Applications of fluorescence spectroscopy in protein conformational changes and intermolecular contacts.

ABSTRACT

Emission fluorescence is one of the most versatile and powerful biophysical techniques used in several scientific subjects. It is extensively applied in the studies of proteins, their conformations, and intermolecular contacts, such as in protein-ligand and protein-protein interactions, allowing qualitative, quantitative, and structural data elucidation. This review, aimed to outline some of the most widely used fluorescence techniques in this area, illustrate their applications and display a few examples. At first, the data on the intrinsic fluorescence of proteins is disclosed, mainly on the tryptophan side chain. Predominantly, research to study protein conformational changes, protein interactions, and changes in intensities and shifts of the fluorescence emission maximums were discussed. Fluorescence anisotropy or fluorescence polarization is a measurement of the changing orientation of a molecule in space, concerning the time between the absorption and emission events. Absorption and emission indicate the spatial alignment of the molecule's dipoles relative to the electric vector of the electromagnetic wave of excitation and emitted light, respectively. In other words, if the fluorophore population is excited with vertically polarized light, the emitted light will retain some polarization based on how fast it rotates in solution. Therefore, fluorescence anisotropy can be successfully used in protein-protein interaction investigations. Then, green fluorescent proteins (GFPs), photo-transformable fluorescent proteins (FPs) such as photoswitchable and photoconvertible FPs, and those with Large Stokes Shift (LSS) are disclosed in more detail. FPs are potent tools for the study of biological systems. Their versatility and wide range of colours and properties allow many applications. Finally, the application of fluorescence in life sciences is exposed, especially the application of FPs in fluorescence microscopy techniques with super-resolution that enables precise in vivo photolabeling to monitor the movement and interactions of target proteins.