Atomic Force Microscopy: Single Molecule Imaging and Force Spectroscopy in the Study of Flavoproteins Ligand Binding and Reaction Mechanisms
Atomic Force Microscopy (AFM) is one of the most versatile tools currently used in nanoscience. AFM allows performing non-destructive imaging of almost any sample in either air or liquid, regardless whether the specimen is insulating, conductive, transparent, or opaque. It also allows measuring interaction forces between a sharp probe and a sample surface, therefore, allowing to probe nanomechanical properties of the specimen by either applying a controlled force or pulling the sample. It can provide topography, mechanical, magnetic and conductive maps for very different type of samples. Transferred to the field of biology, today, AFM is the only microscopy technique able to produce images from biomolecules to bacteria and cells with nanometric resolution in aqueous media. Here, we will focus on the biological applications of AFM to flavoproteins. Despite references in the literature are scarce in this particular field, here it is described how imaging with AFM can contribute to describe catalysis mechanisms of some flavoenzymes, how oxidation states or binding of relevant ligands influence the association state of molecules, the dynamics of functional quaternary assemblies, and even visualize structural differences of individual protein molecules. Furthermore, we will show how force spectroscopy can be used to obtain the kinetic parameters, the dissociation landscape and the mechanical forces that maintain flavoprotein complexes, including the possibility to specifically detect particular flavoproteins on a sample.