Methods in Modern Biophysics

1 The three-dimensional structure of proteins ....................................................... 1 1.1 Structure of the native state ....................................................................... 1 1.2 Protein folding transition states ................................................................. 9 1.3 Structural determinants of the folding rate constants .............................. 12 1.4 Support of structure determination by protein folding simulations ......... 20 2 Liquid chromatography of biomolecules ......................................................... 23 2.1 Ion exchange chromatography ................................................................. 23 2.2 Gel filtration chromatography ................................................................. 28 2.3 Affinity chromatography ......................................................................... 31 2.4 Counter-current chromatography and ultrafiltration ................................ 33 3 Mass spectrometry ........................................................................................... 37 3.1 Principles of operation and types of spectrometers ................................. 37 3.1.1 Sector mass spectrometer ................................................................. 38 3.1.2 Quadrupole mass spectrometer ........................................................ 39 3.1.3 Ion trap mass spectrometer .............................................................. 39 3.1.4 Time-of-flight mass spectrometer .................................................... 40 3.1.5 Fourier transform mass spectrometer ............................................... 43 3.1.6 Ionization, ion transport and ion detection ...................................... 44 3.1.7 Ion fragmentation ............................................................................. 45 3.1.8 Combination with chromatographic methods .................................. 46 3.2 Biophysical applications .......................................................................... 49 4 X-ray structural analysis ................................................................................... 59 4.1 Fourier transform and X-ray crystallography........................................... 59 4.1.1 Fourier transform ............................................................................. 59 4.1.2 Protein X-ray crystallography .......................................................... 69 4.1.2.1 Overview .................................................................................. 69 4.1.2.2 Production of suitable crystals .................................................. 69 4.1.2.3 Acquisition of the diffraction pattern ........................................ 71 XII Contents 4.1.2.4 Determination of the phases: heavy atom replacement ............. 76 4.1.2.5 Calculation of the electron density and refinement .................. 83 4.1.2.6 Cryocrystallography and time-resolved crystallography........... 84 4.2 X-ray scattering ....................................................................................... 85 4.2.1 Small angle X-ray scattering (SAXS) .............................................. 85 4.2.2 X-ray backscattering ........................................................................ 88 5 Protein infrared spectroscopy ........................................................................... 91 5.1 Spectrometers and devices ...................................................................... 92 5.1.1 Scanning infrared spectrometers ...................................................... 92 5.1.2 Fourier transform infrared (FTIR) spectrometers ............................ 92 5.1.3 LIDAR, optical coherence tomography, attenuated total reflection and IR microscopes ................. 96 5.2 Applications .......................................................................................... 102 6 Electron microscopy ...................................................................................... 107 6.1 Transmission electron microscope (TEM)............................................. 107 6.1.1 General design ............................................................................... 107 6.1.2 Resolution ...................................................................................... 109 6.1.3 Electron sources ............................................................................. 110 6.1.4 TEM grids ...................................................................................... 112 6.1.5 Electron lenses ............................................................................... 112 6.1.6 Electron-sample interactions and electron spectroscopy ................ 115 6.1.7 Examples of biophysical applications ........................................... 117 6.2 Scanning transmission electron microscope (STEM) ............................ 118 7 Scanning probe microscopy ........................................................................... 121 7.1 Atomic force microscope (AFM) .......................................................... 121 7.2 Scanning tunneling microscope (STM) ................................................. 133 7.3 Scanning nearfield optical microscope (SNOM) ................................... 135 7.3.1 Overcoming the classical limits of optics ...................................... 135 7.3.2 Design of the subwavelength aperture ........................................... 138 7.3.3 Examples of SNOM applications ................................................... 142 7.4 Scanning ion conductance microscope, scanning thermal microscope and further scanning probe microscopes .................... 143 8 Biophysical nanotechnology .......................................................................... 147 8.1 Force measurements in single protein molecules .................................. 147 8.2 Force measurements in a single polymerase-DNA complex .................. 150 Contents XIII 8.3 Molecular recognition ........................................................................... 152 8.4 Protein nanoarrays and protein engineering .......................................... 155 8.5 Study and manipulation of protein crystal growth ................................. 158 8.6 Nanopipettes, molecular diodes, self-assembled nanotransistors, nanoparticle-mediated transfection and further biophysical nanotechnologies ......... 159 9 Proteomics: high throughput protein functional analysis ............................... 165 9.1 Target discovery ................................................................................... 166 9.2 Interaction proteomics .......................................................................... 168 9.3 Chemical proteomics ............................................................................. 172 9.4 Lab-on-a-chip technology and mass-spectrometric array scanners ....... 173 9.5 Structural proteomics ............................................................................ 174 10 Ion mobility spectrometry ............................................................................ 175 10.1 General design of spectrometers .......................................................... 175 10.2 Resolution and sensitivity .................................................................... 180 10.3 IMS-based “sniffers” ........................................................................... 183 10.4 Design details ...................................................................................... 184 10.5 Detection of biological agents ............................................................. 193 11 Φ-Value analysis .......................................................................................... 197 11.1 The method ......................................................................................... 197 11.2 High resolution of six protein folding transition states ....................... 199 12 Evolutionary computer programming ......................................................... 203 12.1 Reasons for the necessity of self-evolving computer programs .......... 203 12.2 General features of the method ........................................................... 203 12.3 Protein folding and structure simulations ........................................... 206 12.4 Evolution of nanooptical devices made from nanoparticles .............. 207 12.4.1 Materials and methods ................................................................ 207 12.4.2 Results and discussion ................................................................ 208 12.5 Further potential applications .............................................................. 210 13 Conclusions .................................................................................................. 213 References .......................................................................................................... 215 Index .................................................................................................................. 247