Chemical Biology Techniques and Applications

, by Larijani, Banafshe; Rosser, Colin A.; Woscholski, Rudiger

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ISBN: 9780470090640 | 0470090642
Cover: Hardcover
Copyright: 8/25/2006

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This book provides an up-to-date guide to chemical biology. It is a modular textbook that brings together the tools and techniques used by physical scientists and makes them available to biological and biomedical scientists. Tools and techniques are explained at a suitable level and the book addresses topical chemical biology questions within each chapter.

Preface.

List of Contributors.

1. Introduction.

1.1 Chemical biology - the present.

1.2 Chemical biology - the past.

1.3 Chemical biology - the future.

1.4 Chemical biology - mind the interdisciplinary gap.

1.5 An introduction to the following chapters.

1.5.1 Cryo-electron microscopy.

1.5.2 Atomic force microscopy.

1.5.3 Differential scanning calorimetry in the study of lipid structures.

1.5.4 Membrane potentials and membrane probes.

1.5.5 Identification and quantification of lipids using mass spectrometry.

1.5.6 Liquid-state NMR.

1.5.7 Sold-state NMR in biomembranes.

1.5.8 Molecular dynamics.

1.5.9 Two-dimensional infrared studies of biomolecules.

1.5.10 Biological applications of single and two-photon fluorescence.

1.5.11 Optical tweezers.

1.5.12 PET imaging in chemical biology.

1.5.13 Chemical genetics.

2. Cryomicroscopy.

2.1 The need for (electron) microscopy.

2.2 Development of cryomicroscopy.

2.3 Sample-electron interaction.

2.4 Contrast in negatively stained and cryo preparations.

2.5 Image formation.

2.6 Image analysis.

2.7 Software used in the analysis of electron micrographs.

2.8 Examples.

2.8.1 Two-dimensional crystals.

2.8.2 Helical structures.

2.8.3 Single particles with low symmetry.

2.8.4 Cellular tomography.

2.9 Conclusions.

3. Atomic force microscopy: applications in biology.

3.1 A brief history of microscopy.

3.2 The scanning pribe microscope revolution.

3.2.1 The stylus profiler.

3.2.2 The scanning tunnelling microscope.

3.2.3 The atomic force microscope.

3.3 The workings of an AFM instrument.

3.3.1 The imaging probe.

3.3.2 The piezoelectric scanner.

3.3.3 The deflection detection system.

3.3.4 The electronic feedback system.

3.4 Imaging biological molecules with force.

3.4.1 Contact mode.

3.4.2 Oscillating cantilever imaging modes.

3.4.3 Imaging in liquid.

3.5 Factors influencing image quality.

3.5.1 Sample preparation and immobilization.

3.5.2 Tip convolution/broadening.

3.5.3 Double tipping.

3.5.4 Sample roughness.

3.5.5 Temperature variation and vibration isolation.

3.6 Biological applications of AFM and recent developments.

3.6.1 Imaging dynamic processes.

3.6.2 Measuring biomolecular forces.

3.7 Conclusions and future directions.

4. Differential scanning calorimetry in the study of lipid structures.

4.1 Introduction.

4.2 Membranes, lipids and lipid phases.

4.3 Heat exchanges and calorimetry.

4.3.1 Heat and related entities.

4.3.2 Differential scanning calorimetry.

4.4 Phase transitions in pure lipid-water systems.

4.4.1 Gel (Lβ’) to rippled (Pβ’) phase transition (the ‘pre-transition’).

4.4.2 Rippled (Pβ’) to liquid-crystalline (Lα) phase transition.

4.4.3 Gel (Lβ) to liquid-crystalline (Lα) phase transition.

4.4.4 Lamellar (Lα) to inverted hexagonal (HII) phase transition.

4.5 Selected examples of transitions in lipid mixtures.

4.5.1 Phospholipid?cholesterol mixtures.

4.5.2 Lamellar-to-inverted hexagonal transitions.

4.6 Complex systems: lipid-protein mixtures and cell membranes.

4.6.1 Lipid-protein systems.

4.6.2 Cell membranes and cell walls.

4.7 Conclusion.

5. Membrane potentials and membrane probes.

5.1 Introduction: biological membranes; structure and electrical properties.

5.2 Phospholipid membranes as molecular environments.

5.3 The physical origins of the transmembrane (ΔΨ), surface (φS) and dipolar (φD) membrane potentials.

5.3.1 The transmembrane potential difference (ΔΨ).

5.3.2 The membrane surface potential φS).

5.3.3 The membrane dipole potential (φD).

5.4 Measurement of membrane potentials.

5.4.1 Electrodes.

5.4.2 Spectroscopic measurements of the transmembrane potential difference.

5.4.3 Spectroscopic measurements of the membrane surface potential.

5.4.4 Spectroscopic measurements of the membrane dipole potential.

5.5 Problems with Spectroscopic Measurements of Membrane Potentials.

5.6 Spatial Imaging of membrane potentials.

6. Identification and quantification of lipids using mass spectrometry.

6.1 Introduction.

6.2 Lipid analysis by mass spectrometry.

6.2.1 HPLC-ESI-MS.

6.2.2 Tandem mass spectrometry.

6.3 Conclusion.

7. Liquid-state NMR.

7.1 Introduction.

7.2 How NMR works: the basics.

7.3 Some NMR applications in biology.

7.3.1 In vivo cell metabolism made ‘visible’ by NMR.

7.3.2 In vivo phosphorus and nitrogen metabolism in maize roots in response to ammonia.

7.3.3 Identification and quantification of small quantities in a complex mixture by 2D 1H-31P-HMQC-TOCSY NMR.

7.3.4 Simultaneous separation and identification of very complex mixtures: LC-NMR.

7.3.5 Three-dimensional molecular structures in solution.

7.4 Conclusion.

8. Solid-state NMR in biomembranes.

8.1 Introduction.

8.2 NMR basics for membrane systems.

8.2.1 Anisotrpy of NMR interactions in membranes.

8.2.2 Spectra and the effect of motional averages.

8.2.3 A special case of motional averaging: magic angle sample spinning.

8.2.4 Relaxation times: measuring dynamics.

8.3 Applications of wide-line NMR to membrane systems.

8.3.1 Lipid phases: diagrams, peptide-induced fusion-fragmentation.

8.3.2 Bilayer internal dynamics: order parameters, membrane thickness, sterols.

8.3.3 Orientation of molecules (sterols, helical peptides) in membranes.

8.3.4 Membrane dynamics from picoseconds to milliseconds.

8.4 Applications of MAS to biomembranes and natural colloids.

8.4.1 Three-dimensional structure of peptides in membranes.

8.4.2 Three-dimensional structure of wine tannin-salivary protein colloidal complexes.

8.4.3 Distance determination using MAS recoupling techniques.

8.5 Conclusion.

9. Molecular dynamics.

9.1 Introduction.

9.2 The basis of molecular mechanics.

9.2.1 Force fields.

9.2.2 The energy problem.

9.3 The basis of molecular dynamics.

9.3.1 Influence of temperature.

9.4 Factors affecting the length of simulations.

9.5 Problems caused by solvents.

9.6 How to build a lipid bilayer for simulation purposes.

9.7 Special cases of membrane proteins.

9.8 Summary.

10. Two-dimensional infrared studies of biomolecules.

10.1 Introduction.

10.2 Description of the technique.

10.3 Spectral simulations.

10.3.1 Intensity changes.

10.3.2 Band shifting.

10.3.3 Bandwidth.

10.4 Two-dimensional studies of human lipoproteins.

10.5 Summary.

11. Biological applications of single- and two-photon fluorescence.

11.1 Introduction.

11.2 Basic principles of fluorescence.

11.3 Main principles of RET via single-photon excitation.

11.4 Detection of RET.

11.4.1 Steady-state method.

11.4.2 Time-resolved method.

11.5 Biological examples of RET monitored by frequency-domain FLIM.

11.6 Two-photon fluorescence.

11.6.1 Basic concepts.

11.6.2 Two-photon fluorescence.

11.7 Applications of two-photon fluorescence.

11.7.1 Two-photon fluorescence imaging.

11.7.2 Biological example - two-photon time-domain FLIM.

11.8 Photoselection and fluorescence anisotropy.

11.9 Fluorescence anisotropy and isotropic rotational diffusion.

11.10 Fluorescent probes in proteins and membranes.

11.10.1 Ordered molecular systems.

11.11 Future developments.

11.11.1 New chromophores for two-photon fluorescence.

11.11.2 Stimulated emission depletion in two-photon excited states.

11.12 Conclusions.

12. Optical tweezers.

12.1 Introduction.

12.1.1 History of optical tweezers.

12.1.2 Single-molecule studies.

12.2 Theoretical background.

12.3 Apparatus.

12.3.1 Building an optical tweezers transducer.

12.3.2 Creating multiple laser traps: accousto-optical deflectors.

12.3.3 Camera and light path.

12.4 Data collection and analysis.

12.4.1 Collecting data with an optical tweezer.

12.4.2 Calibration of the detectors and of the optical tweezer stiffness.

12.4.3 Stokes calibration.

12.4.4 Equipartition principle.

12.5 A biological application.

12.5.1 Studying acto-myosin interactions.

12.5.2 Identification of events and data analysis.

12.5.3 Measuring the powerstroke.

12.5.4 Lifetime analysis.

12.6 Other biological examples.

12.7 Summary.

13. PET imaging in chemical biology.

13.1 Introduction.

13.2 Positron emission tomography: principles and instrumentation.

13.3 Applications of PET imaging in the biomedical sciences.

13.3.1 A labelled glucose analogue: an indirect probe to measure energy metabolism.

13.3.2 Imaging dopamine metabolism with PET.

13.3.3 PET imaging in gene expression.

13.3.4 Molecular imaging in drug discovery and development.

13.4 Conclusions and outlook.

14. Chemical genetics.

14.1 Introduction.

14.2 Why chemicals?.

14.3 Chemical genetics - why now?.

14.4 The relationship between classical genetics and chemical genetics.

14.5 Forward chemical genetics.

14.5.1 Obtain a compound library.

14.5.2 Screening.

14.5.3 Target identification.

14.6 Reverse chemical genetics.

14.7 Closing remarks.

Index.

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Chemical Biology Techniques and Applications

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Author Biography

Table of Contents

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