Viscoelasticity of a Single Folded Protein

Abstract

The nanomechanical response of a protein, the natural nanomachine responsible for myriad biological processes, provides insight into its function. The conformational flexibility of a folded state, characterised by its viscoelasticity, allows proteins to adopt different conformations to perform their function. We present a direct and simultaneous measurement of the stiffness and internal friction of the Ig27 domain from the giant muscle protein titin using an interferometer based Atomic Force Microscope (AFM). Despite efforts and technological advances in AFM, since its discovery in 1986, accurate viscoelastic measurements on proteins were not possible. Apart from the probe being ∼ 104 times larger than the protein, its own hydrodynamics can cause friction measurements to be riddled with artifacts. To circumvent these issues we performed the experiments at off-resonance regime of the cantilever’s frequency response. This places stringent constraints on the type of cantilevers that can be used. To perform true off-resonance dynamic atomic force microscopy experiments it becomes necessary to use cantilevers with high stiffness and resonance frequency. This reduces their force sensitivity, ie, the bending in the cantilever due to force. To overcome this challenge interferometer based detection system was implemented to measure the cantilever displacement directly. Since the force spectroscopy experiments are done with octamers and linkers in-between them, to extract the viscoelasticity of a folded protein, modelling of the cantilever-protein system was done. The proposed model was validated with the experimental measurements for the different cases one encounters during the experiment. After which, the model was used to extract the viscoelasticity of the folded domains using the new analysis method. We observe that above 95 pN of force, the protein Ig27 transitions from an elastic solid-like native state to a soft viscoelastic intermediate. Finally, we will discussing a two state model that we are currently developing, for explaining the phase lag observed in the response of the protein in terms of the intrinsic rates and energy landscape parameters. This will help us gain insight into the microscopic origin of viscoelasticity observed in our experiments.