificity of ligand binding can be altered.

  14. 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

  15. Evaluation of anisotropic charge carrier mobility of perylene single crystals by time-of-flight method

    NASA Astrophysics Data System (ADS)

    Kougo, Junichi; Ishikawa, Ken

    2016-03-01

    The charge carrier mobilities along the vertical and lateral directions of perylene platelet single crystals were measured by the time-of-flight (TOF) method. In the lateral directional measurement, the entire region between electrodes was irradiated to obtain measurable signals. The transient photocurrent was different from the conventional TOF measurements; hence, we developed an analytic method for lateral directional measurement. The electron mobilities along the thickness and lateral directions were 0.33 and 2.0 cm2·V-1·s-1 and the hole mobilities were 0.12 and 0.6 cm2·V-1·s-1, respectively.

  16. IR laser-induced protein crystal transformation

    PubMed Central

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

    2014-01-01

    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. PMID:24816092

  17. 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.

  18. 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.

  19. 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

  20. Thermal conductivity of heavily doped bulk crystals GaN:O. Free carriers contribution

    NASA Astrophysics Data System (ADS)

    Jeżowski, A.; Churiukova, O.; Mucha, J.; Suski, T.; Obukhov, I. A.; Danilchenko, B. A.

    2015-08-01

    Here we report the results of an experimental study of the thermal conductivity of GaN crystals doped by oxygen with concentrations of 4 × 1016, 2.6 × 1018 and 1.1 × 1020 cm-3, carried out in the temperature interval 7-318 K. We observed the highest thermal conductivity ever reported for GaN, 269 Wm-1 K-1 at 300 K, in the sample with the lowest oxygen content. This result is explained by the renormalization of GaN elastic constants, caused by the effect of spontaneous polarization. Results were analyzed using the Callaway model. The contribution of phonon scattering by free carriers in doped GaN crystals was considered for the first time. We show that free electrons reduce the thermal conductivity by up to 32%-42% at 300 K for a sample with a 1.1 × 1020 cm-3 of oxygen concentration.

  1. 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.

  2. Small Device for Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Dr. Daniel Carter (center), president of New Century Pharmaceuticals, and Dr. Joseph Ho (right), vice president, examine a diffusion Controlled Apparatus for Microgravity (DCAM). At left, Dr. John Ruble, a senior scientist, examines some specimens. The plastic DCAM has two chambers joined by a porous plug through which fluids can diffuse at a controlled rate. This allows researchers to mix protein solutions on Earth and load them aboard the Space Shuttle shortly before launch. The diffusion and crystallization processes are already under way, but at such a slow pace that crystals do not start growing before the DCAM is in orbit aboard the Shuttle or a space station. Dozens of DCAM units can be flown in a small volume and require virtually no crew attention. Specimens are returned to Earth for analysis. Photo credit: NASA/Marshall Space Flight Center

  3. Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis.

    PubMed

    Miyanaga, Akimasa; Iwasawa, Shohei; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2016-02-16

    Acyltransferases (ATs) are key determinants of building block specificity in polyketide biosynthesis. Despite the importance of protein-protein interactions between AT and acyl carrier protein (ACP) during the acyltransfer reaction, the mechanism of ACP recognition by AT is not understood in detail. Herein, we report the crystal structure of AT VinK, which transfers a dipeptide group between two ACPs, VinL and VinP1LdACP, in vicenistatin biosynthesis. The isolated VinK structure showed a unique substrate-binding pocket for the dipeptide group linked to ACP. To gain greater insight into the mechanism of ACP recognition, we attempted to crystallize the VinK-ACP complexes. Because transient enzyme-ACP complexes are difficult to crystallize, we developed a covalent cross-linking strategy using a bifunctional maleimide reagent to trap the VinK-ACP complexes, allowing the determination of the crystal structure of the VinK-VinL complex. In the complex structure, Arg-153, Met-206, and Arg-299 of VinK interact with the negatively charged helix II region of VinL. The VinK-VinL complex structure allows, to our knowledge, the first visualization of the interaction between AT and ACP and provides detailed mechanistic insights into ACP recognition by AT. PMID:26831085

  4. Ultratight crystal packing of a 10 kDa protein

    SciTech Connect

    Trillo-Muyo, Sergio; Chruszcz, Maksymilian; Minor, Wladek; Kuisiene, Nomeda

    2013-03-01

    The crystal structure of the C-terminal domain of a putative U32 peptidase from G. thermoleovorans is reported; it is one of the most tightly packed protein structures reported to date. While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air-sensitive high-solvent-content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C-terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported.

  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. 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

  7. A Pathogenic Fungi Diphenyl Ether Phytotoxin Targets Plant Enoyl (Acyl Carrier Protein) Reductase[W

    PubMed Central

    Dayan, Franck E.; Ferreira, Daneel; Wang, Yan-Hong; Khan, Ikhlas A.; McInroy, John A.; Pan, Zhiqiang

    2008-01-01

    Cyperin is a natural diphenyl ether phytotoxin produced by several fungal plant pathogens. At high concentrations, this metabolite inhibits protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis. However, unlike its herbicide structural analogs, the mode of action of cyperin is not light dependent, causing loss of membrane integrity in the dark. We report that this natural diphenyl ether inhibits Arabidopsis (Arabidopsis thaliana) enoyl (acyl carrier protein) reductase (ENR). This enzyme is also sensitive to triclosan, a synthetic antimicrobial diphenyl ether. Whereas cyperin was much less potent than triclosan on this target site, their ability to cause light-independent disruption of membrane integrity and inhibition of ENR is similar at their respective phytotoxic concentrations. The sequence of ENR is highly conserved within higher plants and a homology model of Arabidopsis ENR was derived from the crystal structure of the protein from Brassica napus. Cyperin mimicked the binding of triclosan in the binding pocket of ENR. Both molecules were stabilized by the π-π stacking interaction between one of their phenyl rings and the nicotinamide ring of the NAD+. Furthermore, the side chain of tyrosine is involved in hydrogen bonding with a phenolic hydroxy group of cyperin. Therefore, cyperin may contribute to the virulence of the pathogens by inhibiting ENR and destabilizing the membrane integrity of the cells surrounding the point of infection. PMID:18467464

  8. Synthesis and Ultrafast Carrier Dynamics of Single-Crystal Two-Dimensional CuInSe2 Nanosheets.

    PubMed

    Tao, Xin; Mafi, Elham; Gu, Yi

    2014-08-21

    We report, for the first time, the synthesis of single-crystal two-dimensional (2D) CuInSe2 nanosheets and the studies of ultrafast carrier dynamics and transport in this 2D material. Particularly, single-crystal 2D CuInSe2 with various thicknesses in the nanometer regime were fabricated by a solid-state chemical reaction between Cu and single-crystal exfoliated In2Se3 nanosheets. Characteristics of transient optical reflectivity, obtained from femtosecond optical pump-probe measurements on single CuInSe2 nanosheets, suggest that the hot carrier cooling process dominates the carrier dynamics within a few picoseconds following the optical excitation. Spatially resolved pump-probe measurements, coupled to simple model calculations, were used to obtain the ambipolar hot carrier diffusion coefficient in single nanosheets. The dependence of the hot carrier diffusion coefficient on the nanosheet thickness provides insight into the limiting mechanisms of hot carrier transport and can be used to gauge the possibility of efficient hot carrier collection in nanostructured CuInSe2 solar cells. PMID:26278089

  9. A Superhydrophobic Surface Templated by Protein Self-Assembly and Emerging Application toward Protein Crystallization.

    PubMed

    Gao, Aiting; Wu, Qian; Wang, Dehui; Ha, Yuan; Chen, Zhijun; Yang, Peng

    2016-01-20

    A proteinaceous superhydrophobic material for facile protein crystallization is reported. The lysozyme phase transition is rationally manipulated to form a reliable superhydrophobic coating on virtually arbitrary material surfaces with good thermostability and mechanical robustness. Such a surface exhibits a fascinating capability to drive protein crystallization, and the protein crystal array can be facilitated in a large area at an ultralow protein concentration. PMID:26607764

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. Imaging of protein crystals with two–photon microscopy†

    PubMed Central

    Padayatti, Pius; Palczewska, Grazyna; Sun, Wenyu; Palczewski, Krzysztof; Salom, David

    2012-01-01

    Second–order non–linear optical imaging of chiral crystals (SONICC), that portrays second harmonic generation (SHG) by non–centrosymmetric crystals, is emerging as a powerful imaging technique for protein crystals in media opaque to visible light because of its high signal–to–noise ratio. Here we report the incorporation of both SONICC and two–photon excited fluorescence (TPEF) into one imaging system that allows visualization of crystals as small as ~10 μm in their longest dimension. Using this system, we then documented an inverse correlation between the level of symmetry in examined crystals and the intensity of their SHG. Moreover, because of blue-green TPEF exhibited by most tested protein crystals, we also could identify and image SHG–silent protein crystals. Our experimental data suggests that the TPEF in protein crystals is mainly caused by the oxidation of tryptophan residues. Additionally, we found that unspecific fluorescent dyes are able to bind to lysozyme crystals and enhance their detection by TPFE. We finally confirmed that the observed fluorescence was generated by a two-photon rather than a three-photon process. The capability for imaging small protein crystals in turbid or opaque media with non–damaging infrared light in a single system, makes the combination of SHG and intrinsic visible TPEF a powerful tool for non–destructive protein crystal identification and characterization during crystallization trials. PMID:22324807

  17. Imaging of Protein Crystals with Two-Photon Microscopy

    SciTech Connect

    Padayatti, Pius; Palczewska, Grazyna; Sun, Wenyu; Palczewski, Krzysztof; Salom, David

    2012-05-02

    Second-order nonlinear optical imaging of chiral crystals (SONICC), which portrays second-harmonic generation (SHG) by noncentrosymmetric crystals, is emerging as a powerful imaging technique for protein crystals in media opaque to visible light because of its high signal-to-noise ratio. Here we report the incorporation of both SONICC and two-photon excited fluorescence (TPEF) into one imaging system that allows visualization of crystals as small as 10 {mu}m in their longest dimension. Using this system, we then documented an inverse correlation between the level of symmetry in examined crystals and the intensity of their SHG. Moreover, because of blue-green TPEF exhibited by most tested protein crystals, we also could identify and image SHG-silent protein crystals. Our experimental data suggest that the TPEF in protein crystals is mainly caused by the oxidation of tryptophan residues. Additionally, we found that unspecific fluorescent dyes are able to bind to lysozyme crystals and enhance their detection by TPEF. We finally confirmed that the observed fluorescence was generated by a two-photon rather than a three-photon process. The capability for imaging small protein crystals in turbid or opaque media with nondamaging infrared light in a single system makes the combination of SHG and intrinsic visible TPEF a powerful tool for nondestructive protein crystal identification and characterization during crystallization trials.

  18. 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.

  19. Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

    PubMed Central

    Miyanaga, Akimasa; Iwasawa, Shohei; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2016-01-01

    Acyltransferases (ATs) are key determinants of building block specificity in polyketide biosynthesis. Despite the importance of protein–protein interactions between AT and acyl carrier protein (ACP) during the acyltransfer reaction, the mechanism of ACP recognition by AT is not understood in detail. Herein, we report the crystal structure of AT VinK, which transfers a dipeptide group between two ACPs, VinL and VinP1LdACP, in vicenistatin biosynthesis. The isolated VinK structure showed a unique substrate-binding pocket for the dipeptide group linked to ACP. To gain greater insight into the mechanism of ACP recognition, we attempted to crystallize the VinK–ACP complexes. Because transient enzyme–ACP complexes are difficult to crystallize, we developed a covalent cross-linking strategy using a bifunctional maleimide reagent to trap the VinK–ACP complexes, allowing the determination of the crystal structure of the VinK–VinL complex. In the complex structure, Arg-153, Met-206, and Arg-299 of VinK interact with the negatively charged helix II region of VinL. The VinK–VinL complex structure allows, to our knowledge, the first visualization of the interaction between AT and ACP and provides detailed mechanistic insights into ACP recognition by AT. PMID:26831085

  20. 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.

  1. Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

    SciTech Connect

    Economou, Nicoleta J.; Zentner, Isaac J.; Lazo, Edwin; Jakoncic, Jean; Stojanoff, Vivian; Weeks, Stephen D.; Grasty, Kimberly C.; Cocklin, Simon; Loll, Patrick J.

    2013-04-01

    Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance.

  2. Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

    SciTech Connect

    Ghadbane, Hemza; Brown, Alistair K.; Kremer, Laurent; Besra, Gurdyal S. Fütterer, Klaus

    2007-10-01

    Binding of Ni{sup 2+} ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution. Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni{sup 2+} ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

  3. 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.

  4. 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

  5. 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.

  6. Carrier scattering processes and low energy phonon spectroscopy in hybrid perovskites crystals

    NASA Astrophysics Data System (ADS)

    Even, Jacky; Paofai, Serge; Bourges, Philippe; Letoublon, Antoine; Cordier, Stéphane; Durand, Olivier; Katan, Claudine

    2016-03-01

    Despite the wealth of research conducted the last three years on hybrid organic perovskites (HOP), several questions remain open including: to what extend the organic moiety changes the properties of the material as compared to allinorganic (AIP) related perovskite structures. To ultimately reach an answer to this question, we have recently introduced two approaches that were designed to take the stochastic molecular degrees of freedom into account, and suggested that the high temperature cubic phase of HOP and AIP is an appropriate reference phase to rationalize HOP's properties. In this paper, we recall the main concepts and discuss more specifically the various possible couplings between charge carriers and low energy excitations such as acoustic and optical phonons. As available experimental or simulated data on low energy excitations are limited, we also present preliminary neutron scattering and ultrasonic measurements obtained and freshly prepared single crystals of CH3NH3PbBr3.

  7. 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. PMID:26574563

  8. Inhibition of the Mycobacterium tuberculosis enoyl acyl carrier protein reductase InhA by arylamides.

    PubMed

    He, Xin; Alian, Akram; Ortiz de Montellano, Paul R

    2007-11-01

    InhA, the enoyl acyl carrier protein reductase (ENR) from Mycobacterium tuberculosis, is one of the key enzymes involved in the type II fatty acid biosynthesis pathway of M. tuberculosis. We report here the discovery, through high-throughput screening, of a series of arylamides as a novel class of potent InhA inhibitors. These direct InhA inhibitors require no mycobacterial enzymatic activation and thus circumvent the resistance mechanism to antitubercular prodrugs such as INH and ETA that is most commonly observed in drug-resistant clinical isolates. The crystal structure of InhA complexed with one representative inhibitor reveals the binding mode of the inhibitor within the InhA active site. Further optimization through a microtiter synthesis strategy followed by in situ activity screening led to the discovery of a potent InhA inhibitor with in vitro IC(50)=90 nM, representing a 34-fold potency improvement over the lead compound. PMID:17723305

  9. 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.

  10. Mycobacterium tuberculosis acyl carrier protein synthase adopts two different pH-dependent structural conformations

    SciTech Connect

    Gokulan, Kuppan; Aggarwal, Anup; Shipman, Lance; Besra, Gurdyal S.; Sacchettini, James C.

    2011-09-20

    The crystal structures of acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) and Corynebacterium ammoniagenes determined at pH 5.3 and pH 6.5, respectively, are reported. Comparison of the Mtb apo-AcpS structure with the recently reported structure of the Mtb AcpS-ADP complex revealed that AcpS adopts two different conformations: the orthorhombic and trigonal space-group structures show structural differences in the {alpha}2 helix and in the conformation of the {alpha}3-{alpha}4 connecting loop, which is in a closed conformation. The apo-AcpS structure shows electron density for the entire model and was obtained at lower pH values (4.4-6.0). In contrast, at a higher pH value (6.5) AcpS undergoes significant conformational changes, resulting in disordered regions that show no electron density in the AcpS model. The solved structures also reveal that C. ammoniagenes AcpS undergoes structural rearrangement in two regions, similar to the recently reported Mtb AcpS-ADP complex structure. In vitro reconstitution experiments show that AcpS has a higher post-translational modification activity between pH 4.4 and 6.0 than at pH values above 6.5, where the activity drops owing to the change in conformation. The results show that apo-AcpS and AcpS-ADP adopt different conformations depending upon the pH conditions of the crystallization solution.

  11. 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.

  12. 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.

  13. 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.

  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. PMID:25700885

  15. Protein Nanoparticles as Drug Delivery Carriers for Cancer Therapy

    PubMed Central

    Lohcharoenkal, Warangkana; Wang, Liying; Chen, Yi Charlie

    2014-01-01

    Nanoparticles have increasingly been used for a variety of applications, most notably for the delivery of therapeutic and diagnostic agents. A large number of nanoparticle drug delivery systems have been developed for cancer treatment and various materials have been explored as drug delivery agents to improve the therapeutic efficacy and safety of anticancer drugs. Natural biomolecules such as proteins are an attractive alternative to synthetic polymers which are commonly used in drug formulations because of their safety. In general, protein nanoparticles offer a number of advantages including biocompatibility and biodegradability. They can be prepared under mild conditions without the use of toxic chemicals or organic solvents. Moreover, due to their defined primary structure, protein-based nanoparticles offer various possibilities for surface modifications including covalent attachment of drugs and targeting ligands. In this paper, we review the most significant advancements in protein nanoparticle technology and their use in drug delivery arena. We then examine the various sources of protein materials that have been used successfully for the construction of protein nanoparticles as well as their methods of preparation. Finally, we discuss the applications of protein nanoparticles in cancer therapy. PMID:24772414

  16. 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.

  17. Quantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State Nanopores.

    PubMed

    Kong, Jinglin; Bell, Nicholas A W; Keyser, Ulrich F

    2016-06-01

    Designed "DNA carriers" have been proposed as a new method for nanopore based specific protein detection. In this system, target protein molecules bind to a long DNA strand at a defined position creating a second level transient current drop against the background DNA translocation. Here, we demonstrate the ability of this system to quantify protein concentrations in the nanomolar range. After incubation with target protein at different concentrations, the fraction of DNA translocations showing a secondary current spike allows for the quantification of the corresponding protein concentration. For our proof-of-principle experiments we use two standard binding systems, biotin-streptavidin and digoxigenin-antidigoxigenin, that allow for measurements of the concentration down to the low nanomolar range. The results demonstrate the potential for a novel quantitative and specific protein detection scheme using the DNA carrier method. PMID:27121643

  18. 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.

  19. Effect of Fe-doping on nonlinear optical responses and carrier trapping dynamics in GaN single crystals

    SciTech Connect

    Fang, Yu; Yang, Junyi; Yang, Yong; Zhou, Feng; Wu, Xingzhi; Xiao, Zhengguo; Song, Yinglin

    2015-08-03

    We presented a quantitative study on the Fe-doping concentration dependence of optical nonlinearities and ultrafast carrier dynamics in Fe-doped GaN (GaN:Fe) single crystals using picosecond Z-scan and femtosecond pump-probe with phase object techniques under two-photon excitation. In contrast to the two-photon absorption that was found to be independent on the Fe-doping, the nonlinear refraction decreased with the Fe concentration due to the fast carrier trapping effect of Fe{sup 3+}/Fe{sup 2+} deep acceptors, which simultaneously acted as an efficient non-radiative recombination channels for excess carriers. Remarkably, compared to that of Si-doped GaN bulk crystal, the free-carrier refraction effect in GaN:Fe crystals was found to be enhanced considerably since Fe-doping and the effective carrier lifetime (∼10 ps) could be tuned over three orders of magnitude at high Fe-doping level of 1 × 10{sup 19 }cm{sup −3}.

  20. 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.

  1. Crystallization of asymmetric patchy models for globular proteins in solution

    NASA Astrophysics Data System (ADS)

    Fusco, Diana; Charbonneau, Patrick

    2013-07-01

    Asymmetric patchy particle models have recently been shown to describe the crystallization of small globular proteins with near-quantitative accuracy. Here, we investigate how asymmetry in patch geometry and bond energy generally impacts the phase diagram and nucleation dynamics of this family of soft matter models. We find the role of the geometry asymmetry to be weak, but the energy asymmetry to markedly interfere with the crystallization thermodynamics and kinetics. These results provide a rationale for the success and occasional failure of the proposal of George and Wilson for protein crystallization conditions as well as physical guidance for developing more effective protein crystallization strategies.

  2. Effect of Column Disorder on Carrier Transport in Columnar Discotic Liquid Crystal Evaluated by Applying Precisely Controlled Shear Stress

    NASA Astrophysics Data System (ADS)

    Kim, Jaeki; Yamasaki, Naoyuki; Hayashi, Takeshi; Katayama, Mitsuyoshi; Yoshida, Hiroyuki; Moritake, Hiroshi; Fujii, Akihiko; Ozaki, Masanori

    2013-10-01

    The effect of column disorder on carrier drift mobility in columnar discotic liquid crystals has been investigated by applying a precisely controlled oscillating shear stress. Drift mobilities on the order of 10-1 cm2.V-1.s-1 were confirmed for positive and negative carriers in the columnar phase of 1,4,8,11,15,18,22,25-octahexylphthalocyanine in a well-aligned homeotropic geometry, in which the columnar axis was perfectly perpendicular to substrates with an electrode. A slight tilt of the columnar axis upon applying shear stress led to a marked decrease in electronic carrier mobility from 10-1 to less than 10-6 cm2.V-1.s-1, and transport was only confirmed for positive ion carriers. This result indicates that a uniform shear stress blocks the carrier transport path in the entire area of the electrode, and one-dimensional carrier transport path along the columns is easily hindered in columnar discotic liquid crystals.

  3. Anisotropy, phonon modes, and free charge carrier parameters in monoclinic β -gallium oxide single crystals

    NASA Astrophysics Data System (ADS)

    Schubert, M.; Korlacki, R.; Knight, S.; Hofmann, T.; Schöche, S.; Darakchieva, V.; Janzén, E.; Monemar, B.; Gogova, D.; Thieu, Q.-T.; Togashi, R.; Murakami, H.; Kumagai, Y.; Goto, K.; Kuramata, A.; Yamakoshi, S.; Higashiwaki, M.

    2016-03-01

    We derive a dielectric function tensor model approach to render the optical response of monoclinic and triclinic symmetry materials with multiple uncoupled infrared and far-infrared active modes. We apply our model approach to monoclinic β -Ga2O3 single-crystal samples. Surfaces cut under different angles from a bulk crystal, (010) and (2 ¯01 ), are investigated by generalized spectroscopic ellipsometry within infrared and far-infrared spectral regions. We determine the frequency dependence of 4 independent β -Ga2O3 Cartesian dielectric function tensor elements by matching large sets of experimental data using a point-by-point data inversion approach. From matching our monoclinic model to the obtained 4 dielectric function tensor components, we determine all infrared and far-infrared active transverse optic phonon modes with Au and Bu symmetry, and their eigenvectors within the monoclinic lattice. We find excellent agreement between our model results and results of density functional theory calculations. We derive and discuss the frequencies of longitudinal optical phonons in β -Ga2O3 . We derive and report density and anisotropic mobility parameters of the free charge carriers within the tin-doped crystals. We discuss the occurrence of longitudinal phonon plasmon coupled modes in β -Ga2O3 and provide their frequencies and eigenvectors. We also discuss and present monoclinic dielectric constants for static electric fields and frequencies above the reststrahlen range, and we provide a generalization of the Lyddane-Sachs-Teller relation for monoclinic lattices with infrared and far-infrared active modes. We find that the generalized Lyddane-Sachs-Teller relation is fulfilled excellently for β -Ga2O3 .

  4. Correlation between Protein Sequence Similarity and Crystallization Reagents in the Biological Macromolecule Crystallization Database

    PubMed Central

    Lu, Hui-Meng; Yin, Da-Chuan; Liu, Yong-Ming; Guo, Wei-Hong; Zhou, Ren-Bin

    2012-01-01

    The protein structural entries grew far slower than the sequence entries. This is partly due to the bottleneck in obtaining diffraction quality protein crystals for structural determination using X-ray crystallography. The first step to achieve protein crystallization is to find out suitable chemical reagents. However, it is not an easy task. Exhausting trial and error tests of numerous combinations of different reagents mixed with the protein solution are usually necessary to screen out the pursuing crystallization conditions. Therefore, any attempts to help find suitable reagents for protein crystallization are helpful. In this paper, an analysis of the relationship between the protein sequence similarity and the crystallization reagents according to the information from the existing databases is presented. We extracted information of reagents and sequences from the Biological Macromolecule Crystallization Database (BMCD) and the Protein Data Bank (PDB) database, classified the proteins into different clusters according to the sequence similarity, and statistically analyzed the relationship between the sequence similarity and the crystallization reagents. The results showed that there is a pronounced positive correlation between them. Therefore, according to the correlation, prediction of feasible chemical reagents that are suitable to be used in crystallization screens for a specific protein is possible. PMID:22949812

  5. Continuous Crystallization of Proteins in a Tubular Plug-Flow Crystallizer

    PubMed Central

    2015-01-01

    Protein crystals have many important applications in many fields, including pharmaceutics. Being more stable than other formulations, and having a high degree of purity and bioavailability, they are especially promising in the area of drug delivery. In this contribution, the development of a continuously operated tubular crystallizer for the production of protein crystals has been described. Using the model enzyme lysozyme, we successfully generated product particles ranging between 15 and 40 μm in size. At the reactor inlet, a protein solution was mixed with a crystallization agent solution to create high supersaturations required for nucleation. Along the tube, supersaturation was controlled using water baths that divided the crystallizer into a nucleation zone and a growth zone. Low flow rates minimized the effect of shear forces that may impede crystal growth. Simultaneously, a slug flow was implemented to ensure crystal transport through the reactor and to reduce the residence time distribution. PMID:25774098

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

    NASA Astrophysics Data System (ADS)

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

    2012-08-01

    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.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. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr31150j

  7. Cardiolipin, a critical determinant of mitochondrial carrier protein assembly and function

    PubMed Central

    Claypool, Steven M.

    2009-01-01

    The ability of phospholipids to act as determinants of membrane protein structure and function is probably best exemplified by cardiolipin (CL), the signature phospholipid of mitochondria. Early efforts to reconstitute individual respiratory complexes and members of the mitochondrial carrier family, most notably the ADP/ATP carrier (AAC), often demonstrated the importance of CL. Over the past decade, the significance of CL in the organization of components of the electron transport chain into higher order assemblies, termed respiratory supercomplexes, has been established. Another protein required for oxidative phosphorylation, AAC, has received comparatively little attention likely stemming from the fact that AACs were thought to function in isolation as either homodimers or monomers. Recently however, AACs have been demonstrated to interact with the respiratory supercomplex, other members of the mitochondrial carrier family, and the TIM23 translocon. Interestingly, many if not all of these interactions depend on CL. As the paradigm for the mitochondrial carrier family, these discoveries with AAC suggest that other members of this large group of important proteins may be more gregarious than anticipated. Moreover, it is proposed that AAC and perhaps additional members of the mitochondrial carrier family might represent downstream targets of pathological states involving alterations in CL. PMID:19422785

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

    SciTech Connect

    Fenglei Li

    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

  9. 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

  10. 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.

  11. 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.

  12. Inorganic and protein crystal growth - Similarities and differences

    NASA Technical Reports Server (NTRS)

    Rosenberger, F.

    1986-01-01

    Transport and interface kinetic concepts for the design and control of inorganic crystal growth experiments are reviewed, and their applications and limitations in protein crystal growth are considered. It is suggested that the interfacial concentration gradients are steeper for faster crystallization, and that the interfacial concentration distributions for the protein and the precipitant can differ significantly. Results show that uniformity in crystal composition and steady-state conditions in growth kinetics are favored by larger sample size, since surface-tension gradients drive strong in microgravity experiments and in small samples on earth.

  13. Mesoporous magnetic hollow nanoparticles—protein carriers for lysosome escaping and cytosolic delivery

    NASA Astrophysics Data System (ADS)

    Huang, Xinglu; Meng, Xianwei; Tang, Fangqiong; Li, Linlin; Chen, Dong; Liu, Huiyu; Zhang, Yanqi; Ren, Jun

    2008-11-01

    It is important for a controlled release system to determine whether nanoparticles can penetrate cell membranes and deliver protein into the nuclear or cytosolic compartments of cells, and thus function as carriers. Here, we prepared different functionalized mesoporous magnetic hollow nanoparticles (MMHs) and chose bovine serum albumin (BSA) as a model protein to detect the intracellular trafficking of MMHs. The results showed that MMHs modified with amino groups (AMMHs) were efficient in protein loading and that the loading was dependent on the pH, temperature and ionic strength. Furthermore, we found that the AMMHs not only transported BSA into the cells but also released the BSA carried into the nuclear or cytosolic compartments of the cells. In addition, the nanoparticles were biocompatible, and the encapsulation of BSA in AMMHs did not affect their bioactivity. Taken together, AMMHs are excellent carriers for releasing protein into the cytosol and nucleus, and they have the potential to be used in a controlled release system.

  14. The biological activity of a-mangostin, a larvicidal botanic mosquito sterol carrier protein-2 inhibitor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alpha-mangostin derived from mangosteen was identified as a mosquito sterol carrier protein-2 inhibitor via high throughput insecticide screening. Alpha-mangostin was tested for its larvicidal activity against 3rd instar larvae of six mosquito species and the LC50 values range from 0.84 to 2.90 ppm....

  15. 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

  16. A new approach to calculate charge carrier transport mobility in organic molecular crystals from imaginary time path integral simulations

    SciTech Connect

    Song, Linze; Shi, Qiang

    2015-05-07

    We present a new non-perturbative method to calculate the charge carrier mobility using the imaginary time path integral approach, which is based on the Kubo formula for the conductivity, and a saddle point approximation to perform the analytic continuation. The new method is first tested using a benchmark calculation from the numerical exact hierarchical equations of motion method. Imaginary time path integral Monte Carlo simulations are then performed to explore the temperature dependence of charge carrier delocalization and mobility in organic molecular crystals (OMCs) within the Holstein and Holstein-Peierls models. The effects of nonlocal electron-phonon interaction on mobility in different charge transport regimes are also investigated.

  17. A new approach to calculate charge carrier transport mobility in organic molecular crystals from imaginary time path integral simulations

    NASA Astrophysics Data System (ADS)

    Song, Linze; Shi, Qiang

    2015-05-01

    We present a new non-perturbative method to calculate the charge carrier mobility using the imaginary time path integral approach, which is based on the Kubo formula for the conductivity, and a saddle point approximation to perform the analytic continuation. The new method is first tested using a benchmark calculation from the numerical exact hierarchical equations of motion method. Imaginary time path integral Monte Carlo simulations are then performed to explore the temperature dependence of charge carrier delocalization and mobility in organic molecular crystals (OMCs) within the Holstein and Holstein-Peierls models. The effects of nonlocal electron-phonon interaction on mobility in different charge transport regimes are also investigated.

  18. Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

    PubMed Central

    Guy, Jodie E.; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

    2011-01-01

    Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

  19. Remote control of regioselectivity in acyl-acyl carrier protein-desaturases.

    PubMed

    Guy, Jodie E; Whittle, Edward; Moche, Martin; Lengqvist, Johan; Lindqvist, Ylva; Shanklin, John

    2011-10-01

    Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals. PMID:21930947

  20. 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.

  1. Exploiting sulphur-carrier proteins from primary metabolism for 2-thiosugar biosynthesis

    PubMed Central

    Sasaki, Eita; Zhang, Xuan; Sun, He G.; Lu, Mei-Yeh Jade; Liu, Tsung-lin; Ou, Albert; Li, Jeng-yi; Chen, Yu-hsiang; Ealick, Steven E.; Liu, Hung-wen

    2014-01-01

    Sulphur is an essential element for life and exists ubiquitously in living systems1,2. Yet, how the sulphur atom is incorporated in many sulphur-containing secondary metabolites remains poorly understood. For C-S bond formation in primary metabolites, the major ionic sulphur sources are the protein-persulphide and protein-thiocarboxylate3,4. In each case, the persulphide and thiocarboxylate group on these sulphur-carrier (donor) proteins are post-translationally generated through the action of a specific activating enzyme. In all bacterial cases reported thus far, the genes encoding the enzyme that catalyzes the actual C-S bond formation reaction and its cognate sulphur-carrier protein co-exist in the same gene cluster5. To study 2-thiosugar production in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action appear similar to those of ThiG, the enzyme catalyzing thiazole formation in thiamin biosynthesis6,7. However, no sulphur-carrier protein gene could be located in the BE-7585A cluster. Subsequent genome sequencing revealed the presence of a few sulphur-carrier proteins likely involved in the biosynthesis of primary metabolites, but surprisingly only a single activating enzyme gene in the entire genome of A. orientalis. Further experiments showed that this activating enzyme is capable of adenylating each of these sulphur-carrier proteins, and likely also catalyzing the subsequent thiolation taking advantage of its rhodanese activity. A proper combination of these sulphur delivery systems is effective for BexX-catalyzed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. These studies represent the first complete characterization of a thiosugar formation in nature and also demonstrate the receptor promiscuity of the sulphur-delivery system in A. orientalis. Our

  2. 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

  3. One-carrier thermally stimulated currents and space-charge-limited currents in naphthalene crystals

    NASA Astrophysics Data System (ADS)

    Campos, M.; Mergulhão, S.

    1980-03-01

    Electron trapping in naphthalene is studied by analysis of the space-charge-limited and thermally stimulated currents as a function of applied voltage and temperature. The two methods are used on naphthalene single crystals, with continuous electron injection from a silver contact. The use of the two techniques allows a quantitative determination of the characteristics of deep and shallow traps. Three monomolecular current peaks were observed. Activation energies of 0.5, 0.79, and 1.1 eV, and cross sections of 8.8 × 10-20, 6.9 × 10-16, and 1.0 × 10-18 cm2, were calculated for the corresponding discrete trapping levels of the charge carriers. The behavior observed for space-charge-limited current was a current-voltage characteristic of a single set of traps, with an activation energy of 0.71 eV, and a cross section of 7.1 × 10-16 cm2. The results showed that the use of only space-charge-limited current is not good enough for a characterization of the traps of the material.

  4. Flow-induced protein crystallization: Macroscopic effects on 2D crystals

    NASA Astrophysics Data System (ADS)

    Young, James; Posada, David; Hirsa, Amir; Lopez, Juan

    2012-11-01

    Proteins must first be crystallized before their molecular structure can be studied in detail. However, crystallizing protein is a challenging task which is often met with limited success. Although 2-D protein crystals at the air/water interface are usually obtained under quiescent conditions, it was recently shown that crystallization can be enhanced by a shearing flow. Here we examine the relationship between Reynolds number and the crystal growth process using the deep-channel surface viscometer geometry. It consists of an annular region bounded by stationary inner and outer cylinders and driven by a constant rotation of the floor. The interfacial velocity measurements are compared to Navier-Stokes computations with the Boussinesq-Scriven surface model. The interfacial film is lifted onto a solid substrate, and the protein crystals are observed via optical and atomic force microscopy. For a particular protein surface concentration, a Reynolds number threshold has been identified for flow-induced crystallization. This flow geometry also allows for the determination of the surface shear viscosity, which provides a quantitative measure of the mesoscale interactions associated with protein crystallization.

  5. 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. PMID:22833181

  6. 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

  7. Pyrrolidine Carboxamides as a Novel Class of Inhibitors of Enoyl Acyl Carrier Protein Reductase (InhA) from Mycobacterium tuberculosis

    PubMed Central

    He, Xin; Alian, Akram; Stroud, Robert; de Montellano, Paul R. Ortiz

    2008-01-01

    In view of the worldwide spread of multidrug resistance of Mycobacterium tuberculosis, there is an urgent need to discover antituberculosis agent with novel structures. InhA, the enoyl acyl carrier protein reductase (ENR) from Mycobacterium tuberculosis is one of the key enzymes involved in the mycobacterial fatty acid elongation cycle and has been validated as an effective antimicrobial target. We report here discovery through high throughput screening of a series of pyrrolidine carboxamides as a novel class of potent InhA inhibitors. Crystal structures of InhA complexed with three inhibitors have been used to elucidate the inhibitor binding mode. The potency of the lead compound was improved over 160-fold by subsequent optimization through iterative microtiter library synthesis followed by in situ activity screening without purification. Resolution of racemic mixtures of several inhibitors indicate that only one enantiomer is active as an inhibitor of InhA. PMID:17034137

  8. Pyrrolidine carboxamides as a novel class of inhibitors of enoyl acyl carrier protein reductase from Mycobacterium tuberculosis.

    PubMed

    He, Xin; Alian, Akram; Stroud, Robert; Ortiz de Montellano, Paul R

    2006-10-19

    In view of the worldwide spread of multidrug resistance of Mycobacterium tuberculosis, there is an urgent need to discover antituberculosis agent with novel structures. InhA, the enoyl acyl carrier protein reductase (ENR) from M. tuberculosis, is one of the key enzymes involved in the mycobacterial fatty acid elongation cycle and has been validated as an effective antimicrobial target. We report here the discovery, through high-throughput screening, of a series of pyrrolidine carboxamides as a novel class of potent InhA inhibitors. Crystal structures of InhA complexed with three inhibitors have been used to elucidate the inhibitor binding mode. The potency of the lead compound was improved over 160-fold by subsequent optimization through iterative microtiter library synthesis followed by in situ activity screening without purification. Resolution of racemic mixtures of several inhibitors indicate that only one enantiomer is active as an inhibitor of InhA. PMID:17034137

  9. 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.

  10. 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. PMID:25849493

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

    DOE PAGESBeta

    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

  12. Chitosan-based nanoparticles as a sustained protein release carrier for tissue engineering applications.

    PubMed

    Hou, Yaping; Hu, Junli; Park, Hyejin; Lee, Min

    2012-04-01

    Chitosan/tripolyphosphate/chondroitin sulfate (Chi/TPP/CS) nanoparticles were prepared by an ionic gelation method to obtain a controlled release of proteins. Using Nel-like molecule-1 (Nell-1), a novel osteogenic protein, as a model protein, it was demonstrated that adjusting the composition of the particles modulated the protein association and release kinetics of incorporated proteins. Increasing the amounts of Chi crosslinking agents, TPP and CS, in the particles achieved sustained protein release. An increase in crosslinking density decreased degradation rates of the particles. Furthermore, the bioactivity of the protein was preserved during the encapsulating procedure into the particles. To demonstrate the feasibility of Chi/TPP/CS nanoparticles as sustained release carriers for tissue engineering scaffold applications, protein-loaded nanoparticles were successfully incorporated into collagen hydrogels or prefabricated porous poly(lactide-co-glycolide) (PLGA) scaffolds without obstructing the integrity of the hydrogels or porous structure of the scaffolds. Thus, we expect that these particles have a potential for efficient protein carriers in tissue engineering applications, and will be further evaluated in vivo. PMID:22275184

  13. 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

  14. Solar cells. Low trap-state density and long carrier diffusion in organolead trihalide perovskite single crystals.

    PubMed

    Shi, Dong; Adinolfi, Valerio; Comin, Riccardo; Yuan, Mingjian; Alarousu, Erkki; Buin, Andrei; Chen, Yin; Hoogland, Sjoerd; Rothenberger, Alexander; Katsiev, Khabiboulakh; Losovyj, Yaroslav; Zhang, Xin; Dowben, Peter A; Mohammed, Omar F; Sargent, Edward H; Bakr, Osman M

    2015-01-30

    The fundamental properties and ultimate performance limits of organolead trihalide MAPbX3 (MA = CH3NH3(+); X = Br(-) or I(-)) perovskites remain obscured by extensive disorder in polycrystalline MAPbX3 films. We report an antisolvent vapor-assisted crystallization approach that enables us to create sizable crack-free MAPbX3 single crystals with volumes exceeding 100 cubic millimeters. These large single crystals enabled a detailed characterization of their optical and charge transport characteristics. We observed exceptionally low trap-state densities on the order of 10(9) to 10(10) per cubic centimeter in MAPbX3 single crystals (comparable to the best photovoltaic-quality silicon) and charge carrier diffusion lengths exceeding 10 micrometers. These results were validated with density functional theory calculations. PMID:25635092

  15. 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.

  16. Effect of carrier selection on immunogenicity of protein conjugate vaccines against Plasmodium falciparum circumsporozoites.

    PubMed Central

    Que, J U; Cryz, S J; Ballou, R; Fürer, E; Gross, M; Young, J; Wasserman, G F; Loomis, L A; Sadoff, J C

    1988-01-01

    Conjugate vaccines against the sporozoite stage of Plasmodium falciparum were synthesized by covalently coupling the recombinant protein R32 [with the one-letter amino acid code of MDP-[(NANP)15NVDP]2LR] to tetanus toxoid, cholera toxin, choleragenoid, and Pseudomonas aeruginosa toxin A. Conjugates were produced by using adipic acid dihydrazide as a spacer molecule and carbodiimide as a coupling agent. The molar ratio of R32 to carrier protein ranged from 2.5:1 to 8.4:1. These conjugates were found to be stable, nontoxic, and nonpyrogenic. When adsorbed onto Al(OH)3, all conjugates were capable of inducing anti-R32 antibody. Conjugates made with either cholera toxin or Pseudomonas aeruginosa toxin A were significantly more immunogenic than those constructed with tetanus toxoid or choleragenoid. However, the magnitude of the immune response to the R32 moiety was not governed by the antibody response to the carrier protein. Images PMID:3047062

  17. Modulation of carrier dynamics and threshold characteristics in 1.3-μm quantum dot photonic crystal nanocavity lasers

    NASA Astrophysics Data System (ADS)

    Xing, Enbo; Tong, Cunzhu; Rong, Jiamin; Shu, Shili; Wu, Hao; Wang, Lijie; Tian, Sicong; Wang, Lijun

    2016-08-01

    A self-consistent all-pathway quantum dot (QD) rate equation model, in which all possible relaxation pathways are considered, is used to investigate the influence of quality (Q) factor on the carrier dynamics of 1.3-μm InAs/GaAs QD photonic crystal (PhC) nanolasers. It is found that Q factor not only affects the photon lifetime, but also modulates the carrier occupation in QDs. About three times increases of carrier injection efficiency in QD ground state can be realized in nanocavity with high Q factor. However, it also reveals that over 90% improvement of threshold current happens when Q factor increases from 2000 to 7000, which means it might be not necessary to pursuit for ultrahigh Q factor for the purpose of low threshold current.

  18. 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.

    1998-01-01

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

  19. Calcium distribution in globoid crystals of cucurbita cotyledon protein bodies.

    PubMed

    Lott, J N; Spitzer, E; Vollmer, C M

    1979-05-01

    Energy-dispersive x-ray analysis was used to investigate the location of globoid crystals with relatively high Ca levels within cotyledons of Cucurbita maxima, Cucurbita mixta, and Cucurbita andreana. The small globoid crystals in both upper and lower epidermal cells commonly contained Ca. Ca was present in globoid crystals of all provascular regions with the exception of the very small provascular regions of C. maxima. In C. maxima and C. mixta cotyledons, some cases were observed where Ca was found in the globoid crystals of the first layer of mesophyll cells surrounding the provascular region, but in general Ca was absent from globoid crystals of palisade and spongy mesophyll cells. In C. andreana, globoid crystals of palisade and spongy mesophyll cells commonly contained at least some Ca. Cell position and cell type are factors affecting the Ca content of globoid crystals in protein bodies. PMID:16660825

  20. 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.

  1. 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.

  2. Effect of temperature and rare-earth doping on charge-carrier mobility in indium-monoselenide crystals

    SciTech Connect

    Abdinov, A. Sh.; Babayeva, R. F.; Amirova, S. I.; Rzayev, R. M.

    2013-08-15

    In the temperature range T = 77-600 K, the dependence of the charge-carrier mobility ({mu}) on the initial dark resistivity is experimentally investigated at 77 K ({rho}d{sub 0}), as well as on the temperature and the level (N) of rare-earth doping with such elements as gadolinium (Gd), holmium (Ho), and dysprosium (Dy) in n-type indium-monoselenide (InSe) crystals. It is established that the anomalous behavior of the dependences {mu}(T), {mu}({rho}d{sub 0}), and {mu}(N) found from the viewpoint of the theory of charge-carrier mobility in crystalline semiconductors is related, first of all, to partial disorder in indium-monoselenide crystals and can be attributed to the presence of random drift barriers in the free energy bands.

  3. Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle

    PubMed Central

    Grabarczyk, Daniel B.; Chappell, Paul E.; Johnson, Steven; Stelzl, Lukas S.; Berks, Ben C.

    2015-01-01

    The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a “suicide enzyme” strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein–protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway. PMID:26655737

  4. Evaluation of Salmonella enterica Type III Secretion System Effector Proteins as Carriers for Heterologous Vaccine Antigens

    PubMed Central

    Hegazy, Wael Abdel Halim; Xu, Xin; Metelitsa, Leonid

    2012-01-01

    Live attenuated strains of Salmonella enterica have a high potential as carriers of recombinant vaccines. The type III secretion system (T3SS)-dependent translocation of S. enterica can be deployed for delivery of heterologous antigens to antigen-presenting cells. Here we investigated the efficacy of various effector proteins of the Salmonella pathogenicity island (SPI2)-encoded T3SS for the translocation of model antigens and elicitation of immune responses. The SPI2 T3SS effector proteins SifA, SteC, SseL, SseJ, and SseF share an endosomal membrane-associated subcellular localization after translocation. We observed that all effector proteins could be used to translocate fusion proteins with the model antigens ovalbumin and listeriolysin into the cytosol of host cells. Under in vitro conditions, fusion proteins with SseJ and SteC stimulated T-cell responses that were superior to those triggered by fusion proteins with SseF. However, in mice vaccinated with Salmonella carrier strains, only fusion proteins based on SseJ or SifA elicited potent T-cell responses. These data demonstrate that the selection of an optimal SPI2 effector protein for T3SS-mediated translocation is a critical parameter for the rational design of effective Salmonella-based recombinant vaccines. PMID:22252866

  5. Imaging and diffraction of protein crystallization using TEM.

    PubMed

    Gomery, Kathryn; Humphrey, Elaine C; Herring, Rodney

    2013-06-01

    Structural biology relies on good-quality protein crystals in order for structure determination. Many factors affect the growth process of a protein crystal including the way it nucleates and the types of damage and contamination during its growth. Although the nucleation process and quality of a crystal is vital to structure determination, they are both under-studied areas of research. Our research begins to explore ways of measuring the quality of protein crystals, using TEM, thus overcoming the problems associated with viewing wet specimens in a vacuum. Our current understanding of nucleation is that it is a two-step mechanism involving the formation of nuclei from dense liquid clusters; however; it is still unclear whether nuclei first start as amorphous aggregates or as crystalline lattices. Potentially, electron diffraction may be capable of uncovering this process. Using TEM imaging and diffraction of lysozyme as a model protein crystal, we report the internal two-dimensional strain and the density of crystallites in a protein crystal, at a resolution never seen before. The TEM diffraction shows unique features of crystal mosaicity that can be directly correlated to TEM images. PMID:23250893

  6. 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

  7. 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.

  8. 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.

  9. (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.

  10. Quantifying Nanomolar Protein Concentrations Using Designed DNA Carriers and Solid-State Nanopores

    PubMed Central

    2016-01-01

    Designed “DNA carriers” have been proposed as a new method for nanopore based specific protein detection. In this system, target protein molecules bind to a long DNA strand at a defined position creating a second level transient current drop against the background DNA translocation. Here, we demonstrate the ability of this system to quantify protein concentrations in the nanomolar range. After incubation with target protein at different concentrations, the fraction of DNA translocations showing a secondary current spike allows for the quantification of the corresponding protein concentration. For our proof-of-principle experiments we use two standard binding systems, biotin–streptavidin and digoxigenin–antidigoxigenin, that allow for measurements of the concentration down to the low nanomolar range. The results demonstrate the potential for a novel quantitative and specific protein detection scheme using the DNA carrier method. PMID:27121643

  11. 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

  12. 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

  13. 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

  14. Intrinsic carrier multiplication efficiency in bulk Si crystals evaluated by optical-pump/terahertz-probe spectroscopy

    SciTech Connect

    Yamashita, G.; Nagai, M. E-mail: ashida@mp.es.osaka-u.ac.jp; Ashida, M. E-mail: ashida@mp.es.osaka-u.ac.jp; Matsubara, E.; Kanemitsu, Y.

    2014-12-08

    We estimated the carrier multiplication efficiency in the most common solar-cell material, Si, by using optical-pump/terahertz-probe spectroscopy. Through close analysis of time-resolved data, we extracted the exact number of photoexcited carriers from the sheet carrier density 10 ps after photoexcitation, excluding the influences of spatial diffusion and surface recombination in the time domain. For incident photon energies greater than 4.0 eV, we observed enhanced internal quantum efficiency due to carrier multiplication. The evaluated value of internal quantum efficiency agrees well with the results of photocurrent measurements. This optical method allows us to estimate the carrier multiplication and surface recombination of carriers quantitatively, which are crucial for the design of the solar cells.

  15. Acyl-acyl carrier protein: Lysomonogalactosyldiacylglycerol acyl transferase in Anabaena variabilis

    SciTech Connect

    Chen, H.H.

    1989-01-01

    Monogalactosyldiacylglycerol was produced when membranes isolated from the cyanobacterium, Anabaena variabilis, and washed free of soluble endogenous constituents, were incubated with ({sup 14}C)acyl-acyl carrier protein. This enzymatic synthesis of monogalactosyldiacylglycerol localized in the membranes was not dependent on any added cofactors, such as ATP, coenzyme A, and dithiothreitol. Palmitoyl-, stearoyl-, and oleoyl-acyl carrier proteins were approximately equally active as substrates with Km of 0.37, 0.36, and 0.23 {mu}M, respectively. The ({sup 14}C)acyl group was exclusively transferred to the sn-1 hydroxyl of the glycerol backbone of monogalactosyldiacylglycerol as demonstrated by hydrolysis of all incorporated acyl groups by the lipase from Rhizopus arrhizus delamar. Using a double labelled ({sup 14}C)acyl-({sup 14}C)acyl carrier protein, this enzyme catalyzed the direct transfer of the acyl group from acyl-acyl carrier protein to an endogenous lysomonogalactosyldiacylglycerol to form monogalactosyldiacylglycerol. The transfer reaction mechanism was also confirmed by the increased activity with the addition of the lysomonogalactosyldiacylglycerol suspension. A specific galactolipid acyl hydrolase activity was released into the soluble protein fraction when the membranes of Anabaena variabilis were treated with 2% Triton X-100. The positional specificity of this acyl hydrolase was demonstrated to be similar to that of Rhizopus lipase, i.e. only the acyl group at the sn-1 position was hydrolyzed. The acyl hydrolase which was also localized in the membrane fraction of Anabaena variabilis was presumably responsible for producing endogenous lysomonogalactosyldiacylglycerol used by the acyltransferase.

  16. Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering.

    PubMed

    Yuan, L; Voelker, T A; Hawkins, D J

    1995-11-01

    The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1. This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression. We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other. Our results show that the C-terminal two-thirds of the protein is critical for the specificity. By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231K, by itself does not effect the specificity. However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant cDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0. Meanwhile the E. coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of Km,app with respect to 14:0-ACP. Inhibitory studies also show that 12:0-ACP is a good

  17. 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.

  18. Containerless protein crystallization in floating drops: application to crystal growth monitoring under reduced nucleation conditions

    NASA Astrophysics Data System (ADS)

    Lorber, Bernard; Giegé, Richard

    1996-10-01

    A micromethod was developed for the batch crystallization of proteins under conditions were the solution has no contact with the container walls. Drops of crystallization solutions (5 to 100 μl) are placed at the interface between two layers of inert and non-miscible silicone fluids contained in square glass or plastic cuvettes. The densities of the fluids are either lower or higher than those of the major precipitating agents of macromolecules, including aqueous solutions containing salts, polyethylene glycols or alcohols. Several proteins and a spherical plant virus were crystallized in the temperature range 4°C-20°C using this set-up. A thermostated device was built for the dynamic control of the temperature of crystallization drops and the monitoring of crystal growth by video-microscopy. In all cases, the habit of the crystals grown in floating drops are identical to those of controls grown in sealed glass tubes without silicone fluid. The comparison of the number of crystals in drops kept under one layer of fluid and in floating drops of the same volume indicates that heterogeneous nucleation is minimized when protein crystallization is performed in floating drops. The advantages and limitations of this novel containerless crystallization method are discussed.

  19. 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.

  20. Three-Dimensional Crystals of Membrane Proteins: Bacteriorhodopsin

    NASA Astrophysics Data System (ADS)

    Michel, Hartmut; Oesterhelt, Dieter

    1980-03-01

    The intrinsic membrane protein bacteriorhodopsin has been crystallized by salt precipitation after solubilization by octyl glucoside. Two different crystal forms were obtained, depending on the nature of the salt used and the pH. Needles formed in the presence of sodium phosphate and in ammonium sulfate solutions above pH 4.8. Cubes appeared in sodium citrate solutions or ammonium sulfate. Unlike the cubic crystals, the birefringent needles showed strong linear dichroism, which allowed determination of the orientation of the chromophore's transition moment. The procedure described here may be of general use in crystallographic studies of membrane proteins.

  1. Protein encapsulated magnetic carriers for micro/nanoscale drug delivery systems.

    SciTech Connect

    Xie, Y.; Kaminski, M. D.; Mertz, C. J.; Finck, M. R.; Guy, S. G.; Chen, H.; Rosengart, A. J.; Chemical Engineering; Univ. of Chicago, Pritzker School of Medicine

    2005-01-01

    Novel methods for drug delivery may be based on nanotechnology using non-invasive magnetic guidance of drug loaded magnetic carriers to the targeted site and thereafter released by external ultrasound energy. The key building block of this system is to successfully synthesize biodegradable, magnetic drug carriers. Magnetic carriers using poly(D,L-lactide-co-glycolide) (PLGA) or poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) as matrix materials were loaded with bovine serum albumin (BSA) by a double-emulsion technique. BSA-loaded magnetic microspheres were characterized for size, morphology, surface charge, and magnetization. The BSA encapsulation efficiency was determined by recovering albumin from the microspheres using dimethyl sulfoxide and 0.05N NaOH/0.5% SDS then quantifying with the Micro-BCA protein assay. BSA release profiles were also determined by the Micro-BCA protein assay. The microspheres had drug encapsulation efficiencies up to 90% depending on synthesis parameters. Particles were spherical with a smooth or porous surface having a size range less than 5 {mu}m. The surface charge (expressed as zeta potential) was near neutral, optimal for prolonged intravascular survival. The magnetization of these BSA loaded magnetic carriers was 2 to 6 emu/g, depending on the specific magnetic materials used during synthesis.

  2. The effects of microgravity on protein crystallization: evidence for concentration gradients around growing crystals

    NASA Astrophysics Data System (ADS)

    McPherson, Alexander; Malkin, Alexander J.; Kuznetsov, Yurii G.; Koszelak, Stan; Wells, Mark; Jenkins, Greg; Howard, Jeff; Lawson, Greg

    1999-01-01

    Atomic force microscopy (AFM) investigations have revealed that macromolecular crystals, during their growth, incorporate an extensive array of impurities. These vary from individual molecules to large particles, and microcrystals in the micron size range. AFM, along with X-ray topology, has further shown that the density of defects and faults in most macromolecular crystals is very high in comparison with conventional crystals. The high defect density is a consequence of the incorporation of impurities, misoriented nutrient molecules, and aggregates of molecules. High defect and impurity density, contributes to a deterioration of both the mechanical and the diffraction properties of crystals. In microgravity, access by impurities and aggregates to growing crystal surfaces is restricted due to altered fluid transport properties. We designed, and have now constructed an instrument, the observable protein crystal growth apparatus (OPCGA) that employs a fused optics, phase shift, Mach-Zehnder interferometer to analyze the fluid environment around growing crystals. Using this device, which will ultimately be employed on the international space station, we have, in thin cells on earth, succeeded in directly visualizing concentration gradients around growing protein crystals. This provides the first direct evidence that quasi-stable depletion zones formed around growing crystals in space may explain the improved quality of macromolecular crystals grown in microgravity. Further application of the interferometric technique will allow us to quantitatively describe the shapes, extent, and magnitudes of the concentration gradients and to evaluate their degree of stability.

  3. 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.

  4. 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.

  5. Two-photon excited UV fluorescence for protein crystal detection

    SciTech Connect

    Madden, Jeremy T.; DeWalt, Emma L.; Simpson, Garth J.

    2011-10-01

    Complementary measurements using SONICC and TPE-UVF allow the sensitive and selective detection of protein crystals. Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging of chiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC.

  6. 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).

  7. An overview on the delivery of antitumor drug doxorubicin by carrier proteins.

    PubMed

    Agudelo, D; Bérubé, G; Tajmir-Riahi, H A

    2016-07-01

    Serum proteins play an increasing role as drug carriers in the clinical settings. In this review, we have compared the binding modalities of anticancer drug doxorubicin (DOX) to three model carrier proteins, human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (β-LG) in order to determine the potential application of these model proteins in DOX delivery. Molecular modeling studies showed stronger binding of DOX with HSA than BSA and β-LG with the free binding energies of -10.75 (DOX-HSA), -9.31 (DOX-BSA) and -8.12kcal/mol (DOX-β-LG). Extensive H-boding network stabilizes DOX-protein conjugation and played a major role in drug-protein complex formation. DOX complexation induced major alterations of HSA and BSA conformations, while did not alter β-LG secondary structure. The literature review shows that these proteins can potentially be used for delivery of DOX in vitro and in vivo. PMID:27037051

  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. Crystal structures of fusion proteins with large-affinity tags.

    PubMed

    Smyth, Douglas R; Mrozkiewicz, Marek K; McGrath, William J; Listwan, Pawel; Kobe, Bostjan

    2003-07-01

    The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest. PMID:12824478

  10. Small-scale batch crystallization of proteins revisited: an underutilized way to grow large protein crystals.

    PubMed

    Rayment, Ivan

    2002-02-01

    Growth of high-quality crystals is a major obstacle in many structural investigations. In recent years, the techniques for screening crystals have improved dramatically, whereas the methods for obtaining large crystals have progressed more slowly. This is an important issue since, although many structures can be solved from small crystals with synchrotron radiation, it is far easier to solve and refine structures when strong data is recorded from large crystals. In an effort to improve the size of crystals, a strategy for a small-scale batch method has been developed that in many cases yields far larger crystals than attainable by vapor diffusion. PMID:11839300

  11. Lateral-flow Immunoassay for the Frataxin Protein in Friedreich’s Ataxia Patients and Carriers

    PubMed Central

    Willis, John H.; Isaya, Grazia; Gakh, Oleksandr; Capaldi, Roderick A.; Marusich, Michael F.

    2008-01-01

    Friedreich’s Ataxia (FA) is an inherited neurodegenerative disease caused by reduction in levels of the mitochondrial protein frataxin. Currently there are no simple, reliable methods to accurately measure the concentrations of frataxin protein. We designed a lateral-flow immunoassay that quantifies frataxin protein levels in a variety of sample materials. Using recombinant frataxin we evaluated the accuracy and reproducibility of the assay. The assay measured recombinant human frataxin concentrations between 40 and 4000 pg/test or approximately 0.1 – 10 nM of sample. The intra and inter-assay error was < 10% throughout the working range. To evaluate clinical utility of the assay we used genetically defined lymphoblastoid cells derived from FA patients, FA carriers and controls. Mean frataxin concentrations in FA patients and carriers were significantly different from controls and from one another (p = 0.0001, p = 0.003, p = 0.005, respectively) with levels, on average, 29% (patients) and 64% (carriers) of the control group. As predicted, we observed an inverse relationship between GAA repeat number and frataxin protein concentrations within the FA patient cohort. The lateral flow immunoassay provides a simple, accurate and reproducible method to quantify frataxin protein in whole cell and tissue extracts, including primary samples obtained by non-invasive means, such as cheek swabs and whole blood. The assay is a novel tool for FA research that may facilitate improved diagnostic and prognostic evaluation of FA patients and could also be used to evaluate efficacy of therapies designed to cure FA by increasing frataxin protein levels. PMID:18485778

  12. 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

  13. 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.

  14. (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.

  15. Induction of the lac carrier and an associated membrane protein in Escherichia coli

    SciTech Connect

    Lagarias, D.M.

    1985-01-01

    Induction of the lac operon in wild type Escherichia coli strains results in synthesis of a 16 kilodalton inner membrane protein in addition to the known products of the lacZ, lacY and lacA genes. Cells carrying the lacY gene on a plasmid over produce this 16 kilodalton polypeptide as well as the Lac carrier, the membrane protein product of the lacY gene. However, (/sup 35/S)methionine labeling of minicells carrying the lacY plasmid shows that the 16 kDa protein is not synthesized from the plasmid DNA. The 16 kDa protein was purified and partially characterized. It is an acidic membrane protein of apparent molecular weight 15,800 whose amino terminal sequence (NH/sub 2/-Met-Arg-Asn-Phe-Asp-Leu-) does not correspond to any nucleotide sequence known in lac operon DNA. Using antibody prepared to the purified 16 kDa protein, a quantitative analysis of conditions under which this protein is made was accomplished, and reveals that the amount of 16 kDa protein which appears in the membrane is proportional to lac operon expression. Hybridization of a synthetic oligonucleotide probe complementary to the 5' end of 16 kDa protein mRNA shows that its synthesis is regulated at the level of transcription. A description of attempts to clone this gene is given. Possible functional roles for the 16 kDa protein are discussed.

  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. PMID:24278480

  19. Photogeneration of charge carrier correlated with amplified spontaneous emission in single crystals of a thiophene/phenylene co-oligomer.

    PubMed

    Saeki, Akinori; Seki, Shu; Shimizu, Yasuhiro; Yamao, Takeshi; Hotta, Shu

    2010-04-01

    Thiophene/phenylene co-oligomers have substantial promise for the use of not only organic electronics but also organic optical devices. However, considerably less is known about the correlation between their optical and optoelectronic properties. We have investigated the charge carrier generation in 1,4-bis(5-phenylthiophen-2-yl)benzene (AC5) single crystals by flash-photolysis time-resolved microwave conductivity (TRMC) and transient absorption spectroscopy (TAS). It was found that the dependence of photocarrier generation efficiency on excitation photon density differed from that of emission efficiency once amplified spontaneous emission (ASE) and resultant spectrally narrowed emission occur upon exposure to 355 nm. In contrast, the dependences of emission and photocarrier generation efficiencies were identical when ASE was not involved at a different excitation wavelength (193 nm). An approximated analytical solution of rate equation considering ASE or singlet-singlet annihilation was applied to the experiments, exhibiting good agreement. On the basis of TRMC, TAS, and extinction coefficient of radical cation assessed by pulse radiolysis, the minimum charge carrier mobility was estimated, without electrodes, to be 0.12 cm(2) V(-1) s(-1). The dynamics of charge carrier and triplet excited state is discussed, accompanying with examination by time-dependent density functional theory. The present work would open the way to a deeper understanding of the fate of excited state in optically robust organic semiconducting crystals. PMID:20387943

  20. Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

    PubMed Central

    Economou, Nicoleta J.; Zentner, Isaac J.; Lazo, Edwin; Jakoncic, Jean; Stojanoff, Vivian; Weeks, Stephen D.; Grasty, Kimberly C.; Cocklin, Simon; Loll, Patrick J.

    2013-01-01

    Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance. PMID:23519660

  1. Influence of Crystal Packing on Global Protein Conformation

    NASA Astrophysics Data System (ADS)

    Ahlstrom, Logan; Miyashita, Osamu

    2011-10-01

    X-ray crystallography is the most robust method for solving protein structure. However, packing forces in the crystal lattice select just a snapshot of a protein's conformational ensemble, whereas proteins are flexible and can adopt different conformations. Here we compare molecular dynamics (MD) simulations in solution and the crystal lattice to add dynamical insight to the static X-ray images of proteins. As a model system, we consider the λ Cro dimer, whose solved X-ray structures range from a ``closed'' to an ``open'' global conformation. Free energy profiles depicting the conformational space sampled by the dimer in solution show some reported structures correspond to stable states. Yet other conformations, while accessible, lie higher in energy, indicating the effect of crystal packing. Subsequent crystal MD simulations estimated the strength of packing interfaces in the lattice, showing the influence of crystal form and mutation in stabilizing different dimer conformations. Our quantitative results will aid analysis of X-ray data in establishing protein structure-function relationships.

  2. 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.

  3. 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.

  4. Crystallization of parasporin-2, a Bacillus thuringiensis crystal protein with selective cytocidal activity against human cells.

    PubMed

    Akiba, Toshihiko; Abe, Yuichi; Kitada, Sakae; Kusaka, Yoshitomo; Ito, Akio; Ichimatsu, Tokio; Katayama, Hideki; Akao, Tetsuyuki; Higuchi, Kazuhiko; Mizuki, Eiichi; Ohba, Michio; Kanai, Ryuta; Harata, Kazuaki

    2004-12-01

    Bacillus thuringiensis is a valuable source of protein toxins that are specifically effective against certain insects and worms but harmless to mammals. In contrast, a protein toxin obtained from B. thuringiensis strain A1547, designated parasporin-2, is not insecticidal but has a strong cytocidal activity against human cells with markedly divergent target specificity. The 37 kDa inactive protein is proteolytically activated to a 30 kDa active form. The active form of the recombinant protein toxin was crystallized in the presence of ethylene glycol and polyethylene glycol 8000 at neutral pH. The crystals belong to the hexagonal space group P6(1) or P6(5), with unit-cell parameters a = b = 134.37, c = 121.24 A. Diffraction data from a native crystal were collected to 2.75 A resolution using a synchrotron-radiation source. PMID:15583389

  5. 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.

  6. Upside-down protein crystallization: designing microbatch experiments for microgravity.

    PubMed

    Khurshid, Sahir; Chayen, Naomi E

    2006-09-01

    The benefits of protein crystal growth in microgravity are well documented. The crystallization vessels currently employed for microgravity crystallization are far from optimal with regards to cost, sample volume, size, and ease of use. The use of microbatch experiments is a favorable alternative in each respect: 96 experiments of 0.5-2 microL volumes can be performed in a single microtiter tray measuring 5 x 8 cm and costing 1 pound sterling each. To date, the use of microbatch has not been pursued on account of concerns of oil leakage. To address this issue, a novel approach to microbatch crystallization experiments is described, where the microbatch plates are inverted throughout the duration of the experiment. The findings intimate the application of the microbatch method to space flight and the potential to drastically increase the output of microgravity crystallization research . PMID:17124125

  7. A new approach to calculate charge carrier transport mobility in organic molecular crystals from imaginary time path integral simulations.

    PubMed

    Song, Linze; Shi, Qiang

    2015-05-01

    We present a new non-perturbative method to calculate the charge carrier mobility using the imaginary time path integral approach, which is based on the Kubo formula for the conductivity, and a saddle point approximation to perform the analytic continuation. The new method is first tested using a benchmark calculation from the numerical exact hierarchical equations of motion method. Imaginary time path integral Monte Carlo simulations are then performed to explore the temperature dependence of charge carrier delocalization and mobility in organic molecular crystals (OMCs) within the Holstein and Holstein-Peierls models. The effects of nonlocal electron-phonon interaction on mobility in different charge transport regimes are also investigated. PMID:25956086

  8. 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.

  9. 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.

  10. 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.

  11. Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells.

    PubMed

    Li, Nancy C; Fan, Jinjiang; Papadopoulos, Vassilios

    2016-01-01

    Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking. PMID:26901662

  12. Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells

    PubMed Central

    Li, Nancy C.; Fan, Jinjiang; Papadopoulos, Vassilios

    2016-01-01

    Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking. PMID:26901662

  13. Containerless protein crystal growth in rotating levitated drops

    NASA Astrophysics Data System (ADS)

    Chung, Sang K.; Trinh, Eugene H.

    1998-01-01

    A method for growing protein crystals in a containerless environment using an ultrasonic-electrostatic hybrid levitator is evaluated. In this approach, a single protein solution droplet bearing a surface charge is electrostatically levitated and acoustically rotated along a horizontal axis during the crystal nucleation and growth phases. Sample rotation is induced by ultrasonic streaming and radiation pressure applied in addition to the electrostatic levitation force. This unique approach is developed in order to create controlled crystal growth conditions which would reproduce some of the aspects of the low-gravity environment. We present the outcome of a development effort and feasibility study showing the successful growth of lysozyme and thaumatin crystals suspended within the bulk of quiescent liquid protein solutions inside rotating droplets also containing a very small concentration of agarose. Even though the crystals are not growing in a completely gelled medium and rotation is required for their long-term suspension, there are indications that a convectionless crystal growth environment has been obtained within the rotating drop, and that artificial flow can be introduced in a controlled manner by imposing drop shape oscillations.

  14. Simulations of nucleation and early growth stages of protein crystals.

    PubMed Central

    Kierzek, A M; Wolf, W M; Zielenkiewicz, P

    1997-01-01

    Analysis of known protein crystal structures reveals that interaction energies between monomer pairs alone are not sufficient to overcome entropy loss related to fixing monomers in the crystal lattice. Interactions with several neighbors in the crystal are required for stabilization of monomers in the lattice. A microscopic model of nucleation and early growth stages of protein crystals, based on the above observations, is presented. Anisotropy of protein molecules is taken into account by assigning free energies of association (proportional to the buried surface area) to individual monomer-monomer contacts in the lattice. Lattice simulations of the tetragonal lysozyme crystal based on the model correctly reproduce structural features of the movement of dislocation on the (110) crystal face. The dislocation shifts with the speed equal to the one determined experimentally if the geometric probability of correct orientation is set to 10(-5), in agreement with previously published estimates. At this value of orientational probability, the first nuclei, the critical size of which for lysozyme is four monomers, appear in 1 ml of supersaturated solution on a time scale of microseconds. Formation of the ordered phase proceeds through the growth of nuclei (rather then their association) and requires nucleations on the surface at certain stages. Images FIGURE 2 PMID:9251778

  15. Drug carriers based on highly protein-resistant materials for prolonged in vivo circulation time

    PubMed Central

    Liu, Ruiyuan; Li, Yan; Zhang, Zhenzhong; Zhang, Xin

    2015-01-01

    Long-circulating drug carriers are highly desirable in drug delivery system. However, nonspecific protein adsorption leaves a great challenge in drug delivery of intravenous administration and significantly affects both the pharmacokinetic profiles of the carrier and drugs, resulting in negatively affect of therapeutic efficiency. Therefore, it is important to make surface modification of drug carriers by protein-resistant materials to prolong the blood circulation time and increase the targeted accumulation of therapeutic agents. In this review, we highlight the possible mechanism of protein resistance and recent progress of the alternative protein-resistant materials and their drug carriers, such as poly(ethylene glycol), oligo(ethylene glycol), zwitterionic materials, and red blood cells adhesion. PMID:26813147

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. Determination of the carrier concentration in CdSe crystals from the effective infrared absorption coefficient measured by means of the photothermal infrared radiometry

    NASA Astrophysics Data System (ADS)

    Pawlak, M.

    2015-01-01

    In this paper, a non-contact method that allows to determine the carrier concentration in CdSe crystals is presented. The method relies on the measurement of the effective infrared absorption coefficient by means of the photothermal infrared radiometry (PTR). In order to obtain the effective infrared absorption coefficient and thermal diffusivity, the frequency characteristics of the PTR signal were analyzed in the frame of a one-dimensional heat transport model for infrared semitransparent crystals. The carrier concentrations were estimated using a theory introduced by Ruda and a recently proposed normalization procedure for the PTR signal. The deduced carrier concentrations of the investigated CdSe crystals are in reasonable agreement with those obtained using Hall measurements and infrared spectroscopy. The method presented in this paper can also be applied to other semiconductors with the carrier concentration in the range of 1014-1017 cm-3.

  1. 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.

  2. Efficiency analysis of sampling protocols used in protein crystallization screening

    NASA Astrophysics Data System (ADS)

    Segelke, Brent W.

    2001-11-01

    In an effort to objectively compare the efficiency of protein crystallization screening techniques, a probability model of sampling efficiency is developed and used to calculate sampling efficiencies from experimental data. Three typical sampling protocols (grid screening, footprint screening, and random screening) are used to crystallize each of five proteins (Phospholipase A 2, Thaumatin, Catalase, Lysozyme, and Ribonuclease B). For each of the three sampling protocols, experiments are chosen from a large set of possible experiments generated by systematic combination of a number of parameters common in crystallization screens. Software has been developed to generate and select from the combinations with each of the three sampling protocols examined in this study. The protocols differ only in the order samples are chosen from the set of possible combinations. Random sampling is motivated by the "Incomplete Factorial" screen (Carter and Carter, J. Biol. Chem. 254 (1979) 12 219); sampling with subsets of four is motivated by the "Footprint" screen (Stura et al., J. Crystal Growth 122 (1992) 273) and sampling with subsets of twenty-four is motivated by the "Grid" screen (McPherson, Prepartion and Analysis of Protein Crystals, Wiley, New York, 1982). For the five proteins examined, random sampling has the greatest average efficiency. Additional benefits of random sampling are discussed.

  3. Ultrafast free-carrier dynamics in Cu{sub 2}ZnSnS{sub 4} single crystals studied using femtosecond time-resolved optical spectroscopy

    SciTech Connect

    Phuong, L. Q.; Kanemitsu, Y.; Okano, M.; Yamada, Y.; Yamashita, G.; Morimoto, T.; Nagai, M.; Ashida, M.; Nagaoka, A.; Yoshino, K.

    2014-12-08

    We studied the dynamics of photogenerated carriers in Cu{sub 2}ZnSnS{sub 4} (CZTS) single crystals using femtosecond transient reflectivity (TR) and optical pump-THz probe transient absorption (THz-TA) spectroscopy. The TR and THz-TA decay dynamics consistently showed that free carriers have long lifetimes of up to a few nanoseconds. The excitation-photon-energy-dependent TR measurements revealed a slow picosecond energy relaxation of free carriers to the band edge in CZTS. The relaxation and recombination dynamics of free carriers were affected by nonradiative recombinations at the surface. Our results revealed a global feature of energy relaxation and recombination processes of free carriers in CZTS single crystals.

  4. 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

  5. Expression and purification of two recombinant sterol-carrier proteins: SCPX and SCP2.

    PubMed

    Manfra, D J; Baum, C L; Reschley, E; Lundell, D; Zavodny, P; Dalie, B

    1995-04-01

    We report the cloning, expression, and purification of the rat sterol carrier proteins SCPX and SCP2. The cDNA's encoding rat SCPX and SCP2 were isolated from a lambda gt11 rat liver cDNA library. To maximize expression and to facilitate the purification of the recombinant proteins, the SCPX and SCP2 proteins were expressed as carboxy-terminal fusion proteins to the glutathione S-transferase (GST). The GST-SCPX and GST-SCP2 fusion proteins contained a thrombin recognition site between the GST and SCPX or SCP2 polypeptides. The expression of the fusion proteins was controlled by the inducible tac promoter. Under optimal conditions, the approximately 85-kDa GST-SCPX and the approximately 41-kDa GST-SCP2 proteins represented approximately 1-2% of the total cell lysate. Both fusion proteins were easily purified under nondenaturing conditions from the soluble fraction of total cell lysate by glutathione-Sepharose 4B affinity chromatography. Thrombin cleavage resulted in the release of the SCPX and SCP2 proteins from the GST-SCPX and GST-SCP2 fusions, respectively. Amino terminal protein sequencing confirmed the authenticity of the recombinant proteins. Furthermore, functional assay revealed that recombinant SCP2 is highly active in facilitating the conversion of 7-dehydrocholesterol to cholesterol. Recombinant SCPX is also active in this assay but only 50% as active as SCP2. We anticipate that the preparation and purification techniques described in this study will facilitate further biochemical characterization of these proteins. PMID:7606169

  6. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis

    PubMed Central

    Nanson, Jeffrey D.; Himiari, Zainab; Swarbrick, Crystall M. D.; Forwood, Jade K.

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

  7. Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis.

    PubMed

    Nanson, Jeffrey D; Himiari, Zainab; Swarbrick, Crystall M D; Forwood, Jade K

    2015-01-01

    Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The β-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis β-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known β-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival. PMID:26469877

  8. 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

  9. Protein Crystal Growth (PCG) experiment aboard mission STS-66

    NASA Technical Reports Server (NTRS)

    2000-01-01

    On the Space Shuttle Orbiter Atlantis' middeck, Astronaut Joseph R. Tarner, mission specialist, works at an area amidst several lockers which support the Protein Crystal Growth (PCG) experiment during the STS-66 mission. This particular section is called the Crystal Observation System, housed in the Thermal Enclosure System (COS/TES). Together with the Vapor Diffusion Apparatus (VDA), housed in Single Locker Thermal Enclosure (SLTES), the COS/TES represents the continuing research into the structure of proteins and other macromolecules such as viruses.

  10. Protein crystal screening and characterization for serial femtosecond nanocrystallography.

    PubMed

    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

  11. A structural dissection of large protein-protein crystal packing contacts

    PubMed Central

    Luo, Jiesi; Liu, Zhongyu; Guo, Yanzhi; Li, Menglong

    2015-01-01

    With the rapid increase in crystal structures of protein-protein complexes deposited in the Protein Data Bank (PDB), more and more crystal contacts have been shown to have similar or even larger interface areas than biological interfaces. However, little attention has been paid to these large crystal packing contacts and their structural principles remain unknown. To address this issue, we used a comparative feature analysis to analyze the geometric and physicochemical properties of large crystal packing contacts by comparing two types of specific protein-protein interactions (PPIs), weak transient complexes and permanent homodimers. Our results show that although large crystal packing contacts have a similar interface area and contact size as permanent homodimers, they tend to be more planar, loosely packed and less hydrophobic than permanent homodimers and cannot form a central core region that is fully buried during interaction. However, the properties of large crystal packing contacts, except for the interface area and contact size, more closely resemble those of weak transient complexes. The large overlap between biological and large crystal packing contacts indicates that interface properties are not efficient indicators for classification of biological interfaces from large crystal packing contacts and finding other specific features urgently needed. PMID:26370141

  12. Screening of Protein Crystallization Trials by Second Order Nonlinear Optical Imaging of Chiral Crystals (SONICC)

    PubMed Central

    Haupert, Levi; Simpson, Garth

    2011-01-01

    Second order nonlinear optical imaging of chiral crystals (SONICC) is a promising new method for the sensitive and selective detection of protein crystals. Relevant general principles of second harmonic generation, which underpins SONICC, are reviewed. Instrumentation and methods for SONICC measurements are described and critically assessed in terms of performance trade-offs. Potential origins of false-positives and false-negatives are also discussed. PMID:22101350

  13. 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.

  14. 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. PMID:26508758

  15. 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

  16. 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.

  17. Purification and characterization of the acyl carrier protein of the Streptomyces glaucescens tetracenomycin C polyketide synthase.

    PubMed Central

    Shen, B; Summers, R G; Gramajo, H; Bibb, M J; Hutchinson, C R

    1992-01-01

    The acyl carrier protein (ACP) of the tetracenomycin C polyketide synthase, encoded by the tcmM gene, has been expressed in both Streptomyces glaucescens and Escherichia coli and purified to homogeneity. Expression of the tcmM gene in E. coli results mainly in the TcmM apo-ACP, whereas expression in S. glaucescens yields solely the holo-ACP. The purified holo-TcmM is active in a malonyl coenzyme A:ACP transacylase assay and is labeled by radioactive beta-alanine, confirming that it carries a 4'-phosphopantetheine prosthetic group. Images PMID:1592832

  18. The detection of tritium-labeled ligands and their carrier proteins using a multiwire proportional counter

    SciTech Connect

    Scott, B.J.; Bateman, J.E.; Bradwell, A.R.

    1982-06-01

    Two-dimensional immunoelectrophoresis combined with autoradiography is a powerful technique for studying the binding of radiolabeled ligands to their carrier proteins. Tritium-labeled compounds are difficult to detect by autoradiography, yet this isotope is often the radiolabel of choice, because it is possible to achieve high specific activity with no loss of biological function. Therefore an electronic detection system called a multiwire proportional counter has been investigated. This has resulted in an increase in detection speed for tritium of several thousand fold over conventional autoradiography and furthermore the results are potentially quantitative.

  19. 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.

  20. Protein crystallization analysis on the World Community Grid

    PubMed Central

    Cumbaa, Christian A.

    2010-01-01

    We have developed an image-analysis and classification system for automatically scoring images from high-throughput protein crystallization trials. Image analysis for this system is performed by the Help Conquer Cancer (HCC) project on the World Community Grid. HCC calculates 12,375 distinct image features on microbatch-under-oil images from the Hauptman-Woodward Medical Research Institute’s High-Throughput Screening Laboratory. Using HCC-computed image features and a massive training set of 165,351 hand-scored images, we have trained multiple Random Forest classifiers that accurately recognize multiple crystallization outcomes, including crystals, clear drops, precipitate, and others. The system successfully recognizes 80% of crystal-bearing images, 89% of precipitate images, and 98% of clear drops. Electronic supplementary material The online version of this article (doi:10.1007/s10969-009-9076-9) contains supplementary material, which is available to authorized users. PMID:20072819

  1. Two-photon excited UV fluorescence for protein crystal detection

    PubMed Central

    Madden, Jeremy T.; DeWalt, Emma L.; Simpson, Garth J.

    2011-01-01

    Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging of chiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC. PMID:21931215

  2. 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.

  3. Solution Structures of Spinach Acyl Carrier Protein with Decanoate and Stearate†

    PubMed Central

    Zornetzer, Gregory A.; Fox, Brian G.; Markley, John L.

    2008-01-01

    Acyl carrier protein (ACP) is a cofactor in a variety of biosynthetic pathways, including fatty acid metabolism. Thus it is of interest to determine structures of physiologically relevant ACP-fatty acid complexes. We report here the NMR solution structures of spinach ACP with decanoate (10:0-ACP) and stearate (18:0-ACP) attached to the 4′ phosphopantetheine prosthetic group. The protein in the fatty acid complexes adopts a single conformer, unlike apo- and holo-ACP, which interconvert in solution between two major conformers. The protein component of both 10:0- and 18:0-ACP adopts the four-helix bundle topology characteristic of ACP, and a fatty acid binding cavity was identified in both structures. Portions of the protein close in space to the fatty acid and the 4′ phosphopantetheine were identified using filtered/edited NOESY experiments. A docking protocol was used to generate protein structures containing bound fatty acid for 10:0- and 18:0-ACP. In both cases, the predominant structure contained fatty acid bound down the center of the helical bundle, in agreement with the location of the fatty acid binding pockets. These structures demonstrate the conformational flexibility of spinach-ACP and suggest how the protein changes to accommodate its myriad binding partners. PMID:16618110

  4. 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

  5. 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.

  6. 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

    2016-01-01

    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

  7. Protein sensing by nanofluidic crystal and its signal enhancement.

    PubMed

    Sang, Jianming; Du, Hongtan; Wang, Wei; Chu, Ming; Wang, Yuedan; Li, Haichao; Alice Zhang, Haixia; Wu, Wengang; Li, Zhihong

    2013-01-01

    Nanofluidics has a unique property that ionic conductance across a nanometer-sized confined space is strongly affected by the space surface charge density, which can be utilized to construct electrical read-out biosensor. Based on this principle, this work demonstrated a novel protein sensor along with a sandwich signal enhancement approach. Nanoparticles with designed aptamer onside are assembled in a suspended micropore to form a 3-dimensional network of nanometer-sized interstices, named as nanofluidic crystal hereafter, as the basic sensing unit. Proteins captured by aptamers will change the surface charge density of nanoparticles and thereby can be detected by monitoring the ionic conductance across this nanofluidic crystal. Another aptamer can further enlarge the variations of the surface charge density by forming a sandwich structure (capturing aptamer/protein/signal enhancement aptamer) and the read-out conductance as well. The preliminary experimental results indicated that human α-thrombin was successfully detected by the corresponding aptamer modified nanofluidic crystal with the limit of detection of 5 nM (0.18 μg/ml) and the read-out signal was enhanced up to 3 folds by using another thrombin aptamer. Being easy to graft probe, facile and low-cost to prepare the nano-device, and having an electrical read-out, the present nanofluidic crystal scheme is a promising and universal strategy for protein sensing. PMID:24404017

  8. Heterogeneous Nucleation of Protein Crystals on Fluorinated Layered Silicate

    PubMed Central

    Ino, Keita; Udagawa, Itsumi; Iwabata, Kazuki; Takakusagi, Yoichi; Kubota, Munehiro; Kurosaka, Keiichi; Arai, Kazuhito; Seki, Yasutaka; Nogawa, Masaya; Tsunoda, Tatsuo; Mizukami, Fujio; Taguchi, Hayao; Sakaguchi, Kengo

    2011-01-01

    Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface. PMID:21818343

  9. Heterogeneous nucleation of protein crystals on fluorinated layered silicate.

    PubMed

    Ino, Keita; Udagawa, Itsumi; Iwabata, Kazuki; Takakusagi, Yoichi; Kubota, Munehiro; Kurosaka, Keiichi; Arai, Kazuhito; Seki, Yasutaka; Nogawa, Masaya; Tsunoda, Tatsuo; Mizukami, Fujio; Taguchi, Hayao; Sakaguchi, Kengo

    2011-01-01

    Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface. PMID:21818343

  10. 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.

  11. 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

  12. [Protein crystals and tubuli bundles in yeast cells. V. Enrichment of crystal and tubuli proteins during O2 limitation and anaerobiosis].

    PubMed

    May, R; Jacob, H E; Müller, H; Meyer, H W

    1976-01-01

    Cells of Saccharomyces carlsbergensis contain proteins assembling to crystals and bundles of tubules (tb) in the cyto- and karyoplasm by osmotic shock with hypertonic solutions. The cellular concentration of these proteins is regulated by oxygen pressure during growth. In cells grown at optimal aeration the protein level is low and crystals and tb cannot be induced. After a short period of O2-limitation or anerobic growth conditions the protein concentration increases and induction of crystals and tb is possible. PMID:969567

  13. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli.

    PubMed

    Pane, Katia; Durante, Lorenzo; Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200-250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15-18 mg of recombinant peptide per liter of culture with 96-98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods. PMID:26808536

  14. Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli

    PubMed Central

    Pizzo, Elio; Varcamonti, Mario; Zanfardino, Anna; Sgambati, Valeria; Di Maro, Antimo; Carpentieri, Andrea; Izzo, Viviana; Di Donato, Alberto; Cafaro, Valeria; Notomista, Eugenio

    2016-01-01

    Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200–250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15–18 mg of recombinant peptide per liter of culture with 96–98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods. PMID:26808536

  15. 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

  16. Preparation and characterization of novel quaternized cellulose nanoparticles as protein carriers.

    PubMed

    Song, Yongbo; Zhou, Jinping; Li, Qian; Guo, Yi; Zhang, Lina

    2009-09-01

    Quaternized cellulose (QC) nanoparticles were prepared in distilled water by ionic crosslinking of QC with sodium tripolyphosphate (TPP) for the first time. BSA as a model protein drug was used to investigate the loading and release features of the nanoparticles. The results indicated that QC nanoparticles had high loading efficiency and capacity for BSA. The in vitro BSA release of the QC nanoparticles displayed a burst effect in the first 2 h and then a slow continuous release. Nanoparticles with a higher DS of QC showed a decrease in particle size, an increase in zeta potential, a higher loading efficiency and a slower drug-release profile. These studies demonstrated that QC nanoparticles are potential protein carriers, and that their physicochemical properties and release profile could be easily adjusted. PMID:19370752

  17. Role of acyl carrier protein isoforms in plant lipid metabolism: Progress report

    SciTech Connect

    Ohlrogge, J.B.

    1989-01-01

    Previous research from my lab has revealed that several higher plant species have multiple isoforms of acyl carrier protein (ACP) and therefore this trait appears highly conserved among higher plants. This level of conservation suggests that the existence of ACP isoforms is not merely the results of neutral gene duplications. We have developed techniques to examine a wider range of species. Acyl carrier proteins can be labelled very specifically and to high specific activity using H-palmitate and the E. coli enzyme acyl-ACP synthetase. Isoforms were then resolved by western blotting and native PAGE of H-palmitate labelled ACP's. Multiple isoforms of ACP were observed the leaf tissue of the monocots Avena sativa and Hordeum vulgare and dicots including Arabidopsis thallina, Cuphea wrightii, and Brassica napus. Lower vascular plants including the cycad, Dioon edule, Ginkgo biloba, the gymnosperm Pinus, the fern Anernia phyllitidis and Psilotum nudum, the most primitive known extant vascular plant, were also found to have multiple ACP isoforms as were the nonvascular liverwort, Marchantia and moss, Polytrichum. Therefore, the development of ACP isoforms occurred early in evolution. However, the uniellular alge Chlamydomonas and Dunaliella and the photosynthetic cyanobacteria Synechocystis and Agmnellum have only a single elecrophotetic form of ACP. Thus, multiple forms of ACP do not occur in all photosynthetic organisms but may be associated with multicellular plants.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. Crystal structure and charge carrier concentration of W 18O 49

    NASA Astrophysics Data System (ADS)

    Viswanathan, K.; Brandt, K.; Salje, E.

    1981-01-01

    The electrical resistivity of the tungsten oxide, W 18O 49, is 1.75 · 10 -3 Ω cm along the needle axis. The charge carrier density, as determined by reflectivity measurements, is 1.87 · 10 22 cm -3, thereby indicating that most of the charge carriers are delocalized. Hence the smaller conductivity along the needle axis than that expected for such charge carrier concentrations must be found in the structure, which has been refined using the data collected with an automatic diffractometer. The structure consists of WO 6 and WO 7 polyhedra which are linked along edges and/or corners. However, as the linkage parallel to b takes place only by sharing corners, an anisotropy in the electrical conductivity may be expected. Another explanation for the smaller conductivity may be found in the occurrence of defects such as tunnels in the structure, which may scatter the electrons. The refinement shows that the tungsten positions, determined by Magneli ( Arkiv Kemi1, 223 (1950)), are essentially correct; but the positions of the oxygens, especially two of them, differ considerably. This results in one of the tungsten atoms getting an additional coordinating oxygen, the coordination number thereby becoming seven.

  3. Minority carrier recombination lifetimes in n-type CdMgSe mixed crystals measured by means of the photothermal infrared radiometry

    NASA Astrophysics Data System (ADS)

    Pawlak, M.; Maliński, M.

    2014-03-01

    Minority recombination lifetimes of n-type CdMgSe mixed crystals were estimated by using infrared photothermal radiometry (PTR) amplitude and phase frequency spectra. The results obtained by the PTR method indicate that the lifetimes of optically generated carriers in CdSe and CdxMg1-xSe crystals are about 0.1 μs. The diffusion length of minority carrier in n-type CdSe single crystal was found to be 4.42 μm and it is in a good agreement with the literature value. It was found that with the increasing thermal-to-plasma component coefficient A the carrier concentration increases as expected from PTR theory.

  4. Laser Scattering Tomography for the Study of Defects in Protein Crystals

    NASA Technical Reports Server (NTRS)

    Feigelson, Robert S.; DeLucas, Lawrence; DeMattei, R. C.

    1997-01-01

    The goal of this research is to explore the application of the non-destructive technique of Laser Scattering Tomography (LST) to study the defects in protein crystals and relate them to the x-ray diffraction performance of the crystals. LST has been used successfully for the study of defects in inorganic crystals and. in the case of lysozyme, for protein crystals.

  5. Studies of Toxoplasma gondii and Plasmodium falciparum enoyl acyl carrier protein reductase and implications for the development of antiparasitic agents

    SciTech Connect

    Muench, Stephen P.; Prigge, Sean T.; McLeod, Rima; Rafferty, John B.; Kirisits, Michael J.; Roberts, Craig W.; Mui, Ernest J.; Rice, David W.

    2007-03-01

    The crystal structures of T. gondii and P. falciparum ENR in complex with NAD{sup +} and triclosan and of T. gondii ENR in an apo form have been solved to 2.6, 2.2 and 2.8 Å, respectively. Recent studies have demonstrated that submicromolar concentrations of the biocide triclosan arrest the growth of the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii and inhibit the activity of the apicomplexan enoyl acyl carrier protein reductase (ENR). The crystal structures of T. gondii and P. falciparum ENR in complex with NAD{sup +} and triclosan and of T. gondii ENR in an apo form have been solved to 2.6, 2.2 and 2.8 Å, respectively. The structures of T. gondii ENR have revealed that, as in its bacterial and plant homologues, a loop region which flanks the active site becomes ordered upon inhibitor binding, resulting in the slow tight binding of triclosan. In addition, the T. gondii ENR–triclosan complex reveals the folding of a hydrophilic insert common to the apicomplexan family that flanks the substrate-binding domain and is disordered in all other reported apicomplexan ENR structures. Structural comparison of the apicomplexan ENR structures with their bacterial and plant counterparts has revealed that although the active sites of the parasite enzymes are broadly similar to those of their bacterial counterparts, there are a number of important differences within the drug-binding pocket that reduce the packing interactions formed with several inhibitors in the apicomplexan ENR enzymes. Together with other significant structural differences, this provides a possible explanation of the lower affinity of the parasite ENR enzyme family for aminopyridine-based inhibitors, suggesting that an effective antiparasitic agent may well be distinct from equivalent antimicrobials.

  6. Protein-Resistant Biodegradable Amphiphilic Graft Copolymer Vesicles as Protein Carriers.

    PubMed

    Wang, Yupeng; Yan, Lesan; Li, Bin; Qi, Yanxin; Xie, Zhigang; Jing, Xiabin; Chen, Xuesi; Huang, Yubin

    2015-09-01

    The protein adsorption and self-assembly behavior of biocompatible graft copolymer, poly(lactide-co-diazidomethyl trimethylene carbonate)-g-poly(ethylene glycol) [P(LA-co-DAC)-g-PEG], were systematically studied. The graft copolymers showed enhanced resistance to non-specific protein adsorption compared with their block copolymer counterparts, indicative of the increased effect of PEG density beyond PEG length. Diverse nanostructures including vesicles can be assembled from the amphiphilic graft copolymers with well-defined nano-sizes. Hemoglobin (Hb), as a model protein, can be entrapped in the formed vesicles and keep the gas-binding capacity. The reduced release rate of Hb from graft copolymer vesicles indicated the relatively stable membrane packing compared with block copolymer counterpart. PMID:26036907

  7. 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…

  8. Evolution of hepatitis B virus surface gene and protein among Iranian chronic carriers from different provinces

    PubMed Central

    Ramezani, Fatemeh; Alavian, Seyed Moayed; Sadeghi, Ahmadreza; Khedive, Abolfazl; Ghalichi, Leila; Norouzi, Mehdi; Karimzadeh, Hadi; Malekzadeh, Reza; Montazeri, Ghodrat; Nejatizadeh, Azim; Ziaee, Masood; Abedi, Farshid; Ataei, Behrooz; Yaran, Majid; Sayad, Babak; Somi, Mohamad Hosein; Sarizadeh, Gholamreza; Sanei-Moghaddam, Ismail; Mansour-Ghanaei, Fariborz; Rafatpanah, Houshang; Keyvani, Hossein; Kalantari, Ebrahim; Saberfiroozi, Mehdi; Rezaee, Reza; Daram, Maryam; Mahabadi, Mostafa; Goodarzi, Zahra; Poortahmasebi, Vahdat; Geravand, Babak; Khamseh, Azam; Mahmoodi, Masoud; Jazayeri, Seyed Mohammad

    2015-01-01

    Background and Objectives: Iranian chronic HBV carrier's population has shown a unique pattern of genotype D distribution all around the country. The aim of this study was to explore more details of evolutionary history of carriers based on structural surface proteins from different provinces. Materials and Methods: Sera obtained from 360 isolates from 12 Different regions of country were used for amplification and sequencing of surface proteins. A detailed mutational analysis was undertaken. Results: The total ratio for Missense/Silent nucleotide substitutions was 0.96. Sistan and Kermanshah showed the lowest rate of evolution between provinces (P = 0.055). On the other hand, Khorasan Razavi and Khoozestan contained the highest ratio (P = 0.055). The rest of regions were laid between these two extremes. Azarbayjan and Guilan showed the highest proportion of immune epitope distribution (91.3% and 96%, respectively). Conversely, Sistan and Tehran harbored the least percentage (66.6% and 68.8%, respectively). Kermanshah province contained only 5.2%, whereas Isfahan had 54.5% of B cell epitope distribution. In terms of T helper epitopes, all provinces showed a somehow homogeneity: 22.58% (Fars) to 46.6% (Khuzestan). On the other hand, distribution of substitutions within the CTL epitopes showed a wide range of variation between 6.6% (Khuzestan) and 63% (Kermanshah). Conclusion: Further to low selection pressure found in Iranian population, the variations between different regions designate random genetic drift within the surface proteins. These finding would have some applications in terms of specific antiviral regimen, design of more efficient vaccine and public health issues. PMID:26697161

  9. Structural correlates of carrier protein recognition in tetanus toxoid-conjugated bacterial polysaccharide vaccines.

    PubMed

    Lockyer, Kay; Gao, Fang; Derrick, Jeremy P; Bolgiano, Barbara

    2015-03-10

    An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8×10(6) g/mol to larger than 20×10(6) g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines. PMID:25640334

  10. Electron and Hydrogen Atom Transfers in the Hydride Carrier Protein EmoB.

    PubMed

    Gillet, Natacha; Lévy, Bernard; Moliner, Vicent; Demachy, Isabelle; de la Lande, Aurélien

    2014-11-11

    In this article, we investigate the mechanism of hydride transfer taking place within the EmoB protein of the Mesorhizobium species. The reaction involves the net transfer of one proton and two electrons from a reduced flavin mononucleotide (FMN) cofactor, which is anchored in the protein scaffold, to a diffusible oxidized FMN cofactor, both being held together by π-stacking interactions. To analyze the formal hydride transfer in terms of more elementary steps, electron transfer (ET), and hydrogen atom transfers (HAT), we employ a combination of classical molecular dynamics simulations and hybrid constrained Density Functional Theory/Molecular Mechanics (cDFT/MM) energy calculations to build the free energy profiles, for the ET before and after HAT occurs between the flavins. The main outcomes of our study are first to highlight the role of the protein in stabilizing the π-stacked FMN dimer and second to reveal the coupling between the ET and HAT. Before HAT has taken place, ET is unfavorable by 8 kcal/mol and become favorable by 8 kcal/mol after HAT. Our simulations show that such a coupling is not present for the analogous process in water (ET is almost athermal). This suggests a functional role for the protein matrix to ensure EmoB a role of hydride carrier in the Mesorhizobium species. PMID:26584385

  11. Maltose-binding protein is a potential carrier for oral immunizations.

    PubMed

    Bellot, P; Tiels, P; Melkebeek, V; Devriendt, B; Goddeeris, B M; Cox, E

    2013-03-15

    Maltose binding protein (MBP) is often fused to a relevant protein to improve its yield and facilitate its purification, but MBP can also enhance the immunogenicity of the fused proteins. Recent data suggest that MBP may potentiate antigen-presenting functions in immunized animals by providing intrinsic maturation stimuli to dendritic cells through TLR4. The aim of this study was to examine if an MBP-specific immune response can be elicited by oral administration of MBP. Therefore, in a first experiment the MBP specific immune response was analyzed after oral immunization with MBP or MBP+CT to piglets and both the systemic and mucosal immune responses were examined Although no high systemic response was observed in the MBP-group, a local mucosal IgM MBP-specific response in the jejunal Peyer's patches was observed. In the second experiment MBPFedF was orally administered to piglets. A significant systemic response against MBP and a weak response against FedF were found after oral administration of MBPFedF+CT. Also the presence of MBP-specific IgA ASC in the lamina propria indicates that a local intestinal immune response against MBP was induced. Our data suggests that MBP can cross the epithelial barrier reaching the gut-associated lymphoid tissue after oral administration to pigs, which implicates that MBP could act as a carrier and delivery system for fused proteins to target the vaccine antigens to intestinal immune cells. PMID:23078905

  12. Protein crystals on phase-separating model membranes

    NASA Astrophysics Data System (ADS)

    Manley, Suliana; Horton, Margaret; Leszczynski, Szymon; Gast, Alice

    2006-03-01

    We study the interplay between the crystallization of proteins tethered to membranes and separation within the membranes of giant unilamellar vesicles (GUVs) composed of DOPC, sphingomyelin (SM), and cholesterol. These model membranes phase separate into coexisting liquid domains below a miscibility transition temperature. This phase separation captures some aspects of the formation of lipid rafts in cell membranes and demonstrates the influence of membrane composition on raft formation. Real cell membranes have a much more complicated structure. There are additional physical constraints present in cell membranes, such as their attachment to the cytoskeleton and the presence of membrane bound proteins. The self-association of membrane proteins can influence the membrane phase behavior. We begin to investigate these effects on model tethered protein- loaded membranes by incorporating a small amount of biotin-X- DPPE into our GUVs. The biotinylated lipid partitions into a cholesterol-poor phase; thus, streptavidin binds preferentially to one of the membrane phases. As streptavidin assembles to form crystalline domains, it restricts the membrane mobility. We examine the effect of this protein association on lipid phase separation, as well as the effect of the lipid phase separation on the crystallization of the tethered proteins.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. Crystal growth of proteins, nucleic acids, and viruses in gels.

    PubMed

    Lorber, Bernard; Sauter, Claude; Théobald-Dietrich, Anne; Moreno, Abel; Schellenberger, Pascale; Robert, Marie-Claire; Capelle, Bernard; Sanglier, Sarah; Potier, Noëlle; Giegé, Richard

    2009-11-01

    Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses. PMID:20005247

  18. Experimental study of osteoinduction using a new material as a carrier for bone morphogenetic protein-2.

    PubMed

    Koyama, Noriaki; Okubo, Yasunori; Nakao, Kazumasa; Osawa, Kenji; Bessho, Kazuhisa

    2011-06-01

    We evaluated the usefulness of artificial collagen as a new carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2) by comparing it with that of atelopeptide collagen, which is derived from porcine skin, and which we have previously shown to be useful for the induction of bone. rhBMP-2 5μg with either atelopeptide collagen 3mg or artificial collagen 3mg was implanted into the calf muscle of 10-week-old Wistar rats (n=3 in each group). Three rats were given artificial collagen alone and acted as controls (n=3). Radiographic evaluation, histological analysis, and biochemical examinations were made on day 21 after implantation. Soft radiographs (wavelength 10-0.10nm) showed opaque shadows in both groups. Histological analysis showed that new bone had formed in both experimental groups. Endochondral ossification was found at the outermost edge of the implanted collagen in the atelopeptide group. However, there was less ossification in the implanted collagen in the artificial collagen group. On biochemical examination, alkaline phosphatase activity and calcium concentrations in both experimental groups were higher than in the control group, and were higher in the atelopeptide group than in the artificial collagen group. Our results suggest that artificial collagen is useful as a carrier for rhBMP-2 designed to promote the formation of new bone. PMID:20554359

  19. Hydrogen- and oxygen-related effects in phthalocyanine crystals: formation of carrier traps and a change in the magnetic state.

    PubMed

    Tsetseris, Leonidas

    2014-02-21

    The performance of organic semiconductors as electronic materials is very sensitive to impurity incorporation and reactions. Here we show using first-principles calculations that hydrogen and oxygen impurities introduce distinct changes in the electronic properties of metal phthalocyanines (MPc), a family of organic semiconductors renowned for their light conversion efficiency. Selective adsorption of hydrogen atoms on pyridinic nitrogen atoms of MPc molecules, namely zinc and copper phthalocyanines, modifies the magnetic state of the latter and generates carrier trap states deep in the band gap of MPc crystals. Reactions with O atoms have a lesser effect on MPc electronic properties, while intercalated oxygen molecules give rise to traps below the conduction band minimum. The results identify H and O impurities as important degradation culprits for MPc-based systems, in agreement with pertinent experiments. PMID:24413162

  20. Protein Crystal Growth Samples Placed Aboard Mir Space Station

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Astronaut Tom Akers places a liquid nitrogen Dewar containing frozen protein solutions aboard Russia's space Station Mir during a visit by the Space Shuttle (STS-79). The protein samples were flash-frozen on Earth and will be allowed to thaw and crystallize in the microgravity environment on Mir Space Station. A later crew will return the Dewar to Earth for sample analysis. Dr. Alexander McPherson of the University of California at Riverside is the principal investigator. Photo credit: NASA/Johnson Space Center.

  1. Protein Crystal Growth Samples Placed Aboard Mir Space Station

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Astronaut Michael Clifford places a liquid nitrogen Dewar containing frozen protein solutions aboard Russia's space station Mir during a visit by the Space Shuttle (STS-76). The protein samples were flash-frozen on Earth and will be allowed to thaw and crystallize in the microgravity environment on Mir Space Station. A later crew will return the Dewar to Earth for sample analysis. Dr. Alexander McPherson of the University of California at Riverside is the principal investigator. Photo credit: NASA/Johnson Space Center.

  2. 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).

  3. 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

  4. 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.

  5. 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

  6. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis

    PubMed Central

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4’-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. DOI: http://dx.doi.org/10.7554/eLife.17828.001 PMID:27540631

  7. Evolutionary, environmental and tissue controls on the occurrence of multiple isoforms of acyl carrier protein

    SciTech Connect

    Battey, J.F.; Ohlrogge, J.B. )

    1989-04-01

    Previous research has revealed that several higher plant species have multiple isoforms of acyl carrier protein (ACP). We have examined the development of this trait in evolutionarily diverse species. Isoforms were resolved by Western blotting and native PAGE of {sup 3}H-palmitate labelled ACP's. Multiple isoforms of ACP were observed in primitive vascular plants including gymnosperms, ferns and Psilotum and the nonvascular liverworts and mosses. Therefore, the development of ACP isoforms occurred early in evolution. However, unicellular algae and bacteria such as Chlamydomonas, Dunaliella, Synechocystis and Agmnellum have only a single electrophoretic form of ACP. Thus, multiple forms of ACP do not occur in all photosynthetic organisms but may be associated with multicellular plants. We have also examined light and tissue control over the expression of ACP isoforms. The expression of multiple forms of ACP in leaf of Spinacia and Avena is altered very little by light. Rather, the different patterns of ACP isoforms are primarily dependant on tissue source.

  8. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis.

    PubMed

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4'-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. PMID:27540631

  9. Rod-shaped hydroxyapatite with mesoporous structure as drug carriers for proteins

    NASA Astrophysics Data System (ADS)

    Zhang, Wandong; Chai, Yamin; Xu, Xianghua; Wang, Yonglan; Cao, Nana

    2014-12-01

    Rod-shaped hydroxyapatite (HAp) with mesoporous structure was synthesized by a hydrothermal method using Pluronic block co-polymer F127 as the template. The rod-shaped HAp was then tested as protein drug carriers by investigating their protein adsorption/release properties. Bovine serum albumin (BSA) and lysozyme (LSZ) were used as the model drugs. Various instrumental methods were used to characterize the structure, morphology, texture and protein drug adsorption/release properties of the samples. The amounts of BSA or LSZ adsorbed onto the rod-shaped HAp and their release profiles were evaluated in a simulated body fluid (SBF). The synthesized rod-shaped HAp had irregular mesostructures with lengths of 75-125 nm and diameters of about 25 nm. The rod-shaped HAp exhibited a higher loading capacity for BSA than for LSZ in the SBF. This adsorption behavior can be explained by the morphology of the rod-shaped HAp, which grew along the c-axis, leading to an a(b)-plane area that is larger than the c-plane area. Consequently, the number of positive charges on the surface of the rod-shaped HAp increased relative to the number of negative charges. The BSA release rate in SBF was slower than that of LSZ which is a result of the HAp surface properties.

  10. Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence†

    PubMed Central

    Martin, Gregory G.; Hostetler, Heather A.; McIntosh, Avery L.; Tichy, Shane E.; Williams, Brad J.; Russell, David H.; Berg, Jeremy M.; Spencer, Thomas A.; Ball, Judith; Kier, Ann B.; Schroeder, Friedhelm

    2008-01-01

    Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2’s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts/caveolae (AF488-CTB), 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488-antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting. PMID:18465878

  11. Recognition of acyl carrier proteins by ketoreductases in assembly line polyketide synthases.

    PubMed

    Ostrowski, Matthew P; Cane, David E; Khosla, Chaitan

    2016-07-01

    Ketoreductases (KRs) are the most widespread tailoring domains found in individual modules of assembly line polyketide synthases (PKSs), and are responsible for controlling the configurations of both the α-methyl and β-hydroxyl stereogenic centers in the growing polyketide chain. Because they recognize substrates that are covalently bound to acyl carrier proteins (ACPs) within the same PKS module, we sought to quantify the extent to which protein-protein recognition contributes to the turnover of these oxidoreductive enzymes using stand-alone domains from the 6-deoxyerythronolide B synthase (DEBS). Reduced 2-methyl-3-hydroxyacyl-ACP substrates derived from two enantiomeric acyl chains and four distinct ACP domains were synthesized and presented to four distinct KR domains. Two KRs, from DEBS modules 2 and 5, displayed little preference for oxidation of substrates tethered to their cognate ACP domains over those attached to the other ACP domains tested. In contrast, the KR from DEBS module 1 showed an ~10-50-fold preference for substrate attached to its native ACP domain, whereas the KR from DEBS module 6 actually displayed an ~10-fold preference for the ACP from DEBS module 5. Our findings suggest that recognition of the ACP by a KR domain is unlikely to affect the rate of native assembly line polyketide biosynthesis. In some cases, however, unfavorable KR-ACP interactions may suppress the rate of substrate processing when KR domains are swapped to construct hybrid PKS modules. PMID:27118242

  12. Isolation and characterization of an enoyl-acyl carrier protein reductase gene from microalga Isochrysis galbana

    NASA Astrophysics Data System (ADS)

    Zheng, Minggang; Liang, Kepeng; Wang, Bo; Sun, Xiuqin; Yue, Yanyan; Wan, Wenwen; Zheng, Li

    2013-03-01

    In most bacteria, plants and algae, fatty acid biosynthesis is catalyzed by a group of freely dissociable proteins known as the type II fatty acid synthase (FAS II) system. In the FAS II system, enoylacyl carrier protein reductase (ENR) acts as a determinant for completing the cycles of fatty acid elongation. In this study, the cDNA sequence of ENR, designated as IgENR, was isolated from the microalga Isochrysis galbana CCMM5001. RACE (rapid amplification of cDNA ends) was used to isolate the full-length cDNA of IgENR (1 503 bp), which contains an open reading frame (ORF) of 1 044 bp and encodes a protein of 347 amino acids. The genomic DNA sequence of IgENR is interrupted by four introns. The putative amino acid sequence is homologous to the ENRs of seed plants and algae, and they contain common coenzymebinding sites and active site motifs. Under different stress conditions, real-time quantitative polymerase chain reaction (RT-qPCR) showed the expression of IgENR was upregulated by high temperature (35°C), and downregulated by depleted nitrogen (0 mol/L). To clarify the mechanism of lipids accumulating lipids, other genes involved in lipids accumulation should be studied.

  13. Reprogramming acyl carrier protein interactions of an acyl-CoA promiscuous trans-acyltransferase

    PubMed Central

    Ye, Zhixia; Musiol, Ewa M; Weber, Tilmann; Williams, Gavin J

    2014-01-01

    SUMMARY Protein interactions between acyl carrier proteins (ACP’s) and trans-acting acyltransferase domains (trans-AT’s) are critical for regioselective extender unit installation by many polyketide synthases. Yet, little is known regarding the specificity of these interactions, particularly for trans-AT’s with unusual extender unit specificities. Currently, the best-studied trans-AT with non-malonyl specificity is KirCII from kirromycin biosynthesis. Here, we developed a new assay to probe ACP interactions based on leveraging the extender unit promiscuity of KirCII. The assay allows us to identify residues on the ACP surface that contribute to specific recognition by KirCII. This information proved sufficient to modify a non-cognate ACP from a different biosynthetic system to be a substrate for KirCII. The findings form a foundation for further understanding the specificity of trans-AT:ACP protein interactions, and for engineering modular polyketide synthases to produce analogues. PMID:24726832

  14. Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence

    SciTech Connect

    Byers, D.M.; Meighen, E.A.

    1985-09-01

    Pulse-chase experiments with (/sup 3/H)tetradecanoic acid and ATP showed that the bioluminescence-related 32-kDa acyltransferase from Vibrio harveyi can specifically catalyze the deacylation of a /sup 3/H-labeled 18-kDa protein observed in extracts of this bacterium. The 18-kDa protein has been partially purified and its physical and chemical properties strongly indicate that it is fatty acyl-acyl carrier protein (acyl-ACP). Both this V. harveyi (/sup 3/H)acylprotein and (/sup 3/H)palmitoyl-ACP from Escherichia coli were substrates in vitro for either the V. harveyi 32-kDa acyltransferase or the analogous enzyme (34K) from Photobacterium phosphoreum. TLC analysis indicated that the hexane-soluble product of the reaction is fatty acid. No significant cleavage of either E. coli or V. harveyi tetradecanoyl-ACP was observed in extracts of these bacteria unless the 32-kDa or 34K acyltransferase was present. Since these enzymes are believed to be responsible for the supply of fatty acids for reduction to form the aldehyde substrate of luciferase, the above results suggest that long-chain acyl-ACP is the source of fatty acids for bioluminescence.

  15. Interactions of the acyl chain with the Saccharomyces cerevisiae acyl carrier protein.

    PubMed

    Perez, Daniel R; Leibundgut, Marc; Wider, Gerhard

    2015-04-01

    Acyl carrier protein (ACP) domains are critical integral components of multifunctional type I fatty acid synthases (FAS I) and polyketide synthases (PKSs), where they shuttle the growing adducts of the synthesis between the catalytic domains. In contrast to ACP of mammalian FAS I, PKSs, and the dissociated fatty acid synthase type II systems (FAS II) of bacteria, fungal FAS I ACP consists of two subdomains, one comprising the canonical ACP fold observed in all FAS systems and the other representing an extra structural subdomain. While ACPs of dissociated FAS II are able to sequester the reaction intermediates during substrate shuttling, such a transport mechanism has not been observed in ACP domains of multifunctional FAS I and PKS systems. For a better understanding of the interaction between the canonical subdomain of fungal ACP with the growing acyl chain and the role of the structural subdomain, we determined the structure of the isolated Saccharomyces cerevisiae acyl carrier protein (ScACP) domain by NMR spectroscopy and investigated the interactions between ScACP and covalently attached substrate acyl chains of varying length by monitoring chemical shift perturbations. The interactions were mapped to the hydrophobic core of the canonical subdomain, while no perturbations were detected in the structural subdomain. A population analysis revealed that only approximately 15% of covalently attached decanoyl chains are sequestered by the ACP core, comparable to the mammalian FAS I and multifunctional PKS systems, which do not sequester their substrates. Finally, denaturation experiments show that both ScACP subdomains unfold cooperatively and that the weak interaction of the acyl chain with the hydrophobic core does not significantly affect the ACP stability. PMID:25774789

  16. Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase

    PubMed Central

    Vance, Steven; Tkachenko, Olga; Thomas, Ben; Bassuni, Mona; Hong, Hui; Nietlispach, Daniel; Broadhurst, William

    2016-01-01

    Type I modular polyketide synthases (PKSs) produce polyketide natural products by passing a growing acyl substrate chain between a series of enzyme domains housed within a gigantic multifunctional polypeptide assembly. Throughout each round of chain extension and modification reactions, the substrate stays covalently linked to an acyl carrier protein (ACP) domain. In the present study we report on the solution structure and dynamics of an ACP domain excised from MLSA2, module 9 of the PKS system that constructs the macrolactone ring of the toxin mycolactone, cause of the tropical disease Buruli ulcer. After modification of apo ACP with 4′-phosphopantetheine (Ppant) to create the holo form, 15N nuclear spin relaxation and paramagnetic relaxation enhancement (PRE) experiments suggest that the prosthetic group swings freely. The minimal chemical shift perturbations displayed by Ppant-attached C3 and C4 acyl chains imply that these substrate-mimics remain exposed to solvent at the end of a flexible Ppant arm. By contrast, hexanoyl and octanoyl chains yield much larger chemical shift perturbations, indicating that they interact with the surface of the domain. The solution structure of octanoyl-ACP shows the Ppant arm bending to allow the acyl chain to nestle into a nonpolar pocket, whereas the prosthetic group itself remains largely solvent exposed. Although the highly reduced octanoyl group is not a natural substrate for the ACP from MLSA2, similar presentation modes would permit partner enzyme domains to recognize an acyl group while it is bound to the surface of its carrier protein, allowing simultaneous interactions with both the substrate and the ACP. PMID:26920023

  17. NMR Solution Structure and Biophysical Characterization of Vibrio harveyi Acyl Carrier Protein A75H

    PubMed Central

    Chan, David I.; Chu, Byron C. H.; Lau, Cheryl K. Y.; Hunter, Howard N.; Byers, David M.; Vogel, Hans J.

    2010-01-01

    Bacterial acyl carrier protein (ACP) is a highly anionic, 9 kDa protein that functions as a cofactor protein in fatty acid biosynthesis. Escherichia coli ACP is folded at neutral pH and in the absence of divalent cations, while Vibrio harveyi ACP, which is very similar at 86% sequence identity, is unfolded under the same conditions. V. harveyi ACP adopts a folded conformation upon the addition of divalent cations such as Ca2+ and Mg2+ and a mutant, A75H, was previously identified that restores the folded conformation at pH 7 in the absence of divalent cations. In this study we sought to understand the unique folding behavior of V. harveyi ACP using NMR spectroscopy and biophysical methods. The NMR solution structure of V. harveyi ACP A75H displays the canonical ACP structure with four helices surrounding a hydrophobic core, with a narrow pocket closed off from the solvent to house the acyl chain. His-75, which is charged at neutral pH, participates in a stacking interaction with Tyr-71 in the far C-terminal end of helix IV. pH titrations and the electrostatic profile of ACP suggest that V. harveyi ACP is destabilized by anionic charge repulsion around helix II that can be partially neutralized by His-75 and is further reduced by divalent cation binding. This is supported by differential scanning calorimetry data which indicate that calcium binding further increases the melting temperature of V. harveyi ACP A75H by ∼20 °C. Divalent cation binding does not alter ACP dynamics on the ps-ns timescale as determined by 15N NMR relaxation experiments, however, it clearly stabilizes the protein fold as observed by hydrogen-deuterium exchange studies. Finally, we demonstrate that the E. coli ACP H75A mutant is similarly unfolded as wild-type V. harveyi ACP, further stressing the importance of this particular residue for proper protein folding. PMID:20659901

  18. 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.

  19. Order induced charge carrier mobility enhancement in columnar liquid crystal diodes.

    PubMed

    Eccher, Juliana; Faria, Gregório C; Bock, Harald; von Seggern, Heinz; Bechtold, Ivan H

    2013-11-27

    Discotic molecules comprising a rigid aromatic core and flexible side chains have been promisingly applied in OLEDs as self-organizing organic semiconductors. Due to their potentially high charge carrier mobility along the columns, device performance can be readily improved by proper alignment of columns throughout the bulk. In the present work, the charge mobility was increased by 5 orders of magnitude due to homeotropic columnar ordering induced by the boundary interfaces during thermal annealing in the mesophase. State-of-the-art diodes were fabricated using spin-coated films whose homeotropic alignment with formation of hexagonal germs was observed by polarizing optical microscopy. The photophysical properties showed drastic changes at the mesophase-isotropic transition, which is supported by the gain of order observed by X-ray diffraction. The electrical properties were investigated by modeling the current-voltage characteristics by a space-charge-limited current transport with a field dependent mobility. PMID:24191748

  20. 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.

  1. 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.

  2. 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

  3. Photosensitizer and polycationic peptide-labeled streptavidin as a nano-carrier for light-controlled protein transduction.

    PubMed

    Minamihata, Kosuke; Maeda, Yasukazu; Yamaguchi, Satoshi; Ishihara, Wataru; Ishiwatari, Akira; Takamori, Satoshi; Yamahira, Shinya; Nagamune, Teruyuki

    2015-12-01

    Transductions of exogenous proteins into cells enable the precise study of the effect of the transduced proteins on cellular functions. Accordingly, the protein transduction technique, which can control the release of proteins into the cytosol with certainty and high-throughput, is highly desired in various research fields. In this study, streptavidin (SA) labeled with a photosensitizer and cell-permeable peptides (CPP) was proposed as a nano-carrier for light-controlled protein transduction. SA was modified with biotinylated oligo-arginine peptides (Rpep), which were functionalized with Alexa Fluor 546 (AF546), to achieve cell penetrating and endosomal escape functionalities. The SA-Rpep complex was efficiently internalized into living HeLa cells corresponding to the length and the modification number of Rpep. SA conjugated with more than three equimolar AF546-modified Rpep consisting of fifteen arginine residues was achieved to diffuse throughout the cytosol without cytotoxicity by irradiation of the excitation light for AF546. The optimized nano-carrier was confirmed to transduce a biotinylated model cargo protein, enhanced green fluorescent protein fused with thioredoxin (tEGFP) into the cytosol at the light-irradiated area. The results provided proof-of-principle that SA possessing multiple AF546-modified Rpep has the potential to be a versatile and facile carrier for light-controlled protein transduction into the cytosol of mammalian cells. PMID:25935501

  4. Dissecting the Structural Elements for the Activation of β-Ketoacyl-(Acyl Carrier Protein) Reductase from Vibrio cholerae

    PubMed Central

    Hou, Jing; Zheng, Heping; Chruszcz, Maksymilian; Zimmerman, Matthew D.; Shumilin, Igor A.; Osinski, Tomasz; Demas, Matt; Grimshaw, Sarah

    2015-01-01

    ABSTRACT β-Ketoacyl-(acyl carrier protein) reductase (FabG) catalyzes the key reductive reaction in the elongation cycle of fatty acid synthesis (FAS), which is a vital metabolic pathway in bacteria and a promising target for new antibiotic development. The activation of the enzyme is usually linked to the formation of a catalytic triad and cofactor binding, and crystal structures of FabG from different organisms have been captured in either the active or inactive conformation. However, the structural elements which enable activation of FabG require further exploration. Here we report the findings of structural, enzymatic, and binding studies of the FabG protein found in the causative agent of cholera, Vibrio cholerae (vcFabG). vcFabG exists predominantly as a dimer in solution and is able to self-associate to form tetramers, which is the state seen in the crystal structure. The formation of the tetramer may be promoted by the presence of the cofactor NADP(H). The transition between the dimeric and tetrameric states of vcFabG is related to changes in the conformations of the α4/α5 helices on the dimer-dimer interface. Two glycine residues adjacent to the dimer interface (G92 and G141) are identified to be the hinge for the conformational changes, while the catalytic tyrosine (Y155) and a glutamine residue that forms hydrogen bonds to both loop β4-α4 and loop β5-α5 (Q152) stabilize the active conformation. The functions of the aforementioned residues were confirmed by binding and enzymatic assays for the corresponding mutants. IMPORTANCE This paper describes the results of structural, enzymatic, and binding studies of FabG from Vibrio cholerae (vcFabG). In this work, we dissected the structural elements responsible for the activation of vcFabG. The structural information provided here is essential for the development of antibiotics specifically targeting bacterial FabG, especially for the multidrug-resistant strains of V. cholerae. PMID:26553852

  5. 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

  6. Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners

    PubMed Central

    2011-01-01

    Background Enzymes in the radical SAM (rSAM) domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis. Evolutionary pressures and relationships to other cellular constituents impose recognizable grammars on each class of rSAM-containing system, shaping patterns in results obtained through various comparative genomics analyses. Results An uncharacterized gene cluster found in many Actinobacteria and sporadically in Firmicutes, Chloroflexi, Deltaproteobacteria, and one Archaeal plasmid contains a PqqE-like rSAM protein family that includes Rv0693 from Mycobacterium tuberculosis. Members occur clustered with a strikingly well-conserved small polypeptide we designate "mycofactocin," similar in size to bacteriocins and PqqA, precursor of pyrroloquinoline quinone (PQQ). Partial Phylogenetic Profiling (PPP) based on the distribution of these markers identifies the mycofactocin cluster, but also a second tier of high-scoring proteins. This tier, strikingly, is filled with up to thirty-one members per genome from three variant subfamilies that occur, one each, in three unrelated classes of nicotinoproteins. The pattern suggests these variant enzymes require not only NAD(P), but also the novel gene cluster. Further study was conducted using SIMBAL, a PPP-like tool, to search these nicotinoproteins for subsequences best correlated across multiple genomes to the presence of mycofactocin. For both the short chain dehydrogenase/reductase (SDR) and iron-containing dehydrogenase families, aligning SIMBAL's top-scoring sequences to homologous solved crystal structures shows signals centered over NAD(P)-binding sites rather than over substrate-binding or active site residues. Previous studies on some of these proteins have revealed a non-exchangeable NAD cofactor, such that enzymatic activity in vitro requires an artificial electron acceptor such as N,N-dimethyl-4

  7. Structural and Functional Analyses of a Sterol Carrier Protein in Spodoptera litura

    PubMed Central

    Xu, Rui; Zheng, Sichun; He, Hongwu; Wan, Jian; Feng, Qili

    2014-01-01

    Backgrounds In insects, cholesterol is one of the membrane components in cells and a precursor of ecdysteroid biosynthesis. Because insects lack two key enzymes, squalene synthase and lanosterol synthase, in the cholesterol biosynthesis pathway, they cannot autonomously synthesize cholesterol de novo from simple compounds and therefore have to obtain sterols from their diet. Sterol carrier protein (SCP) is a cholesterol-binding protein responsible for cholesterol absorption and transport. Results In this study, a model of the three-dimensional structure of SlSCPx-2 in Spodoptera litura, a destructive polyphagous agricultural pest insect in tropical and subtropical areas, was constructed. Docking of sterol and fatty acid ligands to SlSCPx-2 and ANS fluorescent replacement assay showed that SlSCPx-2 was able to bind with relatively high affinities to cholesterol, stearic acid, linoleic acid, stigmasterol, oleic acid, palmitic acid and arachidonate, implying that SlSCPx may play an important role in absorption and transport of these cholesterol and fatty acids from host plants. Site-directed mutation assay of SlSCPx-2 suggests that amino acid residues F53, W66, F89, F110, I115, T128 and Q131 are critical for the ligand-binding activity of the SlSCPx-2 protein. Virtual ligand screening resulted in identification of several lead compounds which are potential inhibitors of SlSCPx-2. Bioassay for inhibitory effect of five selected compounds showed that AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 inhibited the growth of S. litura larvae. Conclusions Compounds AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 selected based on structural modeling showed binding affinity to SlSCPx-2 protein and inhibitory effect on the growth of S. litura larvae. PMID:24454688

  8. 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.

  9. 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.

  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. Abnormalities of ADP/ATP carrier protein in J-2-N cardiomyopathic hamsters.

    PubMed

    Kato, M; Yang, J; Iwai, T; Tanamura, A; Arino, T; Kawashima, O; Takeda, N

    1993-02-17

    ADP/ATP carrier protein (AAC) is located in the mitochondrial inner membrane and has an important function in mitochondrial energy supply. This protein transports ATP to the cytoplasm and counter transports ADP into the mitochondria. J-2-N cardiomyopathic hamsters were investigated to determine the AAC content in cardiac mitochondria. After recording an electrocardiogram and collecting blood, the cardiac mitochondria were isolated. The mitochondrial membranes were labelled with eosin-5-maleimide (EMA) and separated on SDS polyacrylamide gels. The position of the AAC component was identified by exposing the gel under UV light, and the AAC content was determined by densitometry after staining with Coomassie blue. The AAC content ratio was significantly decreased in both 10-week-old and 1-year survived J-2-N hamsters when compared to control Golden hamster. Among 10-week-old J-2-N hamsters, the decrease in the AAC content ratio was more marked for the animals with more severe myocardial damage. The H(+)-ATPase activities of mitochondrial membrane were higher in 10-week-old J-2-N hamsters than in control hamsters. These results suggest that the decrease of AAC in J-2-N hamster plays an important role in the pathogenesis of cardiomyopathy in J-2-N hamsters. PMID:8455591

  12. Cloning of a palmitoyl-acyl carrier protein thioesterase from oil palm.

    PubMed

    Othman, A; Lazarus, C; Fraser, T; Stobart, K

    2000-12-01

    A palmitoyl-acyl carrier protein (ACP) thioesterase cDNA clone was isolated from an oil palm cDNA library. The cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein and a crude bacterial extract was assayed for acyl-CoA-hydrolysing activity. The recombinant enzyme was able to hydrolyse medium- and long-chain acyl-CoAs. Northern-blot analysis showed a high level of gene expression in leaf, flower and 15-, 17- and 18-week mesocarp tissues. Low-level gene expression was detected in germinated seedlings and 8- and 12-week mesocarp tissues, but no transcript was detected in any kernel tissues. Southern-blot analysis indicated the presence of a single gene and we have also isolated a genomic clone using the cDNA as a probe. Two genomic fragments were subcloned and a 7 kb contiguous stretch of the oil palm genome was sequenced. Comparison of this sequence with the cDNA sequence identified a putative 93 amino acid transit peptide, most of which is missing from the cDNA. The coding region of the gene consisted of seven exons and six introns. PMID:11171146

  13. Haem carrier protein 1 (HCP1): Expression and functional studies in cultured cells.

    PubMed

    Latunde-Dada, Gladys O; Takeuchi, Ken; Simpson, Robert J; McKie, Andrew T

    2006-12-22

    Haem released from digestion and breakdown of meat products provides an important source of dietary iron, which is readily absorbed in the proximal intestine. The recent cloning and characterization of a haem carrier protein 1 (HCP 1) has provided a candidate intestinal haem transporter. The current studies describe the expression and functional analysis of HCP1 in cultured Caco-2 cells, a commonly used model of human intestinal cells. HCP1 mRNA expression in other cell types was also studied. The uptake of (55)Fe labeled haem was determined in cells under different experimental conditions and HCP1 expression was measured by RT-PCR and immunohistochemistry. mRNA and protein expressions increased in Caco-2 cells transduced with HCP1 adenoviral plasmid, and consequently (55)Fe haem uptake was higher in these cells. Haem uptake was also increased in fully differentiated Caco-2 cells compared to undifferentiated cells. Preincubation of cells with desferrioxamine (DFO, to deplete cells of iron) had no effect on HCP1 expression or haem uptake. Treatment with CdCl(2) (to induce haem oxygenase, HO-1) enhanced HCP1 expression and increased haem uptake into the cells. HCP1 expression and function were found to be adaptive to the rate of haem degradation by HO-1. Furthermore, HCP1 expression in different cells implies a functional role in tissues other than the duodenum. PMID:17156779

  14. Tragacanth as an oral peptide and protein delivery carrier: Characterization and mucoadhesion.

    PubMed

    Nur, M; Ramchandran, L; Vasiljevic, T

    2016-06-01

    Biopolymers such as tragacanth, an anionic polysaccharide gum, can be alternative polymeric carrier for physiologically important peptides and proteins. Characterization of tragacanth is thus essential for providing a foundation for possible applications. Rheological studies colloidal solution of tragacanth at pH 3, 5 or 7 were carried out by means of steady shear and small amplitude oscillatory measurements. Tragacanth mucoadhesivity was also analyzed using an applicable rheological method and compared to chitosan, alginate and PVP. The particle size and zeta potential were measured by a zetasizer. Thermal properties of solutions were obtained using a differential scanning calorimetry. The solution exhibited shear-thinning characteristics. The value of the storage modulus (G') and the loss modulus (G″) increased with an increase in angular frequency (Ω). In all cases, loss modulus values were higher than storage values (G″>G') and viscous character was, therefore, dominant. Tragacanth and alginate showed a good mucoadhesion. Tragacanth upon dispersion created particles of a submicron size with a negative zeta potential (-7.98 to -11.92mV). These properties were pH dependant resulting in acid gel formation at pH 3.5. Tragacanth has thus a potential to be used as an excipient for peptide/protein delivery. PMID:27083363

  15. Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

    PubMed Central

    Fice, D; Shen, Z; Byers, D M

    1993-01-01

    A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism. Images PMID:8384617

  16. SMc01553 is the sixth acyl carrier protein in Sinorhizobium meliloti 1021.

    PubMed

    Dávila-Martínez, Yadira; Ramos-Vega, Ana Laura; Contreras-Martínez, Sandra; Encarnación, Sergio; Geiger, Otto; López-Lara, Isabel M

    2010-01-01

    Acyl carrier proteins (ACPs) are required for the transfer of acyl intermediates during fatty acid and polyketide syntheses. In Sinorhizobium meliloti 1021 there are five known ACPs: AcpP, NodF, AcpXL, the ACP domain in RkpA and SMb20651. The genome sequence of S. meliloti 1021 also reveals the ORF SMc01553, annotated as a putative ACP. smc01553 is part of a 6.6 kb DNA region that is duplicated in the chromosome and in the pSymb plasmid, the result of a recent duplication event. SMc01553 overexpressed in Escherichia coli was labelled in vivo with [(3)H]beta-alanine, a biosynthetic building block of the 4'-phosphopantetheine prosthetic group of ACPs. The purified SMc01553 was modified with 4'-phosphopantetheine in the presence of S. meliloti holo-ACP synthase, and this modification resulted in a major conformational change of the protein structure, since the holo-form runs faster in native PAGE than the apo-form. SMc01553 could not be loaded with a malonyl group by malonyl-CoA-ACP transacylase from S. meliloti. Using RT-PCR we could show the presence of mRNA for SMc01553 and of the duplicated ORF SMb22007 in cultures of S. meliloti. However, a mutant in which the two duplicated regions were deleted did not show any different phenotype with respect to the wild-type in the free-living or symbiotic lifestyle. PMID:19797355

  17. Competitive Adsorption of Plasma Proteins Using a Quartz Crystal Microbalance.

    PubMed

    Felgueiras, Helena P; Murthy, N Sanjeeva; Sommerfeld, Sven D; Brás, M Manuela; Migonney, Véronique; Kohn, Joachim

    2016-06-01

    Proteins that get adsorbed onto the surfaces of biomaterials immediately upon their implantation mediate the interactions between the material and the environment. This process, in which proteins in a complex mixture compete for adsorption sites on the surface, is determined by the physicochemical interactions at the interface. Competitive adsorption of bovine serum albumin (BSA), fibronectin (Fn), and collagen type I (Col I), sequentially and from mixtures, was investigated so as to understand the performances of different surfaces used in biomedical applications. A quartz crystal microbalance with dissipation was used to monitor the adsorption of these proteins onto two materials used in functional bone replacement, a titanium alloy (Ti6Al4V) and Ti6Al4V physisorbed with poly(sodium styrenesulfonate) [poly(NaSS)], and three controls, gold, poly(desaminotyrosyltyrosine ethyl ester carbonate) [poly(DTEc)], and polystyrene (PS). In experiments with individual proteins, the adsorption was the highest with Fn and Col I and the least with BSA. Also, protein adsorption was the highest on poly(NaSS) and Ti6Al4V and the least on poly(DTEc). In sequential adsorption experiments, protein exchange was observed in BSA + Fn, Fn + Col I, and BSA + Col I sequences but not in Fn + BSA and Col I + BSA because of the lower affinity of BSA to surfaces relative to Fn and Col I. Protein adsorption was the highest with Col I + Fn on hydrophobic surfaces. In experiments with protein mixtures, with BSA & Fn, Fn appears to be preferentially adsorbed; with Fn & Col I, both proteins were adsorbed, probably as multilayers; and with Col I & BSA, the total amount of protein was the highest, greater than that in sequential and individual adsorption of the two proteins, probably because of the formation of BSA and Col I complexes. Protein conformational changes induced by the adsorbing surfaces, protein-protein interactions, and affinities of proteins appear to be the important factors that

  18. Protein crystal growth on the Russian segment of the International Space Station

    NASA Astrophysics Data System (ADS)

    Smirnova, E. A.; Kislitsyn, Yu. A.; Sosfenov, N. I.; Lyashenko, A. V.; Popov, A. N.; Baĭdus', A. N.; Timofeev, V. I.; Kuranova, I. P.

    2009-09-01

    Experiments on protein crystallization on the Russian segment of the International Space Station were started in 2005. These experiments were performed in the Modul’-1 protein crystallization apparatus specially designed for crystal growth by the free-interface-diffusion method. This paper describes experiments on the crystallization of lysozyme, carboxypeptidase B, and recombinant human insulin on Earth and in microgravity using the Modul’-1 protein crystallization apparatus during the ISS-11-ISS-14 space flights. Crystals of all proteins grown in microgravity have larger sizes than those grown on Earth. Space-grown crystals of lysozyme and insulin characterized by X-ray diffraction were shown to diffract to higher resolution than the Earth-grown crystals. The three-dimensional structures of Zn-insulin crystals grown both on Earth and in microgravity were established. The conformation of the Zn-insulin hexamer in the crystalline state is described.

  19. 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

  20. Modulation of phase behaviors and charge carrier mobilities by linkage length in discotic liquid crystal dimers.

    PubMed

    Wang, Yi-Fei; Zhang, Chun-Xiu; Wu, Hao; Zhang, Ao; Wang, Jian-Chuang; Zhang, Shuai-Feng; Pu, Jia-Ling

    2015-01-28

    A clear structure-property relationship was revealed in a series of triphenylene-based dimers, which contained two triphenylene nuclei each bearing five β-OC4H9 substituents and are linked through a flexible O(CH2)nO polymethylene chain (n=6-12). Dimers with the linkage close to twice the length of the free side chains (n=8, 9) exhibited a single Colhp phase, while others with the linkage shorter (n=6, 7) or longer (n=10, 11, 12) showed multiphase behaviors with a transition from the Colhp phase to Colh phase; hole mobilities of Colhp phases reached 1.4×10(-2) cm2 V(-1) s(-1) in the dimer for which the linkage is exactly twice the length of the free side chains (n=8), and decreased regularly both with linkage length becoming shorter or longer. This modulation of phase behaviors and charge carrier mobilities was demonstrated to be generated by various steric perturbations introduced by linkages with different lengths, which result in different degrees of lateral fluctuations of discotic moieties in the columns. PMID:25467212

  1. Carrier transport property of truxene discotic liquid crystals with three different ring substituents

    NASA Astrophysics Data System (ADS)

    Monobe, Hirosato; Ni, Hai-Liang; Hu, Ping; Wang, Bi-Qin; Zhao, Ke-Qing; Shimizu, Yo

    2016-03-01

    In this study, the charge carrier transport property of 3,8,13-trioctyloxytruxene [Trx(OC8)3] and its analogues, to which two different ring substituents of hydroxyl [Trx(OH)3(OC8)3] and methoxy [Trx(OMe)3(OC8)3] groups are introduced, has been studied relative to mesomorphism. Three analogues exhibit a hexagonal columnar (Colh) mesophase and their thermal stability increases with the introduction of hydroxyl and methoxy groups. The drift mobility measurements of Trx(OC8)3 and Trx(OH)3(OC8)3 reveal that the drift mobility is on the order of 5 × 10-2 cm2 V-1 s-1 in the Colh phase and it increases to 10-1 cm2 V-1 s-1 at the Colh-metastable phase transition, although Trx(OMe)3(OC8)3 shows a drift mobility of 1 × 10-2 cm2 V-1 s-1 in the Colh phase with temperature dependence. These results indicate that truxene with three alkoxy chains is an interesting molecular core for mesophase semiconductors.

  2. PEGylated Dendritic Unimolecular Micelles as Versatile Carriers for Ligands of G Protein-Coupled Receptors

    PubMed Central

    Kim, Yoonkyung; Hechler, Béatrice; Gao, Zhan-Guo; Gachet, Christian; Jacobson, Kenneth A.

    2009-01-01

    Despite its widespread application in nanomedicine, poly(ethylene glycol) (PEG) is seldom used for covalent modification of ligands for G protein-coupled receptors (GPCRs) due to potential steric complications. In order to study the influence of PEG chains on the biological activity of GPCR ligands bound to a common macromolecular carrier, we prepared a series of G3 polyamidoamine (PAMAM) dendrimers derivatized with Alexa Fluor 488, varying numbers of PEG550/PEG750/PEG2000, and nucleoside moieties derived from the A2A adenosine receptor (AR) agonist CGS21680 (2-[4-(2-carboxylethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine). These dendrimer conjugates were purified by size exclusion chromatography and characterized by 1H NMR and MALDI MS. In radioligand binding assays, some PAMAM-PEG conjugates showed enhanced subtype-selectivity at the human A2A AR compared to monomeric ligands of comparable affinity. The functional potency was measured in the A2A AR-mediated activation of adenylate cyclase and inhibition of ADP-induced platelet aggregation. Interestingly, the dendrimer conjugate 10c bearing 11 PEG750 chains (out of theo. 32 amino end groups) and 14 nucleoside moieties was 5-fold more potent in A2A AR–mediated stimulation of cyclic AMP formation than 10d with four PEG2000 chains and 21 nucleosides, although the binding affinities of these two compounds were similar. Thus, a relatively small (≤10 nm) multivalent ligand 10c modified for water solubility maintained high potency and displayed increased A2A AR binding selectivity over the monomeric nucleosides. Longer PEG chains reduced affinity at the A2A AR. The current study demonstrates the feasiblity of using short PEG chains in the design of carriers that target ligand-receptor interactions. PMID:19785401

  3. PEGylated dendritic unimolecular micelles as versatile carriers for ligands of G protein-coupled receptors.

    PubMed

    Kim, Yoonkyung; Hechler, Béatrice; Gao, Zhan-Guo; Gachet, Christian; Jacobson, Kenneth A

    2009-10-21

    Despite its widespread application in nanomedicine, poly(ethylene glycol) (PEG) is seldom used for covalent modification of ligands for G protein-coupled receptors (GPCRs) due to potential steric complications. In order to study the influence of PEG chains on the biological activity of GPCR ligands bound to a common macromolecular carrier, we prepared a series of G3 polyamidoamine (PAMAM) dendrimers derivatized with Alexa Fluor 488, varying numbers of PEG(550)/PEG(750)/PEG(2000), and nucleoside moieties derived from the A(2A) adenosine receptor (AR) agonist CGS21680 (2-[4-(2-carboxylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine). These dendrimer conjugates were purified by size exclusion chromatography and characterized by (1)H NMR and MALDI MS. In radioligand binding assays, some PAMAM-PEG conjugates showed enhanced subtype-selectivity at the human A(2A) AR compared to monomeric ligands of comparable affinity. The functional potency was measured in the A(2A) AR-mediated activation of adenylate cyclase and inhibition of ADP-induced platelet aggregation. Interestingly, the dendrimer conjugate 10c bearing 11 PEG(750) chains (out of theoretical 32 amino end groups) and 14 nucleoside moieties was 5-fold more potent in A(2A) AR-mediated stimulation of cyclic AMP formation than 10d with 4 PEG(2000) chains and 21 nucleosides, although the binding affinities of these 2 compounds were similar. Thus, a relatively small (≤10 nm) multivalent ligand 10c modified for water solubility maintained high potency and displayed increased A(2A) AR binding selectivity over the monomeric nucleosides. The current study demonstrates the feasibility of using short PEG chains in the design of carriers that target ligand-receptor interactions. PMID:19785401

  4. Powder diffraction from a continuous microjet of submicrometer protein crystals.

    PubMed

    Shapiro, D A; Chapman, H N; Deponte, D; Doak, R B; Fromme, P; Hembree, G; Hunter, M; Marchesini, S; Schmidt, K; Spence, J; Starodub, D; Weierstall, U

    2008-11-01

    Atomic-resolution structures from small proteins have recently been determined from high-quality powder diffraction patterns using a combination of stereochemical restraints and Rietveld refinement [Von Dreele (2007), J. Appl. Cryst. 40, 133-143; Margiolaki et al. (2007), J. Am. Chem. Soc. 129, 11865-11871]. While powder diffraction data have been obtained from batch samples of small crystal-suspensions, which are exposed to X-rays for long periods of time and undergo significant radiation damage, the proof-of-concept that protein powder diffraction data from nanocrystals of a membrane protein can be obtained using a continuous microjet is shown. This flow-focusing aerojet has been developed to deliver a solution of hydrated protein nanocrystals to an X-ray beam for diffraction analysis. This method requires neither the crushing of larger polycrystalline samples nor any techniques to avoid radiation damage such as cryocooling. Apparatus to record protein powder diffraction in this manner has been commissioned, and in this paper the first powder diffraction patterns from a membrane protein, photosystem I, with crystallite sizes of less than 500 nm are presented. These preliminary patterns show the lowest-order reflections, which agree quantitatively with theoretical calculations of the powder profile. The results also serve to test our aerojet injector system, with future application to femtosecond diffraction in free-electron X-ray laser schemes, and for serial crystallography using a single-file beam of aligned hydrated molecules. PMID:18955765

  5. Potential of Translationally Controlled Tumor Protein-Derived Protein Transduction Domains as Antigen Carriers for Nasal Vaccine Delivery.

    PubMed

    Bae, Hae-Duck; Lee, Joohyun; Jin, Xing-Hai; Lee, Kyunglim

    2016-09-01

    Nasal vaccination offers a promising alternative to intramuscular (i.m.) vaccination because it can induce both mucosal and systemic immunity. However, its major drawback is poor absorption of large antigens in the nasal epithelium. Protein transduction domains (PTDs), also called cell-penetrating peptides, have been proposed as vehicles for nasal delivery of therapeutic peptides and proteins. Here, we evaluated the potential of a mutant PTD derived from translationally controlled tumor protein (designated TCTP-PTD 13) as an antigen carrier for nasal vaccines. We first compared the l- and d-forms of TCTP-PTD 13 isomers (l- or d-TCTP-PTD 13) as antigen carriers. Studies in mice demonstrated that nasally administered mixtures of the model antigen ovalbumin (OVA) and d-TCTP-PTD 13 induced higher plasma IgG titers and secretory IgA levels in nasal washes than nasally administered OVA alone, OVA/l-TCTP-PTD 13, or i.m.-injected OVA. Plasma IgG subclass responses (IgG1 and IgG2a) of mice nasally administered OVA/d-TCTP-PTD 13 showed that the predominant IgG subclass was IgG1, indicating a Th2-biased immune response. We also used synthetic CpG oligonucleotides (CpG) as a Th1 immune response-inducing adjuvant. Nasally administered CpG plus OVA/d-TCTP-PTD 13 was superior in eliciting systemic and mucosal immune responses compared to those induced by nasally administered OVA/d-TCTP-PTD 13. Furthermore, the OVA/CpG/d-TCTP-PTD 13 combination skewed IgG1 and IgG2a profiles of humoral immune responses toward a Th1 profile. These findings suggest that TCTP-derived PTD is a suitable vehicle to efficiently carry antigens and to induce more powerful antigen-specific immune responses and a more balanced Th1/Th2 response when combined with a DNA adjuvant. PMID:27454469

  6. 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.

  7. A Polyketide Synthase Acyltransferase Domain Structure Suggests a Recognition Mechanism for Its Hydroxymalonyl-Acyl Carrier Protein Substrate

    PubMed Central

    Park, Hyunjun; Kevany, Brian M.; Dyer, David H.; Thomas, Michael G.; Forest, Katrina T.

    2014-01-01

    We have previously shown that the acyl transferase domain of ZmaA (ZmaA-AT) is involved in the biosynthesis of the aminopolyol polyketide/nonribosomal peptide hybrid molecule zwittermicin A from cereus UW85, and that it specifically recognizes the precursor hydroxymalonyl-acyl carrier protein (ACP) and transfers the hydroxymalonyl extender unit to a downstream second ACP via a transacylated AT domain intermediate. We now present the X-ray crystal structure of ZmaA-AT at a resolution of 1.7 Å. The structure shows a patch of solvent-exposed hydrophobic residues in the area where the AT is proposed to interact with the precursor ACP. We addressed the significance of the AT/ACP interaction in precursor specificity of the AT by testing whether malonyl- or methylmalonyl-ACP can be recognized by ZmaA-AT. We found that the ACP itself biases extender unit selection. Until now, structural information for ATs has been limited to ATs specific for the CoA-linked precursors malonyl-CoA and (2S)-methylmalonyl-CoA. This work contributes to polyketide synthase engineering efforts by expanding our knowledge of AT/substrate interactions with the structure of an AT domain that recognizes an ACP-linked substrate, the rare hydroxymalonate. Our structure suggests a model in which ACP interaction with a hydrophobic motif promotes secondary structure formation at the binding site, and opening of the adjacent substrate pocket lid to allow extender unit binding in the AT active site. PMID:25340352

  8. Protein-protein binding detection with nanoparticle photonic crystal enhanced microscopy (NP-PCEM).

    PubMed

    Zhuo, Yue; Tian, Limei; Chen, Weili; Yu, Hojeong; Singamaneni, Srikanth; Cunningham, Brian T

    2014-01-01

    We demonstrate a novel microscopy-based biosensing approach that utilizes a photonic crystal (PC) surface to detect protein-protein binding with the functionalized nanoparticles as tags. This imaging approach utilizes the measurement of localized shifts in the resonant wavelength and resonant reflection magnitude from the PC biosensor in the presence of individual nanoparticles. Moreover, it substantially increases the sensitivity of the imaging approach through tunable localized surface plasmon resonant frequency of the nanoparticle matching with the resonance of the PC biosensor. Experimental demonstrations of photonic crystal enhanced microscopy (PCEM) imaging with single nanoparticle resolution are supported by Finite-Difference Time-Domain (FDTD) computer simulations. The ability to detect the surface adsorption of individual nanoparticles as tags offers a route to single molecule biosensing with photonic crystal biosensor in the future. PMID:25570391

  9. Isoelectric focusing of human parotid salivary proteins in hybrid carrier ampholyte-immobilized pH gradient polyacrylamide gels.

    PubMed

    Khoo, K S; Beeley, J A

    1990-06-01

    Isoelectric focusing of human salivary proteins with carrier ampholyte-isoelectric focusing systems requires prior desalting and concentration of samples, a procedure which is time-consuming and requires relatively large volumes of samples. By contrast, immobilized pH gradient gels are more tolerant to salt loads. Thus pretreatment of samples consists only of centrifugation prior to isoelectric focusing. If larger loads (greater than 50 micrograms) are required, the samples may be concentrated by lyophilization and reconstitution in a smaller volume of water or by dialysis against 30% w/v polyethylene glycol. Immobilized pH gradient polyacrylamide gels (incorporating a hybrid carrier ampholyte system) of two pH ranges (pH 4-9 and pH 3.5-5.0) have been used to separate the proteins in human parotid saliva. The effects of urea on focused patterns were studied; in pH 4-9 gels it gave improved resolution of protein bands, whereas in pH 3.5-5.0 gels it prevented protein precipitation. The salivary proteins were then visualized by staining with Coomassie Brilliant Blue G-250 or a silver procedure. Using the latter, 25-30 well-resolved bands were formed on a pH 4-9 gel loaded with 20 micrograms of proteins. The method offers considerable advantages compared with carrier ampholyte-isoelectric focusing. PMID:1697536

  10. 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.

  11. 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.

  12. Self-assembled multicompartment liquid crystalline lipid carriers for protein, peptide, and nucleic acid drug delivery.

    PubMed

    Angelova, Angelina; Angelov, Borislav; Mutafchieva, Rada; Lesieur, Sylviane; Couvreur, Patrick

    2011-02-15

    Lipids and lipopolymers self-assembled into biocompatible nano- and mesostructured functional materials offer many potential applications in medicine and diagnostics. In this Account, we demonstrate how high-resolution structural investigations of bicontinuous cubic templates made from lyotropic thermosensitive liquid-crystalline (LC) materials have initiated the development of innovative lipidopolymeric self-assembled nanocarriers. Such structures have tunable nanochannel sizes, morphologies, and hierarchical inner organizations and provide potential vehicles for the predictable loading and release of therapeutic proteins, peptides, or nucleic acids. This Account shows that structural studies of swelling of bicontinuous cubic lipid/water phases are essential for overcoming the nanoscale constraints for encapsulation of large therapeutic molecules in multicompartment lipid carriers. For the systems described here, we have employed time-resolved small-angle X-ray scattering (SAXS) and high-resolution freeze-fracture electronic microscopy (FF-EM) to study the morphology and the dynamic topological transitions of these nanostructured multicomponent amphiphilic assemblies. Quasi-elastic light scattering and circular dichroism spectroscopy can provide additional information at the nanoscale about the behavior of lipid/protein self-assemblies under conditions that approximate physiological hydration. We wanted to generalize these findings to control the stability and the hydration of the water nanochannels in liquid-crystalline lipid nanovehicles and confine therapeutic biomolecules within these structures. Therefore we analyzed the influence of amphiphilic and soluble additives (e.g. poly(ethylene glycol)monooleate (MO-PEG), octyl glucoside (OG), proteins) on the nanochannels' size in a diamond (D)-type bicontinuous cubic phase of the lipid glycerol monooleate (MO). At body temperature, we can stabilize long-living swollen states, corresponding to a diamond cubic phase

  13. A potential carrier based on liquid crystal nanoparticles for ophthalmic delivery of pilocarpine nitrate.

    PubMed

    Li, Jing; Wu, Lin; Wu, Weijun; Wang, Baoyan; Wang, Zhongyuan; Xin, Hongliang; Xu, Qunwei

    2013-10-15

    Poor corneal penetration and short preocular retention of a clinical hydrophilic drug, pilocarpine nitrate (PN), for the treatment of open-angle glaucoma and acute angle-closure glaucoma, limit its ocular application. The purpose of this study was to investigate the potential of liquid crystal nanoparticles (LCNPs) for ocular delivery of PN. LCNPs were developed by a top-down method using glyceryl monoolein (GMO) and water in the presence of stabilizer Poloxamer 407. They were characterized by transmission electron microscopy (TEM) and small angle X-ray diffraction (SAXS). The size of LCNP is 202.28±19.32 nm and the encapsulation efficiency reached 61.03%. The in vitro release profiles indicated that PN could keep sustained release from PN-loaded LCNPs for 8h. An ex vivo corneal permeation study revealed that the apparent permeability coefficient of PN-loaded LCNPs was 2.05-fold higher than that of commercial eye drops. In addition, the topical administration test showed that PN-loaded LCNPs had a prolonged effect on decreasing intraocular pressure (IOP) of rabbits compared with commercial drug and physiological saline. In conclusion, LCNPs had been demonstrated to be potential for controlled-release ocular drug delivery. PMID:23916822

  14. Plasma protein corona modulates the vascular wall interaction of drug carriers in a material and donor specific manner.

    PubMed

    Sobczynski, Daniel J; Charoenphol, Phapanin; Heslinga, Michael J; Onyskiw, Peter J; Namdee, Katawut; Thompson, Alex J; Eniola-Adefeso, Omolola

    2014-01-01

    The nanoscale plasma protein interaction with intravenously injected particulate carrier systems is known to modulate their organ distribution and clearance from the bloodstream. However, the role of this plasma protein interaction in prescribing the adhesion of carriers to the vascular wall remains relatively unknown. Here, we show that the adhesion of vascular-targeted poly(lactide-co-glycolic-acid) (PLGA) spheres to endothelial cells is significantly inhibited in human blood flow, with up to 90% reduction in adhesion observed relative to adhesion in simple buffer flow, depending on the particle size and the magnitude and pattern of blood flow. This reduced PLGA adhesion in blood flow is linked to the adsorption of certain high molecular weight plasma proteins on PLGA and is donor specific, where large reductions in particle adhesion in blood flow (>80% relative to buffer) is seen with ∼60% of unique donor bloods while others exhibit moderate to no reductions. The depletion of high molecular weight immunoglobulins from plasma is shown to successfully restore PLGA vascular wall adhesion. The observed plasma protein effect on PLGA is likely due to material characteristics since the effect is not replicated with polystyrene or silica spheres. These particles effectively adhere to the endothelium at a higher level in blood over buffer flow. Overall, understanding how distinct plasma proteins modulate the vascular wall interaction of vascular-targeted carriers of different material characteristics would allow for the design of highly functional delivery vehicles for the treatment of many serious human diseases. PMID:25229244

  15. 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

  16. Homodimeric Intrinsic Membrane Proteins. Identification and Modulation of Interactions between Mitochondrial Transporter (Carrier) Subunits

    PubMed Central

    Wohlrab, Hartmut

    2010-01-01

    Transporter (carrier) proteins of the inner mitochondrial membrane link metabolic pathways within the matrix and the cytosol with transport/exchange of metabolites and inorganic ions. Their strict control of these fluxes is required for oxidative phosphorylation. Understanding the ternary complex transport mechanism with which most of these transporters function requires an accounting of the number and interactions of their subunits. The phosphate transporter (PTP, Mir1p) subunit readily forms homodimers with intersubunit affinities changeable by mutations. Cys28, likely at the subunit interface, is a site for mutations yielding transport inhibition or a channel-like transport mode. Such mutations yield a small increase or decrease in affinity between the subunits. The PTP inhibitor N-ethylmaleimide decreases subunit affinity by a small amount. PTP mutations that yield the highest (40%) and the lowest (2%) liposome incorporation efficiencies (LIE) are clustered near Cys28. Such mutant subunits show the lowest and highest subunit affinities respectively. The oxaloacetate transporter (Oac1p) subunit has an almost 2-fold lower affinity than the PTP subunit. The Oac1p, dicarboxylate (Dic1p) and PTP transporter subunits form heterodimers with even lower affinities. These results form a firm basis for detailed studies to establish the effect of subunit affinities on transport mode and activity and for the identification of the mechanism that prevents formation of heterodimers that surely will negatively impact oxidative phosphorylation and ATP levels with serious consequences for the cell. PMID:20171189

  17. Stearoyl-acyl carrier protein desaturases are associated with floral isolation in sexually deceptive orchids

    SciTech Connect

    Schluter, P.M.; Shanklin, J.; Xu, S.; Gagliardini, V.; Whittle, E.; Grossniklaus, U.; Schiestl, F. P.

    2011-04-05

    The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both as an 18:0-ACP {Delta}{sup 9} and a 16:0-ACP {Delta}{sup 4} desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection.

  18. Identification and Characterization of Inhibitors of Bacterial Enoyl-Acyl Carrier Protein Reductase

    PubMed Central

    Ling, Losee L.; Xian, Jun; Ali, Syed; Geng, Bolin; Fan, Jun; Mills, Debra M.; Arvanites, Anthony C.; Orgueira, Hernan; Ashwell, Mark A.; Carmel, Gilles; Xiang, Yibin; Moir, Donald T.

    2004-01-01

    Bacterial enoyl-acyl carrier protein reductase (ENR) catalyzes an essential step in fatty acid biosynthesis. ENR is an attractive target for narrow-spectrum antibacterial drug discovery because of its essential role in metabolism and its sequence conservation across many bacterial species. In addition, the bacterial ENR sequence and structural organization are distinctly different from those of mammalian fatty acid biosynthesis enzymes. High-throughput screening to identify inhibitors of Escherichia coli ENR yielded four structurally distinct classes of hits. Several members of one of these, the 2-(alkylthio)-4,6-diphenylpyridine-3-carbonitriles (“thiopyridines”), inhibited both purified ENR (50% inhibitory concentration [IC50] = 3 to 25 μM) and the growth of Staphylococcus aureus and Bacillus subtilis (MIC = 1 to 64 μg/ml). The effect on cell growth is due in part to inhibition of fatty acid biosynthesis as judged by inhibition of incorporation of [14C]acetate into fatty acids and by the increased sensitivity of cells that underexpress an ENR-encoding gene (four- to eightfold MIC shift). Synthesis of a variety of compounds in this chemical series revealed a correlation between IC50 and MIC, and the results provided initial structure-activity relationships. Preliminary structure-activity relationships, potency on purified ENR, and activity on bacterial cells indicate that members of the thiopyridine chemical series are effective fatty acid biosynthesis inhibitors suitable for further antibacterial development. PMID:15105103

  19. A conserved motif flags Acyl Carrier Proteins for β-branching in polyketide synthesis

    PubMed Central

    Song, Zhongshu; Farmer, Rohit; Williams, Christopher; Hothersall, Joanne; Płoskoń, Eliza; Wattana-amorn, Pakorn; Stephens, Elton R.; Yamada, Erika; Gurney, Rachel; Takebayashi, Yuiko; Masschelein, Joleen; Cox, Russell J.; Lavigne, Rob; Willis, Christine L.; Simpson, Thomas J.; Crosby, John; Winn, Peter J.; Thomas, Christopher M.; Crump, Matthew P.

    2015-01-01

    Type I PKSs often utilise programmed β-branching, via enzymes of an “HMG-CoA synthase (HCS) cassette”, to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in Acyl Carrier Proteins (ACPs) where β-branching is known. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. While these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modelling and mutagenesis identified ACP Helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality while ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering novel polyketides and lays a basis for determining specificity rules. PMID:24056399

  20. Tailoring enzymes acting on carrier protein-tethered substrates in natural product biosynthesis.

    PubMed

    Lin, Shuangjun; Huang, Tingting; Shen, Ben

    2012-01-01

    Carrier proteins (CPs) are integral components of fatty acid synthases, polyketide synthases, and nonribosomal peptide synthetases and play critical roles in the biosynthesis of fatty acids, polyketides, and nonribosomal peptides. An emerging role CPs play in natural product biosynthesis involves tailoring enzymes that act on CP-tethered substrates. These enzymes provide a new opportunity to engineer natural product diversity by exploiting CPs to increase substrate promiscuity for the tailoring steps. This chapter describes protocols for in vitro biochemical characterization of SgcC3 and SgcC that catalyze chlorination and hydroxylation of SgcC2-tethered (S)-β-tyrosine and analogues in the biosynthesis of the enediyne chromophore of the chromoprotein C-1027. These protocols are applicable to mechanistic characterization and engineered exploitation of other tailoring enzymes that act on CP-tethered substrates in natural product biosynthesis and structural diversification. The ultimate goal is to use the in vitro findings to guide in vivo engineering of designer natural products. PMID:23034236

  1. 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.

  2. 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

  3. Relaxation of stored charge carriers in a Zn sub 0. 3 Cd sub 0. 7 Se mixed crystal

    SciTech Connect

    Lin, J.Y.; Jiang, H.X. )

    1990-03-15

    Persistent photoconductivity (PPC) has been investigated in detail in a Zn{sub 0.3}Cd{sub 0.7}Se mixed crystal. Two different temperature conductivity states have been observed. Relaxation of stored charge carriers, which contribute to PPC, has been studied at different conditions. We find that the decay of PPC follows the stretched-exponential'' function that is usually observed in a wide class of disordered materials. At {ital T}{lt}220 K, the relaxation time increases with increase of temperature. At a constant temperature, the relaxation time increases with increase of excitation photon dose, which is a consequence of the presence of the two different conductivity states. However, at a constant temperature, the decay exponent is excitation-photon-dose independent, while the characteristic decay time constant depends on excitation photon dose. The PPC observed here thus exhibits characteristic phenomena of disordered systems, which suggests that the random local-potential fluctuations, which arise from the compositional fluctuations, are responsible for PPC. PPC-decay behavior is also analyzed for some of the previously published data on other materials. We find that the stretched-exponential function describes the PPC decay in various materials at low temperatures.

  4. 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

  5. Some practical guidelines for UV imaging in the protein crystallization laboratory

    PubMed Central

    Desbois, Sebastien; Seabrook, Shane A.; Newman, Janet

    2013-01-01

    High-throughput imaging of protein crystallization experiments with ultraviolet (UV) light has recently become commercially available and can enable crystallographers to differentiate between crystals of protein and those of salt, as the visualization of protein crystals is based on intrinsic tryptophan fluorescence. Unfortunately, UV imaging is not a panacea, as some protein crystals will not fluoresce under UV excitation and some salt crystals are UV-fluorescently active. As a new technology, there is little experience within the general community on how to use this technology effectively and what caveats to look out for. Here, an attempt is made to identify some of the common problems that may arise using UV-imaging technology by examining test proteins, common crystallization reagents and a range of proteins by assessing their UV–Vis absorbance spectra. Some pointers are offered as to which systems may not be appropriate for this methodology. PMID:23385768

  6. Some practical guidelines for UV imaging in the protein crystallization laboratory.

    PubMed

    Desbois, Sebastien; Seabrook, Shane A; Newman, Janet

    2013-02-01

    High-throughput imaging of protein crystallization experiments with ultraviolet (UV) light has recently become commercially available and can enable crystallographers to differentiate between crystals of protein and those of salt, as the visualization of protein crystals is based on intrinsic tryptophan fluorescence. Unfortunately, UV imaging is not a panacea, as some protein crystals will not fluoresce under UV excitation and some salt crystals are UV-fluorescently active. As a new technology, there is little experience within the general community on how to use this technology effectively and what caveats to look out for. Here, an attempt is made to identify some of the common problems that may arise using UV-imaging technology by examining test proteins, common crystallization reagents and a range of proteins by assessing their UV-Vis absorbance spectra. Some pointers are offered as to which systems may not be appropriate for this methodology. PMID:23385768

  7. 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.

  8. 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.

  9. Primary structure of a cerulenin-binding. beta. -ketoacyl-(acyl carrier protein) synthase from barley chloroplasts

    SciTech Connect

    Siggaard-Andersen, M.; Kauppinen, S. ); von Wettstein-Knowles, P. Univ. of Copenhagen )

    1991-05-15

    The radioactively labeled {beta}-ketoacyl thioester synthase inhibitor ({sup 3}H)cerulenin was used to tag three dimeric barley chloroplast proteins ({alpha}{alpha}, {alpha}{beta}, and {beta}{beta}) from the stromal fraction. Oligonucleotides corresponding to amino acid sequences obtained from the purified proteins were used to generate with the polymerase chain reaction a probe for cDNAs encoding the {beta} subunit. cDNA sequencing revealed an open reading frame for 462 residues comprising the mature protein and a 35-amino acid transit peptide. The deduced amino acid sequence of the mature protein is homologous to the {beta}-ketoacyl-(acyl carrier protein) (ACP) synthase I (3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3.1.41) of Escherichia coli. Under analogous experimental conditions ({sup 3}H)cerulenin tagged a single dimeric protein from spinach chloroplasts.

  10. 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.

  11. 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.

  12. The use of heterogeneous and epitaxial nucleants to promote the growth of protein crystals

    NASA Technical Reports Server (NTRS)

    Mcpherson, Alexander; Shlichta, P.

    1988-01-01

    Fifty different mineral samples were tested as potential heterogeneous or epitaxial nucleants for four commonly crystallized proteins. It was found, using conventional protein crystallization techniques, that for each protein there was a set of mineral substrates that promoted nucleation of crystals at lower critical levels of supersaturation than required for spontaneous growth. In at least one case, the growth of lysozyme on the mineral apophyllite, it was shown by lattice analysis and X-ray diffraction that the nucleation and growth of the protein crystal on the mineral was likely to be truly epitaxial.

  13. Protein structure. Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism.

    PubMed

    Li, Fei; Liu, Jian; Zheng, Yi; Garavito, R Michael; Ferguson-Miller, Shelagh

    2015-01-30

    The 18-kilodalton translocator protein (TSPO), proposed to be a key player in cholesterol transport into mitochondria, is highly expressed in steroidogenic tissues, metastatic cancer, and inflammatory and neurological diseases such as Alzheimer's and Parkinson's. TSPO ligands, including benzodiazepine drugs, are implicated in regulating apoptosis and are extensively used in diagnostic imaging. We report crystal structures (at 1.8, 2.4, and 2.5 angstrom resolution) of TSPO from Rhodobacter sphaeroides and a mutant that mimics the human Ala(147)→Thr(147) polymorphism associated with psychiatric disorders and reduced pregnenolone production. Crystals obtained in the lipidic cubic phase reveal the binding site of an endogenous porphyrin ligand and conformational effects of the mutation. The three crystal structures show the same tightly interacting dimer and provide insights into the controversial physiological role of TSPO and how the mutation affects cholesterol binding. PMID:25635101

  14. 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.

  15. 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°.

  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. The substrate promiscuity of a phosphopantetheinyl transferase SchPPT for coenzyme A derivatives and acyl carrier proteins.

    PubMed

    Wang, Yue-Yue; Luo, Hong-Dou; Zhang, Xiao-Sheng; Lin, Tao; Jiang, Hui; Li, Yong-Quan

    2016-03-01

    Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of acyl carrier proteins (ACPs) in fatty acid synthases (FASs), ACPs in polyketide synthases, and peptidyl carrier proteins (PCPs) in nonribosomal peptide synthetases (NRPSs) in all organisms. Some bacterial PPTases have broad substrate specificities for ACPs/PCPs and/or coenzyme A (CoA)/CoA analogs, facilitating their application in metabolite production in hosts and/or labeling of ACPs/PCPs, respectively. Here, a group II PPTase SchPPT from Streptomyces chattanoogensis L10 was characterized to accept a heterologous ACP and acetyl-CoA. Thus, SchPPT is a promiscuous PPTase and may be used on polyketide production in heterologous bacterial host and labeling of ACPs. PMID:26748983

  18. 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.

  19. Cloning and expression of a cDNA encoding human sterol carrier protein 2

    SciTech Connect

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III ); Billheimer, J.T. )

    1991-01-15

    The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP{sub 2}). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP{sub 2} amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP{sub 2}. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A){sup +} RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP{sub 2} gene in the human genome or that the SCP{sub 2} gene is very large. Coexpression of the SCP{sub 2} cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP{sub 2} plays a role in regulating steroidogenesis, among other possible functions.

  20. Relations between coenzyme A and presumptive acyl carrier protein in different conditions of streptococcal growth.

    PubMed

    Das, D N; Toennies, G

    1969-06-01

    Exploration of the specific role of cystine in the postexponential growth of Streptococcus faecalis led to an inquiry into the fate of cellular coenzyme A (CoA) and acyl carrier protein (ACP), both of which depend for their biosynthesis on cystine and pantothenate as precursors. In S. faecalis cells labeled by growth in the presence of (14)C-pantothenate, the label could be separated on the basis of solubility at pH 2.1 into two fractions of sharply differing metabolic characteristics. The fractions were not purified, but the soluble (14)C behaved analytically like CoA, and the insoluble (14)C was considered to represent an ACP-like entity on the basis of circumstantial evidence. The fate of these two fractions under various conditions of growth was studied. When the medium contained an excess of the needed precursors, the cellular content of CoA and ACP appeared to remain constant during exponential growth, and in a molar ratio of about 4 CoA to 1 ACP. Cellular ACP, once formed, appeared to be stable under these conditions, but CoA was degraded and replaced at the rate of approximately 20% per division period. With restrictive levels of pantothenate in the medium, initially formed CoA disappeared during growth, as a result, apparently of being converted to ACP. However, when the resulting CoA-depleted cells were returned to a medium containing enough pantothenate, resumption of normal growth was preceded by a lag period, during which rapid conversion of ACP to CoA appeared to take place. PMID:4977991

  1. Slow onset inhibition of bacterial beta-ketoacyl-acyl carrier protein synthases by thiolactomycin.

    PubMed

    Machutta, Carl A; Bommineni, Gopal R; Luckner, Sylvia R; Kapilashrami, Kanishk; Ruzsicska, Bela; Simmerling, Carlos; Kisker, Caroline; Tonge, Peter J

    2010-02-26

    Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the beta-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices alpha5 and alpha6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100-200 microg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton. PMID:20018879

  2. The effect of CdSe/ZnS quantum dots on the rotational viscosity and charge carrier concentration of a nematic liquid crystal

    NASA Astrophysics Data System (ADS)

    Shcherbinin, D. P.; Konshina, E. A.; Solodkov, D. E.

    2015-08-01

    The addition of CdSe/ZnS quantum dots (QDs) with a core diameter of 3.5 nm at a concentration of 10 wt % leads to a 2.5-fold increase in the dynamic rotational viscosity of a 5CB nematic liquid crystal (NLC). A comparison of the diffusion currents in NLC cells filled with pure 5CB and a suspension with QDs shows evidence of an increase in the concentration of charge carriers in the latter case.

  3. Spin dynamics of charge carriers in the process of their localization in {alpha}'-(BEDT-TTF){sub 2}IBr{sub 2} single crystals

    SciTech Connect

    Morgunov, R. B. Dmitriev, A. I.; Chernen'kaya, A. S.; Yakushi, K.; Yamamoto, K.; Tanimoto, Y.

    2010-11-15

    Sharp changes in the integral intensity and linewidth of the ESR spectrum that accompany the localization of the charge carriers have been revealed in {alpha}'-(BEDT-TTF){sub 2}IBr{sub 2} crystals. It has been found that the types of localization in two compounds under investigation are different: charge carriers in {beta}''-(BEDT-TTF){sub 4}NH{sub 4}[Cr(C{sub 2}O{sub 4}){sub 3}] are localized on irregular defects of the crystal lattice, whereas charge carriers in {alpha}'-(BEDT-TTF){sub 2}IBr{sub 2} are localized at the regular positions of the unit cell. The exchange narrowing of the ESR line and a sharp decrease in the dc and ac magnetic susceptibilities are observed in {alpha}'-(BEDT-TTF){sub 2}IBr{sub 2} at low temperatures T < 50 K. The dc and ac magnetic susceptibilities observed in {alpha}'-(BEDT-TTF){sub 2}IBr{sub 2} at high temperatures T > 50 K differ from each other, because the thermally activated hopping frequency of the charge carriers is higher than the frequency of the measuring UHF field of an ESR spectrometer.

  4. 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.

  5. Discovery of a potent enoyl-acyl carrier protein reductase (FabI) inhibitor suitable for antistaphylococcal agent.

    PubMed

    Kim, Yun Gyeong; Seo, Jae Hong; Kwak, Jin Hwan; Shin, Kye Jung

    2015-10-15

    We report the discovery, synthesis, and biological activities of phenoxy-4-pyrone and phenoxy-4-pyridone derivatives as novel inhibitors of enoyl-acyl carrier protein reductase (FabI). Pyridone derivatives showed better activities than pyrone derivatives against FabI and Staphylococcus aureus strains, including methicillin-resistant Staphylococcus aureus (MRSA). Among the pyridone derivatives, compound 16l especially exhibited promising activities against the MRSA strain and good pharmacokinetic profiles. PMID:26343826

  6. Sulfhydryl-based tumor antigen-carrier protein conjugates stimulate superior antitumor immunity against B cell lymphomas.

    PubMed

    Betting, David J; Kafi, Kamran; Abdollahi-Fard, Alireza; Hurvitz, Sara A; Timmerman, John M

    2008-09-15

    Therapeutic vaccination of B cell lymphoma patients with tumor-specific Ig (idiotype, or Id) chemically coupled to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde has shown promising results in early clinical trials, and phase III trials are underway. However, glutaraldehyde Id-KLH vaccines fail to elicit anti-Id immune and clinical responses in many patients, possibly because glutaraldehyde reacts with lysine, cysteine, tyrosine, and histidine residues, damaging critical immunogenic epitopes. A sulfhydryl-based tumor Ag-carrier protein conjugation system using maleimide chemistry was used to enhance the efficacy of Id-KLH vaccines. Maleimide Id-KLH conjugates eradicated A20 lymphoma from most tumor-bearing mice, whereas glutaraldehyde Id-KLH had little efficacy. Maleimide Id-KLH elicited tumor-specific IgG Abs and T cells, with CD8(+) T cells being the major effectors of antilymphoma immunity. Maleimide Id-KLH vaccines also demonstrated superior efficacy in 38C13 and BCL-1 lymphoma models, where Abs were shown to be critical for protection. Importantly, standard glutaraldehyde Id-KLH conjugation procedures could result in "overconjugation" of the tumor Ag, leading to decreased efficacy, whereas the heterobifunctional maleimide-based conjugation yielded potent vaccine product regardless of conjugation duration. Under lysosomal processing conditions, the Id-carrier protein linkage was cleavable only after maleimide conjugation. Maleimide KLH conjugation was easily performed with human Igs analogous to those used in Id-KLH clinical trials. These data support the evaluation of sulfhydryl-based Id-KLH vaccines in lymphoma clinical trials and possibly the use of tumor Ag-carrier protein vaccines for other cancers. PMID:18768870

  7. A profile of protein-protein interaction: Crystal structure of a lectin-lectin complex.

    PubMed

    Surya, Sukumaran; Abhilash, Joseph; Geethanandan, Krishnan; Sadasivan, Chittalakkottu; Haridas, Madhathilkovilakathu

    2016-06-01

    Proteins may utilize complex networks of interactions to create/proceed signaling pathways of highly adaptive responses such as programmed cell death. Direct binary interactions study of proteins may help propose models for protein-protein interaction. Towards this goal we applied a combination of thermodynamic kinetics and crystal structure analyses to elucidate the complexity and diversity in such interactions. By determining the heat change on the association of two galactose-specific legume lectins from Butea monosperma (BML) and Spatholobus parviflorus (SPL) belonging to Fabaceae family helped to compute the binding equilibrium. It was extended further by X-ray structural analysis of BML-SPL binary complex. In order to chart the proteins interacting mainly through their interfaces, identification of the nature of forces which stabilized the association of the lectin-lectin complex was examined. Comprehensive analysis of the BMLSPL complex by isothermal titration calorimetry and X-ray crystal structure threw new light on the lectin-lectin interactions suggesting of their use in diverse areas of glycobiology. PMID:26945504

  8. 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.

  9. Effect of conjugation methodology, carrier protein, and adjuvants on the immune response to Staphylococcus aureus capsular polysaccharides.

    PubMed

    Fattom, A; Li, X; Cho, Y H; Burns, A; Hawwari, A; Shepherd, S E; Coughlin, R; Winston, S; Naso, R

    1995-10-01

    Conjugate vaccines were prepared with S. aureus type 8 capsular polysaccharide (CP) using three carrier proteins: Pseudomonas aeruginosa exotoxin A (ETA), a non-toxic recombinant ETA (rEPA), and diphtheria toxoid (DTd). Adipic acid dihydrazide (ADH) or N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was used as a spacer to link the CP to carrier protein. All conjugates gave a high immune response with a boost after the second immunization. Conjugates prepared with ADH gave higher antibody titers than conjugates prepared with SPDP. IgG1 was the primary subclass elicited by all conjugates regardless of the carrier protein or the conjugation method used to prepare the vaccines. The non-immunogenic CP and the conjugates were formulated with either monophosphoryl lipid A (MPL), QS21, or in Novasomes and evaluated in mice. While the adjuvants failed to improve the immunogenicity of the nonconjugated CP, a more than fivefold increase in the antibody levels was observed when these adjuvants were used with the conjugates. Significant rises in IgG2b and IgG3 were observed with all formulations. The enhancement of the immunogenicity and the IgG subclass shift, as seen with some adjuvants, may prove to be important in immunocompromised patients. PMID:8585282

  10. 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

  11. Ultrahigh-resolution optical coherence tomography imaging of protein crystals using gel inclusion technique

    NASA Astrophysics Data System (ADS)

    Nishizawa, N.; Ishida, S.; Hirose, M.; Sugiyama, S.; Inoue, T.; Mori, Y.; Itoh, K.; Matsumura, H.

    2013-03-01

    Protein crystals are required for X-ray crystallography to determine three-dimensional structures of proteins at atomic resolution. The conventional microscopy is currently used for observation and screening of protein crystals. However, the three-dimensional imaging, which is important for automated treatment of protein crystals, is generally difficult by light microscopy. In addition, the protein crystals in the media are frequently difficult to identify by conventional light microscopy owing to the appearance of salt crystals or amorphous materials. In this work, we successfully demonstrated micro-scale, non-invasive, three-dimensional cross-sectional imaging of protein crystals using ultrahigh resolution optical coherence tomography (UHR-OCT). A low noise, Gaussian like, high power supercontinuum at wavelength of 800 nm was used as the light source. The axial resolution of 2 um in sample and the sensitivity of 95 dB were achieved. Since the protein crystal has homogeneous nano-structure, the optical scattering is negligibly small. Therefore, we used gel-inclusion technique to enhance the intensity of scattered signals, and clear, sharp 3D cross-sectional images of protein crystals were successfully observed. As the gel concentration was increased, the OCT signal intensity was increased. Using this method, the protein crystals surrounded by substantial amount of precipitates could be visualized, which is difficult by conventional light microscopy. The discrimination of protein and salt crystals was also demonstrated by the OCT signal intensity. The wavelength dependence of OCT imaging for protein crystal was examined at wavelength of 800-1700 nm regions. It was confirmed that the finest images were observed using 800 nm wavelength system.

  12. 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.

  13. 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

  14. A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase.

    PubMed

    Kolek, Stefan Andrew; Bräuning, Bastian; Shaw Stewart, Patrick Douglas

    2016-04-01

    Random microseed matrix screening (rMMS), in which seed crystals are added to random crystallization screens, is an important breakthrough in soluble protein crystallization that increases the number of crystallization hits that are available for optimization. This greatly increases the number of soluble protein structures generated every year by typical structural biology laboratories. Inspired by this success, rMMS has been adapted to the crystallization of membrane proteins, making LCP seed stock by scaling up LCP crystallization conditions without changing the physical and chemical parameters that are critical for crystallization. Seed crystals are grown directly in LCP and, as with conventional rMMS, a seeding experiment is combined with an additive experiment. The new method was used with the bacterial integral membrane protein OmpF, and it was found that it increased the number of crystallization hits by almost an order of magnitude: without microseeding one new hit was found, whereas with LCP-rMMS eight new hits were found. It is anticipated that this new method will lead to better diffracting crystals of membrane proteins. A method of generating seed gradients, which allows the LCP seed stock to be diluted and the number of crystals in each LCP bolus to be reduced, if required for optimization, is also demonstrated. PMID:27050265

  15. Phase Sensitive X-Ray Diffraction Imaging Study of Protein Crystals

    NASA Technical Reports Server (NTRS)

    Hu, Z. W.

    2003-01-01

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

  16. 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

  17. Isolation of a crystal matrix protein associated with calcium oxalate precipitation in vacuoles of specialized cells.

    PubMed

    Li, Xingxiang; Zhang, Dianzhong; Lynch-Holm, Valerie J; Okita, Thomas W; Franceschi, Vincent R

    2003-10-01

    The formation of calcium (Ca) oxalate crystals is considered to be a high-capacity mechanism for regulating Ca in many plants. Ca oxalate precipitation is not a stochastic process, suggesting the involvement of specific biochemical and cellular mechanisms. Microautoradiography of water lettuce (Pistia stratiotes) tissue exposed to 3H-glutamate showed incorporation into developing crystals, indicating potential acidic proteins associated with the crystals. Dissolution of crystals leaves behind a crystal-shaped matrix "ghost" that is capable of precipitation of Ca oxalate in the original crystal morphology. To assess whether this matrix has a protein component, purified crystals were isolated and analyzed for internal protein. Polyacrylamide gel electrophoresis revealed the presence of one major polypeptide of about 55 kD and two minor species of 60 and 63 kD. Amino acid analysis indicates the matrix protein is relatively high in acidic amino acids, a feature consistent with its solubility in formic acid but not at neutral pH. 45Ca-binding assays demonstrated the matrix protein has a strong affinity for Ca. Immunocytochemical localization using antibody raised to the isolated protein showed that the matrix protein is specific to crystal-forming cells. Within the vacuole, the surface and internal structures of two morphologically distinct Ca oxalate crystals, raphide and druse, were labeled by the antimatrix protein serum, as were the surfaces of isolated crystals. These results demonstrate that a specific Ca-binding protein exists as an integral component of Ca oxalate crystals, which holds important implications with respect to regulation of crystal formation. PMID:14555781

  18. Protein crystallization in a 100 nl solution with new stirring equipment.

    PubMed

    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

    2008-05-01

    To investigate quantitatively the effects of stirring on protein crystallization, a new stirring system which can agitate a protein solution, approximately 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

  19. Design Principles for Broad-Spectrum Protein-Crystal Nucleants with Nanoscale Pits

    NASA Astrophysics Data System (ADS)

    van Meel, Jacobus A.; Sear, Richard P.; Frenkel, Daan

    2010-11-01

    Growing high-quality crystals is a bottleneck in the determination of protein structures by x-ray diffraction. Experiments find that materials with a disordered pitted surface seed the growth of protein crystals. Here we report computer simulations of rapid crystal nucleation in nanoscale pits. Nucleation is rapid, as the crystal forms in pits that have filled with liquid via capillary condensation. Surprisingly, we find that pits whose surfaces are rough are better than pits w