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
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.