Single Molecule Methods to Study Flavoproteins
A variety of single-molecule techniques have been developed that provide new and important information on the mechanisms of biological systems. The trajectory of an individual molecule can be observed by following the fluorescence of the molecule itself, or that of a label tagged to it. Individual molecules can also be observed and manipulated by attaching them to an external probe so as to determine its position (or to act upon it) by using mechanical forces. Methods such as Atomic Force Microscopy or Optical Tweezers can be used for this purpose.
The emission of fluorescence of the flavin cofactor in flavoproteins can be used to provide information regarding structural changes that take place during the turnover process of an enzyme. It can also indicate the oxidation-reduction state of the protein during the catalytic cycle.
Molecular recognition between biological molecules is an area in which single-molecule manipulation techniques find especial interest. These methods can be used to analyze association patterns and to measure the forces that hold together molecular structures. These include protein-ligand interactions, such as those present in enzyme-substrate, protein-cofactor, protein-DNA, protein-protein or, even, protein folding.
Although flavoenzymes are good candidates to be studied with these techniques, the references in the literature on this area of research are scarce. This review intends to describe the importance and interest of these techniques, and illustrate their application to flavoenzymes specifically. An attempt is also made to point at specific aspects of processes involving flavoproteins in which single-molecule techniques can provide answers to specific mechanistic questions.