This chapter elaborates on centrifugation used in almost all molecular biology laboratories. It primarily focuses on analytical ultracentrifuge (AUC) as a powerful tool to measure and visualize the behaviour of the sedimenting macromolecule. The AUC is a powerful tool for characterizing macromolecules and their binding properties to themselves and other ligands. In a sedimentation equilibrium experiment, diffusion and sedimentation forces are balanced, which results in the concentration distribution in the centrifuge cell depending on the effective molecular weight. The chapter cites that the Svedberg equation describes the measurement of the sedimentation velocity, which correlates to the size, shape, and interactions of macromolecules. It then details the formation of density gradients and equilibrium density gradients.
Atomic and molecular orbitals, their energy states, and transitions
This chapter outlines a tutorial on atomic and molecular orbitals, their energy states, and transitions. It details the aim of introducing ideas about the shapes and energies of wave functions that describe the distribution of electrons in atoms and molecules. The spatial distribution and energy of electrons in atoms can be characterized by a three-dimensional wave function or orbitals. In molecules, the orbitals describing the energy levels of the electrons can be assumed to arise from a combination of atomic orbitals. The chapter then presents figures of electronic transitions that involve organic molecules and d-block metals. It notes worked examples on d-block elements and octahedral symmetry.
This chapter presents a tutorial concerning biological molecules. It elaborates on the notion of nucleic acids, proteins, polysaccharides, and lipids. The tutorial's purpose revolves around reminding readers about some of the molecules of life, their nomenclaturec, and some of their properties. The chapter then provides several figures to provide a visual representation of RNA with four bases, a protein with four amino acids and sidechains, a branched polysaccharide, and a lipid bilayer. It also presents a working example of six cysteines forming three cystine disulfide bridges, which concerns the amino-acid table and the amino-acid composition of a small trypsin inhibitor.
Iain D. Campbell
Biophysical Techniques starts off by introducing the topics that come under the umbrella of biophysical techniques. It then considers molecular principles in the biophysical sphere. It looks at transport and heat. Then it considers scattering, refraction, and diffraction. The text also looks at electronic and vibrational spectroscopy. Next, it moves on to magnetic resonance. Microscopy and single molecule studies are also examined in detail. Finally, computational biology is presented as a topic to end with.
This chapter considers the key concepts of calorimetry. It explains that calorimetry measures the heat produced by chemical reactions or physical changes. Thus, calorimetry can be used as a universal detection system so unmodified and unlabeled material can be monitored in almost any solution conditions. The chapter then provides an overview into the differences of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC), which are the main types of calorimetry used to study biological systems. ITC measures heat changes when a complex is formed at a constant temperature, while DSC measures the heat flow associated with molecular changes brought about by changing the temperature.
This chapter focuses on chromatograph, which is a broad range of methods that separate and analyze mixtures of molecules. Chromatography is an essential tool in the purification of a wide range of macromolecules but it can also be used to characterize molecular weights and to identify interactions. Size exclusion, ion exchange, and affinity chromatography are the main types of chromatography used in biological applications. The chapter explains that an understanding of practical chromatography is aided by the concept of theoretical plates and the van Deemter plot. It also considers more chromatography techniques, such as immobilized metal ion affinity chromatography (IMAC) and tandem affinity purification (TAP).
This chapter provides an overview of computational biology and its three major areas: modeling of systems, bioinformatics, and molecular modeling. It defines computational biology as the application of computational methods to all levels of exploration from molecules to ecosystems. The World Wide Web gives ready access to a wide range of databases and analysis software that forms the basis of bioinformatics. Thus, the visualization of molecules with software that operates on personal computers is a powerful aid to understanding molecules. The chapter then explains that molecular dynamics (MD) simulations are often incorporated in structure determination procedures for nuclear magnetic resonance (NMR) and X-ray crystallography.
This chapter elaborates on the concept and principles of diffraction. Diffraction arises from the elastic scattering of radiation in directions other than the incident direction. Moreover, diffracted waves can interact with each other and produce interference patterns which are most informative when the wavelength of the applied radiation is less than or equal to the dimensions of the diffracting object. The diffraction patterns can be interpreted directly to give information about the size of the unit cell, the symmetry of the molecule, and information about periodicity. The chapter then discusses the experiments, techniques, and achievements of crystallography while also detailing the interpretation of diffraction data that observe molecular structures.
Diffusion, osmosis, viscosity, and friction
This chapter discusses the notion of diffusion, osmosis, viscosity, and friction. It explains that analysing the movement provides valuable information about many macromolecular properties including ion channels in membranes. Diffusion results in a net flux of molecules that reduce a concentration gradient to zero after some time. Meanwhile, osmosis is diffusional flow in a direction that reduces a concentration gradient across a semi-permeable barrier. In the Donnan effect, changes in the impermeable molecules can influence osmotic pressure. The chapter defines viscosity as a fluid's internal resistance to flow, referencing how the viscosity coefficient is sensitive to macromolecular size and shape.
Dipoles, dipole-dipole interactions, and spectral effects
This chapter contains a tutorial surrounding dipoles, dipole-dipole interactions, and spectral effects. It explains that an electric dipole consists of two charges separated by a distance before considering a general interaction between two dipoles and their correlating energy. Interactions between transition dipole moments have important effects on optical spectra and magnetic resonance spectra, which can be computed from equations for dipolar interactions. The chapter then looks into the frequency of the chromophore-containing monomer spectrum and the optical activity of a molecule involving both electric and magnetic transition dipole moments. It also considers the worked examples that involved proteins and angles.
This chapter provides a tutorial on electrical circuits that features several basic ideas and nomenclature. It looks into a figure that features some of the nomenclature of an electrical circuit that consists of a battery with positive and negative poles, a resistor, and a capacitor. The unit used to measure the electric potential difference is the volt, while the resistance of a conductor is represented as a resistor. The chapter considers how circuit analysis can be done with simple rules, which include resistors being in parallel. It also considers a worked example of the total resistance and current in a provided circuit figure.
This chapter provides a tutorial that showcases the basic properties of electromagnetic radiation. It explains how James Clerk Maxwell combined the laws of electricity and magnetism with those of light to develop the theory of electromagnetic radiation before proposing that electromagnetic radiation behaves as two wave motions at right angles. Moreover, electromagnetic waves are generated by oscillating electric or magnetic dipoles and are propagated through a vacuum at the velocity of light. The chapter discusses the notion of polarization and types of electromagnetic radiation, such as radiofrequency and X-rays. It then looks into interconversion between scales, which involves frequency, wavelength, velocity of propagation, and electron volt.
Electron paramagnetic resonance
This chapter explores the key concepts of electron paramagnetic resonance (EPR). It notes that EPR, also known as electron spin resonance (ESR), detects unpaired electrons, which are relatively rare in biological systems but they occur in important processes like free radicals in photosynthesis. Additionally, the utilization of EPR helps in the study of the structure and function of numerous paramagnetic metalloproteins. The chapter then details the spectral parameters of an EPR signal, which ranges between intensity, linewidth, g-value, and multiplet structure. It considers several biological applications of EPR, such as the exploration of the ligand environment around a metal site in a metalloprotein.
This chapter discusses electrophoresis that exploits the differential mobility of molecular ions in an applied electric field. Due to its versatility, electrophoresis became a major tool for the separation and characterization of proteins and nucleic acids with a wide range of migration media. Meanwhile, gel electrophoresis is in widespread use for separating electrically charged molecules while also separating and purifying DNA and RNA and characterizing protein preparations. The capture cites polyacrylamide and agarose as useful gel support media. It also mentions that capillary electrophoresis is useful for analyzing small molecules, while isoelectric focusing (IEF) separates proteins based on their pI value.
This chapter covers the application of electrophysiology, which is the study of the electrical properties of biological cells and tissues. It details the key concepts of membrane potential, action potentials, voltage clamp, and patch clamp. Electrical activity in cells and tissues results from the movement of ions through ion channels in membranes. Meanwhile, an action potential is generated by the opening and closing of gated ion channels. Moreover, different cells have different patterns of action potential firing. The chapter explains how the firing rate of a neuron depends on the stimulus level, while the propagation of signal along a neuron is modelled through cable theory.
This chapter notes how fluorescence occurs when light is emitted from an excited state. It considers the measurable parameters in fluorescence, such as excitation and emission spectra. Since fluorescence is very sensitive to the environment and molecular motion, fluorescence probes are widely used as sensitive indicators, such as in DNA sequencing, immunofluorescence, cell sorting, and microscopy. The chapter then explains that the physical picture of fluorescence comes from considering the energy levels involved in electronic transitions. It also looks into the emission process called phosphorescence which can be used to measure relatively slow motions while also having a longer lifetime than fluorescence.
Fourier series, Fourier transforms, and convolution
This chapter examines the mathematical concepts of Fourier series, Fourier transforms, and convolution. In 1804, Joseph Fourier introduced the idea that a periodic (repeating) function F(t) may be represented by an infinite series of sine and cosine functions. The chapter then presents several illustrations to highlight changes and representations of the Fourier series and Fourier transform (FT), which also includes a decaying oscillation known as a Lorentzian function. It also considers the Lorentizian function in correlation to the convolution used to change line shapes in spectroscopy. The tutorial's worked example involves an exponentially decaying cosine wave with a decay constant.
Infrared and Raman spectroscopy
This chapter details the features and application of infrared and Raman spectroscopy. It mentions how molecules undergo vibrational motion when their bonds stretch or bend, which corresponds to the infrared (IR) region of the electromagnetic spectrum. Thus, vibrations can be detected directly by measuring an IR spectrum or indirectly by Raman light scattering. In biological applications, IR and Raman spectra are utilized in many ways, such as identifying molecules, giving information about protein conformation, and defining ionization states. The chapter also considers the application of both spectroscopy to achieve the measurement of ligand binding to macromolecules and probing hydrogen bonds and hydrogen-deuterium exchange.
This chapter talks about the identification of molecules, the measurement of their concentrations and interaction partners, and the definition of their structure and location in the cell. It reviews extraordinarily powerful sets of physical and mathematical tools that can be used to study molecules. It also analyses molecular assemblies which need appropriate dynamic and energetic properties and the correct location and structure in order to function properly. The chapter describes diverse, powerful, and complementary biophysical tools, which exploit developments in technology and apply insights from the laws of physics and chemistry to all aspects of molecular and cellular biology. It reviews the key role of biophysical techniques in the exploration of other properties of cellular components.
Introduction to absorption and emission spectra
This chapter provides an overview of absorption and emission spectra. It starts with the quantum mechanics needed to understand the transitions between energy levels. Absorption can be measured by plotting the amount of energy absorbed by the sample as a function of the kind of energy applied. Meanwhile, the emission of radiation can occur when a molecule changes from an excited energy state to a lower energy state. Transitions between energy levels depend on the transition dipole moment, a property related to the change in charge distribution. The chapter then explains that molecules are distributed among energy levels at equilibrium achieved through relation processes, according to the Boltzmann law.