Single Molecule Analysis

Miguel Cantero, María Jesús Rodríguez-Espinosa, Klara Strobl, Pablo Ibáñez, Alejandro Díez-Martínez, Natalia Martín-González, Manuel Jiménez-Zaragoza, Alvaro Ortega-Esteban, Pedro José de Pablo

In atomic force microscopy (AFM), the probe is a nanometric tip located at the end of a microcantilever which palpates the specimen under study as a blind person manages a walking stick. In this way, AFM allows obtaining nanometric resolution images of individual protein shells, such as viruses, in liquid milieu. Beyond imaging, AFM also enables not only the manipulation of single protein cages but also the evaluation of each physicochemical property which is able of inducing any measurable mechanical perturbation to the microcantilever that holds the tip. In this chapter, we start revising some recipes for adsorbing protein shells on surfaces and how the geometrical dilation of tips can affect to the AFM topographies. This work also deals with the abilities of AFM to monitor TGEV coronavirus under changing conditions of the liquid environment. Subsequently, we describe several AFM approaches to study cargo release, aging, and multilayered viruses with single indentation and fatigue assays. Finally, we comment on a combined AFM/fluorescence application to study the influence of crowding on GFP packed within individual P22 bacteriophage capsids.