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This study by Prof. Eugenia Pechkova delves into the crucial role of LB thin protein films in protein crystal growth, utilizing LB nanotemplate and robotic technologies. The research, conducted at the Laboratories of Biophysics and Nanobiotechnology at the University of Genova, Italy, employs advanced techniques such as Langmuir-Blodgett (LB) assembly for protective plate layers and layer-by-layer self-assembly. Various methods like solid phase surface pressure measurements, spreading compression isotherms, and nanogravimetric analyses are employed to study protein crystal growth processes. The study includes AFM images of LB films, experimental setups involving stepper motors, Wilhelmy balances, Langmuir troughs, and compartments for adsorption, among others. The research also explores the effects of LB templates on crystal growth, as well as comparison studies between LB-based and classical methods for protein crystallography. This comprehensive research sheds light on innovative approaches in structural proteomics and protein crystallization, offering insights into key protein crystallization conditions and critical concentrations for effective crystal growth.
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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) 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
The Langmuir-Blodgett Technique p - measurements Spreading Compression p - isotherm
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.
Isotherm and Atomic Force Microscopy of Lysozyme Langmuir-Blodgett film.
AFM images (in air): LB film (2 monolayers) of thaumatin (24 kDa) at 200 nm scale
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
Test proteins for classical vs LB nanotemplate crystallization
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.
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.
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
Classical Proteinase K crystal LB Proteinase K crystal
Screening test for thaumatin “critical” concentration. A: screening test for ribonuclease A “critical” concentration. B: screening test for proteinase K “critical” concentration
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.
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.
Phase Diagrammes of Proteinase K, Ribonuclease K, Thaumatin and Lysozyme (clockvise)
European Synchrotron Radiation Facility (ESRF), Grenoble, France
LB and classical protein crystals analyzed at the different ESRF beamlines.
High throughput protein crystallization
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.
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
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.
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
