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Summary

GENERAL PRINCIPLES

The term electrophoresis describes the migration of a charged particle under the influence of an electric field. Many important biological molecules, such as amino acids, peptides, proteins, nucleotides and nucleic acids, possess ionisable groups and, therefore, at any given pH, exist in solution as electrically charged species, either cations (positively charged) or anions (negatively charged). Under the influence of an electric field these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.

The equipment required for electrophoresis consists basically of two items, a power pack and an electrophoresis unit. Electrophoresis units are available for running either vertical or horizontal gel systems. Vertical slab gel units are commercially available and routinely used to separate proteins in acrylamide gels (Section 6.2). The gel is formed between two glass plates that are clamped together, but held apart by plastic spacers. A commonly used equipment in this context is the so-called minigel apparatus (Figure 6.1). Gel dimensions are typically 8.5 cm wide × 5 cm high, with a thickness of 0.5–1 mm. A plastic comb is placed in the gel solution and is removed after polymerisation to provide loading wells for up to 10 samples. When the apparatus is assembled, the lower electrophoresis tank buffer surrounds the gel plates and affords some cooling of the gel plates. A typical horizontal gel system is shown in Figure 6.2. The gel is cast on a glass or plastic sheet and placed on a cooling plate (an insulated surface through which cooling water is passed to conduct away generated heat). Connection between the gel and electrode buffer is made using a thick wad of wetted filter paper (Figure 6.2); note, however, that agarose gels for DNA electrophoresis are run submerged in the buffer

(Section 6.4.1). The power pack supplies a direct current between the electrodes in the electrophoresis unit. All electrophoresis is carried out in an appropriate buffer, which is essential to maintain a constant state of ionisation of the molecules being separated. Any variation in pH would alter the overall charge and hence the mobilities (rate of migration in the applied field) of the molecules being separated.

Summary

INTRODUCTION

Determining the structure of molecules and the interactions between molecules is critical for understanding biochemical processes. All experimental techniques discussed in the following sections revolve around investigations of the three-dimensional structure of molecules as well as the elucidation of interactions between different groups of molecules. As such, some of these techniques are characterised by a somewhat higher level of complexity in undertaking and are often employed at a later stage of biochemical characterisation.

Techniques probing the thermodynamics of a system (such as isothermal titration calorimetry) or atomic/molecular structure and interactions without requirements for washing or flow (NMR spectrosocopy, magnetic resonance imaging, X-ray diffraction, light scattering) can be summarised as direct techniques. Alternatively, molecular interactions can be analysed by applying tracers to the molecules themselves, resulting in molecular switch techniques. Last, there are in vitro methods that combine the use of analytical techniques (such as the spectroscopic techniques outlined in Chapter 13) with physical sampling – these can be classified as indirect techniques.

The analysis of molecular interactions can result in the development of a biosensor: a device that is composed of a biological element and a physico-chemical transduction part that converts signal reception by the biological entity into a physical quantifiable response. Such technology gives rise to the field of biosensing.

ISOTHERMAL TITRATION CALORIMETRY

Principles

Isothermal titration calorimetry (ITC) enables study of the thermodynamics of molecules binding to each other. This is a general method for studying the thermodynamics of any binding (association) process in solution. It detects and quantifies small heat changes associated with the binding and has the advantages of speed, accuracy and no requirement for either of the reacting species to be chemically modified or immobilised. The apparatus consists of a pair of matched cells (sample and reference) of approximately 2 cm 3 volume contained in a microcalorimeter (Figure 14.1a). One of the reactants (say the enzyme preparation) is added to the sample cell and the ligand (substrate, inhibitor or effector) is added via a stepper-motor-driven syringe. The mixture is stirred to ensure homogeneity. The reference cell contains an equal volume of reference liquid. A constant power of less than 1 mW is applied to the reference cell.

Summary

INTRODUCTION

An enormous cache of human biology remains to be explored as discoveries and new insights emerge from the ‘-omics’ sciences. The goal for many pharmaceutical companies (and increasingly also for academic organisations) is to find selective small molecules that can modulate these newly identified targets (mainly proteins), so that they can ultimately be developed into therapeutics. In this context, the term small molecules is used for organic compounds, typically with a molecular weight < 1000 dalton (Da) comprised mainly of carbon, hydrogen, nitrogen and oxygen atoms, but frequently also sulfur and fluorine, and less often chlorine, bromine and phosphorus. The modulation of new biological targets may require novel structural frameworks (called scaffolds), which contain various functional groups in strategic positions. The correlation between the chemical structure of compounds and their biological activities constitutes the so-called structure–activity relationships and their analysis is an integral part of drug development. Indeed, the identification of new structural motifs of small molecules is one of the many drivers en route to understanding biological systems and developing innovative, safer therapeutics with novel modes of action.

The task of finding a selective molecule is truly daunting considering there is an estimated 10 20 –10 200 compounds with a molecular weight below 500 Da comprised of the atoms that make up current small-molecule therapies. To put this into perspective, approximately 2.7 × 10 7 molecules have been reported to date, which equates roughly to the ratio of the mass of the Sun compared to the mass of a proton, if the Bohecek number (6 × 10 62) is used to represent the set of small molecules with drug-like properties (see Section 24.2.2). Moreover, it would be impossible to screen this large number of molecules across multiple assays if we were limited to using the available resources on our planet, given the Earth is estimated to contain ‘only’ 10 51 atoms. The real challenge, which will be the primary focus of this chapter, lies in:

Summary

BIOLOGICALLY IMPORTANT MOLECULES

Molecules of biological interest can be classified into ions, small molecules and macromolecules. Typical organic small molecules include the ligands of enzymes, substrates such as adenosine triphosphate (ATP) and effector molecules (inhibitors, drugs). Ions such as Ca 2+ play a key role in signalling events. Biological macromolecules are polymers which, by definition, consist of covalently linked monomers, the building blocks. The four types of biologically relevant polymers are summarised in Table 2.1.

Proteins

Proteins are formed by a condensation reaction of the α-amino group of one amino acid (or the imino group of proline) with the α-carboxyl group of another. Concomitantly, a water molecule is lost and a peptide bond is formed. The peptide bond possesses partial double-bond character and thus restricts rotation around the C–N bond. The progressive condensation of many amino acids gives rise to an unbranched polypeptide chain. Since biosynthesis of proteins proceeds from the N- to the C-terminal amino acid, the N-terminal amino acid is taken as the beginning of the chain and the C-terminal amino acid as the end. Generally, chains of amino acids containing fewer than 50 residues are referred to as peptides, and those with more than 50 are referred to as proteins. Most proteins contain many hundreds of amino acids; ribonuclease, for example, is considered an extremely small protein with only 103 amino-acid residues. Many biologically active peptides contain 20 or fewer amino acids, such as the mammalian hormone oxytocin (nine amino-acid residues) which is clinically used to induce labour since it causes contraction of the uterus, and the neurotoxin apamin (18 amino-acid residues) found in bee venom.

THE IMPORTANCE OF STRUCTURE

Three main factors determine the three-dimensional structure of a macromolecule:

Summary

INTRODUCTION: FROM EDMAN SEQUENCING TO MASS SPECTROMETRY

Prior to mass spectrometry, E dman degradation was the only technique to obtain the sequence information of proteins. Edman sequencing was based on the chemical reaction of the N-terminal amine with phenyl isothiocyanate, leading to a phenylthiocarbamoyl derivative, which was cleaved upon acidification and determined based on chromatography or electrophoresis. This was a slow process identifying one amino acid per reaction cycle. In addition, it required that the N-terminus of the proteins of interest was not blocked. However, most intact proteins, if they are not processed from a secretory or pro-peptide form, are blocked at the N-terminus, most commonly with an acetyl group. Other amino-terminal blocking includes fatty acylation, such as myristoylation or palmitoylation. Cyclisation of glutamine to a pyroglutamyl residue and other post-translational modification to N-termini also occur. In short, all these modifications leave the N-terminal residue without a free proton on the alpha nitrogen, thus Edman chemistry cannot proceed. Nowadays, Edman sequencing plays a minor role in protein analyses and has been surpassed by biological mass spectrometry techniques.

This revolution of biological mass spectrometry was largely enabled by the soft ionisation techniques ESI and MALDI (see Sections 15.2.4 and 15.2.5) that allowed the largescale analysis of biomolecules (proteins, peptides, oligonucleotides, oligosaccharides and lipids) and thereby revolutionised the areas of proteomics and metabolomics (Chapter 22). In contrast to electron impact (EI), these soft ionisation techniques produce molecular ions and only insignificant amounts of fragment ions. Therefore, in order to obtain structural sequence information on biomolecules, tandem MS (or MS/ MS) has been developed. Furthermore, the faster speed and sensitivity of tandem MS soon dwarfed the sequencing turnaround available by Edman degradation.

DIGESTION

The identification of proteins by mass spectrometry usually involves protease cleavage, mostly by trypsin. Owing to the specificity of this protease, tryptic peptides usually have basic groups at the N- and C-termini. Trypsin cleaves after lysine and arginine residues, both of which have basic side chains (an amino and a guanidino group, respectively). This results in a large proportion of high-energy doubly charged positive ions that are easily fragmented. The digestion of the protein into peptides is followed by identification of the peptides by tandem mass spectrometry (Section 21.3). This is commonly referred to as bottom-up or shotgun proteomics.

Summary

INTRODUCTION

A biologist will often turn to computer programming in situations where the amount or the complexity of data is too much to be sensibly handled by spreadsheets, and where no other, more specialised, software exists. Often only a relatively simple program needs to be written to get something useful from biological data, which would otherwise not be available. For biologists, the task of writing a computer program can sometimes seem like a significant barrier, but once the basic programming skills are learned then many possibilities are enabled. This chapter offers an introduction to the Python language and gives some concrete examples of programs that may be useful in molecular biology. However, there is not space to cover all aspects of the language and many of its finer details. For this we recommend further reading, but nonetheless hope this chapter serves to illustrate the basics and to show what is possible.

Python is one of the most popular programming languages and is becoming an increasingly attractive option for the biologist. It is a high-level, general-purpose language that is well supported and relatively easy to learn; indeed it is now taught in mainstream UK schools. Also, it has a large number of external modules, including many relating to mathematics, science and biology. Python is easy to install, if it isn't already installed as standard, and runs on almost all kinds of computer system. In this chapter we will show some of the features and capabilities of Python 3 and then apply this to several example programs to illustrate the sort of things that can be achieved for molecular biology. Python version 2, should you need to work with that instead, is very, very similar and most programs are easily transferred (e.g. using the conversion program 2to3 supplied with Python), although the two versions are not 100% compatible.

Even if you don't intend to use Python in the long-run or for all programming work, it nonetheless serves as a good starting point to learn some of the major principles of many modern computing languages. Python is a high-level language like Perl, Matlab and R, which is directly interpreted when a program is run; there is no distinct compilation step.

Summary

It has been a tremendous honour being asked by Keith Wilson, John Walker and Cambridge University Press to take on the role of editorship for this eighth edition of Principles and Techniques. In designing the content, we extend the long and successful tradition of this text to introduce relevant methodologies in the biochemical sciences by uniquely integrating the theories and practices that drive the fields of biology, biotechnology and medicine. Methodologies have improved tremendously over the past ten years and new strategies and protocols that once were just applied by a few pioneering laboratories are now applied routinely, accompanied by ever more fluent boundaries between core disciplines – leading to what is now called the life sciences.

In this eighth edition, all core methodologies covered in the previous edition have been kept and appropriately updated and consolidated. New chapters have been added to address the requirements of today's students and scientists, who operate in a much broader area than did the typical biochemists one or two decades ago. The contents of this text are thus structured into six areas, all of which play pivotal roles in current research: basic principles, biochemistry and molecular biology, biophysical methods, information technology, -omics methods and chemical biology. Of course, due to space restraints that help to keep this text accessible, the addition of new material required us to consolidate and carefully select content to be kept from the previous edition. These decisions have been guided by the over-arching theme of this text, namely to present principles and techniques. Importantly, our experience as teachers has always been that many undergraduates are challenged by quantitative calculations based on these principles, and hence we have continued the tradition of this text by including relevant mathematical and numerical tools as well as examples.

Indeed, new chapters on data processing, visualisation and Python have example applications that can be accessed from the CUP website to aid understanding.

Sadly, two authors of previous editions have passed away: John Fyffe died shortly after the seventh edition was launched and Alastair Aitken passed away in 2014. Also, Keith Wilson, John Walker and Robert Burns decided to retire, and we thus invited several new authors to contribute to this eighth edition.

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Index

22 - Fundamentals of Metabolomics

6 - Electrophoretic Techniques

Summary

14 - Basic Techniques Probing Molecular Structure and Interactions

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Contents

Contents

24 - Drug Discovery and Development

Summary

Foreword to the Eighth Edition

Contents

2 - Basic Principles

Summary

12 - Centrifugation and Ultracentrifugation

Contents

1 - Biochemical and Molecular Biological Methods in Life Sciences Studies

Contents

21 - Fundamentals of Proteomics

Summary

18 - The Python Programming Language

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Contents

13 - Spectroscopic Techniques

16 - Fundamentals of Bioinformatics

Preface

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