Proteomics and Nanocrystallography

ISBN-13: 9780306479021 / Angielski / Twarda / 2003 / 190 str.

Eugenia Pechkova; Claudio Nicolini; C. Nicolini

Opis

The book addresses the most recent developments in structural and functional proteomics underlying the recent contributions given in these areas by our laboratory to the instrumentations, the methods and the procedures as mutuated from the nanoscale sciences and technologies. These developments introduced in the last few years make now possible protein massive identification (mass spectrometry and biomolecular arrays down to nanoamounts) and protein structural characterization in solution and in crystals down to the atomic scale to an extent and to a degree so far unmatched. Emphasis is placed in the growth by nanobiofilm template of protein crystals of any type and size from millimeter to micron, leading in combination with microfocus synchrotron technology and atomic force microscopy to the definition of a new field called nanocrystallography. The few useful examples being shown, concerning yet structurally unsolved proteins, point this very promising approach nanotechnology-based in structural proteomics using highly focused X-rays. This has not to be confused with the important study of nanocrystals, both organic and inorganic, and novel diamond like nanocomposite materials and devices having 3D protein crystals as matrices to be equilibrated with nanoparticles/gold/silver to be utilized in the most diversified electronic applications here also summarized. vii Acknowledgments We are particularly grateful to Giuseppe Zanotti at the University of Padova for his fundamental collaboration during all the crystallographic studies.

Spis treści

1 The New Frontier at the Crossing of life and Physical Sciences.- 2 State of the Art in Proteomics, Crystallography and Nanobiotechnology.- 2.1 Methods of Protein Production.- 2.1.1 Recombinant DNA Technology: expression, purification and mutagenesis.- 2.2 Methods for Protein Identification.- 2.2.1 Two-dimensional electrophoresis.- 2.2.2 Mass spectrometry and its principles.- 2.3 Methods of Protein Crystallization.- 2.3.1. Importance of protein sample preparation.- 2.3.2 Protein solubility and supersaturation. Nucleation and crystal growth.- 2.3.3 Classical methods.- 2.3.4 Less common techniques.- 2.3.5 Advanced methods.- 2.4 Methods of Crystal Structure Determination.- 2.4.1 Protein X-ray crystallography.- 2.4.2 Crystal structure determination.- 2.4.3 Data collection and data processing.- 2.4.4 Traditional X-ray diffractometer.- 2.4.5 Electron microscopy.- 2.5 Other Methods of Protein Structure Investigation.- 2.5.1 Circular Dichroism measurements.- 2.5.2 High Resolution Nuclear Magnetic Resonance.- 2.6 Methods of Protein Immobilization In Solid Thin Films.- 2.6.1 Pressure-area isotherms.- 2.6.2 Surface potential.- 2.6.3 Methods of thin film deposition.- 2.6.4 Methods of thin film characterization.- 3 Nanocrystallography.- 3.1 Nanobiofilms Template.- 3.1.1 Substrate preparation.- 3.1.2 Protein thin film formation.- 3.1.3 Protein thin film characterization.- 3.2 Atomic Force Microscopy.- 3.2.1 Design of the chamber.- 3.2.2 The hardware and software apparatus.- 3.2.3 Performances of the system.- 3.3 Synchrotron Radiation.- 3.3.1 Principles of Synchrotron functioning.- 3.3.2 Microfocus beamline and ultramicrocrystal diffraction.- 3.3.3 Protein crystals synchrotron radiation analysis.- 3.3.4 Structure determination and refinement.- 3.4 From Art to Science With Protein Nanocrystallography.- 4 From Art to Science in Protein Crystallography.- 4.1 New Method Validation With Lysozyme as a Model Protein.- 4.1.1 Effect of the thin film template: kinetics of the lysozyme crystal growth.- 4.1.2 Thin film surface pressure influence on the template-induced crystallization.- 4.1.3 X-ray diffraction of lysozyme crystals.- 4.1.4 Synchrotron radiation analysis of lysozyme crystals.- 4.2 Yet Unresolved Proteins: Bovine Cytochrome P450scc.- 4.2.1 Cytochrome P450scc (Side-Chain Cleavage).- 4.2.2 Sample homogeneity.- 4.2.3 Pressure-area isotherm.- 4.2.4 Quartz crystal nanobalance.- 4.2.5 Cytochrome P450scc crystallization.- 4.2.6 Atomic Force Microscopy of cytochrome microcrystals.- 4.3 Yet unresolved proteins: Human kinase CK2? Catalytic Subunit.- 4.3.1 Protein kinase CK2? catalytic subunit.- 4.3.2 Sample homogenity.- 4.3.3 Pressure-area isotherm.- 4.3.4 Quartz crystal nanobalance.- 4.3.5 Human protein kinase CK2? crystallization.- 4.3.6 Synchrotron radiation: analysis, data processing and structure determination.- 4.4 Conclusions.- 5 From Science Technology in Proteomics.- 5.1 Automated Mass Spectrometry.- 5.2 Biomolecular Arrays.- 5.2.1 The surface patterning.- 5.2.2 The instrumental layout and data analysis.- 5.3 Unsolved Protein Structures of Scientific and Industrial Interest.- 6 Future Trends.- 6.1 Novel Composite Biomaterials and Nanodevices.- 6.2 nanocrystals.- References.