Structure plays a key role in protein’s function. Characterization of newly isolated or genetically modified proteins therefore often begins by verification of their proper folding. Spectroscopic techniques represent the first-choice methods used to analyse the conformational behaviour of biomolecules under different conditions. They are also used for comparison of the structural properties of related molecules such as homologous or mutant forms of proteins. Furthermore, spectroscopic methods are used to determine the structural stability of proteins and to monitor the kinetics of their structural transitions under different physicochemical conditions (temperature, pH, chemical denaturants or organic co-solvents). An overview of spectroscopic techniques used for analysis of secondary and tertiary structure of proteins will be presented in the lecture. Main attention will be paid to the circular dichroism (CD) spectroscopy [1-3].
CD spectroscopy is a fast, quantitative and non-destructive spectroscopic technique where the CD of molecules is measured over a range of wavelengths. CD spectroscopy is used to study chiral molecules of all types and sizes. A primary use is in analysing the secondary structure or conformation of macromolecules, particularly proteins. As protein secondary structure is sensitive to its environment, CD can be used to observe how secondary structure changes with environmental conditions or interaction with other molecules. Structural, kinetic and thermodynamic information about macromolecules can be derived from CD spectroscopy. The principle of CD spectroscopy, attainable types of structural information, experimental aspects, advantages/disadvantages and specific examples of applications will be introduced and discussed in this lecture.