Viscoelasticity of single folded proteins using dynamic atomic force microscopy

The advent of atomic force microscopy, along with optical tweezers, ushered in a new field of single molecule force spectroscopy, wherein the response of a single protein or a macromolecule to external mechanical perturbations is measured. Controlled forces ranging from pN to nN are applied to measure the unfolding force distribution of a single protein domain. In a clamp type experiment, the folded protein is subjected to a constant force to measure the unfolding time distribution. Simultaneously, there were efforts to measure the elastic and viscous response of a single domain by applying sinusoidal forces and measuring the resulting deformations produced in a bid to quantify its viscoelasticity. The deformation's phase lag with respect to the applied force provides the elastic and viscous response of the protein, akin to oscillatory rheology. Despite numerous technical advances in AFM, an artefact-free measurement of a folded protein's viscoelasticity largely remains a challenge. In this perspective, we review efforts to measure the viscoelasticity of proteins using dynamic AFM, identifying pitfalls that make these measurements elusive. Finally, we discuss a new promising method, which reported viscoelasticity of a folded protein and its implications for our understanding of protein dynamics and structural flexibility.