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The role of LB thin protein film in protein crystal growth by LB nanotemlate and robot

The role of LB thin protein film in protein crystal growth by LB nanotemlate and robot Prof. Eugenia Pechkova Laboratories of Biophysics and Nanobiotechnology University of Genova Italy. Solid phase. Surface pressure (mN/m). Liquid phase. Gas phase. Barrier position (mm).

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The role of LB thin protein film in protein crystal growth by LB nanotemlate and robot

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  1. The role of LB thin protein film in protein crystal growth by LB nanotemlate and robot Prof. Eugenia Pechkova Laboratories of Biophysics and Nanobiotechnology University of Genova Italy

  2. Solid phase Surface pressure (mN/m) Liquid phase Gas phase Barrier position (mm) M1-M6 - stepper motors; BA1, BA2 - Wilhelmy balances; B1,B2 - barriers; T1,T2 –Langmuir troughs; A1, A2 - compartments for adsorption; W - compartment for washing; S - substrate; MB-mobile block Thin Film Technologies Langmuir-Blodgett Protective plate Layer-by-Layer self assembly

  3. The Langmuir-Blodgett Technique p - measurements Spreading Compression p - isotherm

  4. LB nanotemplate protein crystallization method LB nanotemplate protein crystallization method. Pechkova E, Nicolini C., Protein nanocrystallography: a new approach to structural proteomics, Trends in Biotechnology 22, 117-122, 2004.

  5. Isotherm and Atomic Force Microscopy of Lysozyme Langmuir-Blodgett film.

  6. AFM images (in air): LB film (2 monolayers) of thaumatin (24 kDa) at 200 nm scale

  7. Nanogravimetric measuarments Protein surface density Human protein kinase CK2 (MW 45 kDa) Cytochrome P450 scc (MW 56 kDa) Film packing (area per molecule) 20,36 nm2 Experimental Close parking system 29,52 nm2 17,8 nm2 30 nm2

  8. Test proteins for classical vs LB nanotemplate crystallization

  9. Dependence of the surface density of deposited ribonuclease A, thaumatin, proteinase K and lysozyme nanostructured films upon the number of transferred protein mono-layers, at 20 mN/m of pressure.

  10. Energy ΔG CSalt CProtein Specific aggregates VDrop Precipitant Precipitant Critical nuclei II I Crystals Specific aggregates Non-specific aggregates Protein solution Time Protein solution with precipitant LB nanotemplate Classical hanging drop Vacuum grease Pechkova E, Nicolini C, From art to science in protein crystallization by means of thin film technology, Nanotechnology 13, 460-464, 2002.

  11. Protein crystallization conditions used for both LB and classical hanging drop vapour diffusion method. The cryo-protectant used during the X ray diffraction data collection is also indicated

  12. Classical Proteinase K crystal LB Proteinase K crystal

  13. Screening test for thaumatin “critical” concentration. A: screening test for ribonuclease A “critical” concentration. B: screening test for proteinase K “critical” concentration

  14. A: critical concentration for proteinase K with classical vapor diffusion method. B: critical concentration for thaumatin with classical vapor diffusion method. C: critical concentration for ribonuclease A with classical vapor diffusion method.

  15. Effects of LB template in crystal growth (in brackets number of crystals), Effect A: template facilitates nucleation step. LB crystals grow earlier than classical, Effect B: template facilitates crystal growth. LB crystals grow larger than classical.

  16. Phase Diagrammes of Proteinase K, Ribonuclease K, Thaumatin and Lysozyme (clockvise)

  17. European Synchrotron Radiation Facility (ESRF), Grenoble, France

  18. LB and classical protein crystals analyzed at the different ESRF beamlines.

  19. High throughput protein crystallization

  20. Electron density of crystals grown using robotic technique for LB-based nanotemplate method and classical method.. A: comparison of electron density map for carboxilic acid group of aspartic acid at 55 position contoured at 2.01sigma. B: comparison of electron density map of disulphide bond of cystein residue at 71 position contoured at 2.5 sigma. C: comparison of electron density map of disulphide bond of cystein residue at 149 position contoured at 2.5 sigma. D: comparison of electron density map of proline residue at 135 position of classical and LB crystal contoured at 2.5 sigma. E: comparison of electron density map of cysteine residue at 159 position of classical and LB crystal contoured at 2.5 sigma. From left to right and from top down.

  21. microGISAX in situ Protein nanotemplate Detector MAR CCD Sealed camera i X ray beam f Protein and salt solution, Cd Kapton window Pump1 Salt solution , Cs T1 Pump 2 T2 The tubes connected with pamps fo the salt solution exchange: Cs=Cd “stop solution” for aliement procedure Cs»Cd for accelerated nucleation Cs=2Cd for controlled grouth

  22. Setup of GISAXS setup at ID13 beamline/ESRF. The flow through crystallisation cell is tilted by y to adjust a fixed angle of incidence (i). As typical features the Yoneda Peak (Y) at c and the specular peak at f=i are shown in the 2d GISAXS pattern.

  23. Figure 3. Actual experimental configuration

  24. MODEL for reaction pathways on LB-film Pechkova, Gebhardt, Riekel, Nicolini, Biophysical Journal, Part I, 2010 Gebhardt, Pechkova, Rielel, Nicolini, Biophysical Journal, Part II, 2010

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