Modern Biophysical Chemistry

Detection and Analysis of Biomolecules

Peter Jomo Walla

• Publisher: Wiley-VCH
• Year: 2015
• ISBN:
  9783527683550
• Edition: 2
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

This updated and up-to-date version of the first edition continues with the really interesting stuff to spice up a standard biophysics and biophysical chemistry course. All relevant methods used in current cutting edge research including such recent developments as super-resolution microscopy and next-generation DNA sequencing techniques, as well as industrial applications, are explained. The text has been developed from a graduate course taught by the author for several years, and by presenting a mix of basic theory and real-life examples, he closes the gap between theory and experiment.
The first part, on basic biophysical chemistry, surveys fundamental and spectroscopic techniques as well as biomolecular properties that represent the modern standard and are also the basis for the more sophisticated technologies discussed later in the book. The second part covers the latest bioanalytical techniques such as the mentioned super-resolution and next generation sequencing methods, confocal fluorescence microscopy, light sheet microscopy, two-photon microscopy and ultrafast spectroscopy, single molecule optical, electrical and force measurements, fluorescence correlation spectroscopy, optical tweezers, quantum dots and DNA origami techniques. Both the text and illustrations have been prepared in a clear and accessible style, with extended and updated exercises (and their solutions) accompanying each chapter.
Readers with a basic understanding of biochemistry and/or biophysics will quickly gain an overview of cutting edge technology for the biophysical analysis of proteins, nucleic acids and other biomolecules and their interactions. Equally, any student contemplating a career in the chemical, pharmaceutical or bio-industry will greatly benefit from the technological knowledge presented. Questions of differing complexity testing the reader's understanding can be found at the end of each chapter with clearly described solutions available on the Wiley-VCH textbook homepage under: www.wiley-vch.de/textbooks

Part One
Basic Methods in Biophysical Chemistry

In Part 1 of this book the more established modern techniques are presented that are nowadays very broadly applied in many labs throughout the world. It starts with a chapter that introduces the basic principles governing the interactions of electromagnetic radiation (“light”) with molecules and biomolecules. These interactions are the basis for many of the methods presented not only in Part 1 but also in some chapters of Part 2. Important examples are general methods such as absorption spectroscopy and a plethora of advanced fluorescence techniques (Chapters 2 and 3) as well as very important techniques such as NMR studies of biomolecules (Chapter 5), but also methods such as super-resolution microscopy (Chapter 8), single-biomolecule detection (Chapter 9), nonlinear spectroscopy (Chapter 10) and next-generation DNA-sequencing methods (Chapter 11).

The introductory chapter (Chapter 1) aims at providing a brief and concise but still thorough review of the interaction of electromagnetic radiation with molecules for readers with a more chemical or physical background and serves as an introduction into these interactions for readers with a more biological background. Readers who require a rather quick overview and who have at least some basic knowledge about orbitals of molecules might focus on Sections 1.1, 1.3, 1.5 and the beginning of Section 1.6 only. These parts should suffice to have enough background information to understand the subsequent chapters. For example, they will already give an indepth idea about transition dipole moments and the polarization of light, which are central for quite a number of the methods presented in this book.

In Chapters 2 and 3, absorption and fluorescence properties in the visible spectral range of important biomolecules and fluorescence markers are presented as well as the application of the corresponding techniques to investigate biomolecules and biomolecular processes. Important examples are the determination of biomolecule concentrations or purities as well as investigations of receptor–ligand equilibria, enzymatic conversions and activators and inhibitors. In Chapter 3 some very central concepts in modern biophysical chemistry are also discussed in more detail, such as labelling and linking techniques, Förster resonance energy transfer (FRET) and fluorescence polarization assays. Also, the mathematical framework for the latter two is discussed in more detail, but readers who require a quick overview can also focus only on the description of the equations that are marked by boxes, which represent, in general, the central equations throughout this book.

Chapter 4 summarizes some very important optical methods that do not primarily rely on the absorption and emission of unpolarized or linearly polarized light in the visible spectral range but rather on scattering of light, the use of left- and right-handed circular light or light in the infrared spectral region. These techniques are, in general, of great use to derive information about the size, shape and secondary structural elements of biomolecules without any labelling of the samples.

Chapter 5 gives an overview of the application of nuclear magnetic resonance (NMR) techniques, which work with electromagnetic radiation in the radiowave range. NMR allows for the analysis of biomolecular structures under more physiological conditions than is possible with classical electron or X-ray microscopies. Chapter 5 also aims at providing an insight into the physical basics behind multidimensional NMR that are essential for such studies and how the information derived from such techniques can be used to derive a final 3D structure of proteins. Also, the use of electron paramagnetic resonance (EPR) for investigating biomolecu...