Viscoelasticity of single macromolecules using Atomic Force Microscopy

Abstract

We measured viscoelasticity of single protein molecules using two types of Atomic Force Microscopes (AFM) which employ different detection schemes to measure the cantilever response. We used a commonly available deflection detection scheme in commercial AFMs which measures cantilever bending and a fibre-interferometer based home-built AFM which measures cantilever displacement. For both methods, the dissipation coefficient of a single macromolecule is immeasurably low. The upper bound on the dissipation coefficient is 5 x 10-7 kg/s whereas the entropic stiffness of single unfolded domains of protein measured using both methods is in the range of 10 mN/m. We show that in a conventional deflection detection measurement, the phase of bending signal can be a primary source of artefacts in the dissipation estimates. It is recognized that the measurement of cantilever displacement, which does not have phase lag due to hydrodynamics of the cantilever, is better suited for ensuring artefact-free measurement of viscoelasticty compared to the measurement of the cantilever bending. We confirmed that the dissipation coefficient in single macromolecules is below the detection limit of AFM by measuring dissipation in water layers confined between the tip and the substrate using similar experimental parameters. Further, we experimentally determined the limits in which the simple point-mass approximation of the cantilever works in off-resonance operation. Significance StatementSingle Macromolecules, including unfolded proteins bear rubber-like entropic elasticity and internal friction characterized by finite dissipation coefficient. Direct measurement of this viscoelastic response is important since it plays a significant role, both in polymer physics as well as protein folding dynamics. The viscoelastic response of single polymer chain is difficult and prone to artefacts owing to the complications of hydrodynamics of macroscopic probe itself in the liquid environment. Using a special atomic force microscope, which allows quantitative estimate of viscoelasticity in liquid environments, we measured viscoelastic response of single molecule of Titin. We report here that the dissipation coefficient is below the detection limit of our experiments - with upper bound which is less than reported values in the literature.