Methods in Modern Biophysics, 2/e

Description

Incorporating dramatic recent advances, "Methods in Modern Biophysics" presents a fresh and timely introduction to modern biophysical methods. This innovative text surveys and explains the ten key biophysical methods, including those related to biophysical nanotechnology, scanning probe microscopy, X-ray crystallography, ion mobility spectrometry, mass spectrometry, and proteomics. Containing much information previously unavailable in tutorial form, "Methods in Modern Biophysics" employs worked examples and more than 260 illustrations to fully detail the techniques and their underlying mechanisms. The book was written for advanced undergraduate and graduate students, postdocs, researchers, lecturers and professors in biophysics, biochemistry, general biology and related fields.

Table of contents

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