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.

Direct and Simultaneous Measurement of the Stiffness and Internal Friction of a Single Folded Protein.

The nanomechanical response of a folded single protein, the natural nanomachine responsible for myriad biological processes, provides insight into its function. The conformational flexibility of a folded state, characterized by its viscoelasticity, allows proteins to adopt different shapes to perform their function. Despite efforts, its direct measurement has not been possible so far. We present a direct and simultaneous measurement of the stiffness and internal friction of the folded domains of the protein titin using a special interferometer based atomic force microscope. We analyzed the data by carefully separating different contributions affecting the response of the experimental probe to obtain the folded state's viscoelasticity. Above ∼95 pN of force, the individual immunoglobulins of titin transition from an elastic solid-like native state to a soft viscoelastic intermediate.

Quantitative Elasticity of Flexible Polymer Chains Using Interferometer-Based AFM.

We estimate the elasticity of single polymer chains using atomic force microscope (AFM)-based oscillatory experiments. An accurate estimate of elasticity using AFM is limited by assumptions in describing the dynamics of an oscillating cantilever. Here, we use a home-built fiber-interferometry-based detection system that allows a simple and universal point-mass description of cantilever oscillations. By oscillating the cantilever base and detecting changes in cantilever oscillations with an interferometer, we extracted stiffness versus extension profiles for polymers. For polyethylene glycol (PEG) in a good solvent, stiffness-extension data showed significant deviation from conventional force-extension curves (FECs) measured in constant velocity pulling experiments. Furthermore, modeling stiffness data with an entropic worm-like chain (WLC) model yielded a persistence length of (0.5 ± 0.2 nm) compared to anomaly low value (0.12 nm ± 0.01) in conventional pulling experiments. This value also matched well with equilibrium measurements performed using magnetic tweezers. In contrast, polystyrene (PS) in a poor solvent, like water, showed no deviation between the two experiments. However, the stiffness profile for PS in good solvent (8M Urea) showed significant deviation from conventional force-extension curves. We obtained a persistence length of (0.8 ± 0.2 nm) compared to (0.22 nm ± 0.01) in pulling experiments. Our unambiguous measurements using interferometer yield physically acceptable values of persistence length. It validates the WLC model in good solvents but suggests caution for its use in poor solvents.

Validity of point-mass model in off-resonance dynamic atomic force microscopy.

The quantitative measurement of viscoelasticity of nano-scale entities is an important goal of nanotechnology research and there is considerable progress with advent of dynamic atomic force microscopy. The hydrodynamics of cantilever, the force sensor in AFM measurements, plays a pivotal role in quantitative estimates of nano-scale viscoelasticity. The point-mass (PM) model, wherein the AFM cantilever is approximated as a point-mass with mass-less spring is widely used in dynamic AFM analysis and its validity, particularly in liquid environments, is debated. It is suggested that the cantilever must be treated as a continuous rectangular beam to obtain accurate estimates of nano-scale viscoelasticity of materials it is probing. Here, we derived equations, which relate stiffness and damping coefficient of the material under investigation to measured parameters, by approximating cantilever as a point-mass and also considering the full geometric details. These equations are derived for both tip-excited as well as base-excited cantilevers. We have performed off-resonance dynamic atomic force spectroscopy on a single protein molecule to investigate the validity of widely used PM model. We performed measurements with AFMs equipped with different cantilever excitation methods as well as detection schemes to measure cantilever response. The data was analyzed using both, continuous beam model and the PM model. We found that both models yield same results when the experiments are performed in truly off-resonance regime with small amplitudes and the cantilever stiffness is much higher than the interaction stiffness. Our findings suggest that a simple PM approximation based model is adequate to describe the dynamics, provided care is taken while performing experiments so that the approximations used in these models are valid.

The nano-scale viscoelasticity using atomic force microscopy in liquid environment.

We measured viscoelasticity of two nanoscale systems, single protein molecules and molecular layers of water confined between solid walls. In order to quantify the viscoelastic response of these nanoscale systems in liquid environment, the measurements are performed using two types of atomic force microscopes (AFMs), which employ different detection schemes to measure the cantilever response. We used a deflection detection scheme, available in commercial AFMs, that measures cantilever bending and a fibre-interferometer based detection which measures cantilever displacement. The hydrodynamics of the cantilever is modelled using Euler-Bernoulli equation with appropriate boundary conditions which accommodate both detection schemes. In a direct contradiction with many reports in the literature, the dissipation coefficient of a single octomer of titin I278 is found to be immeasurably low. The upper bound on the dissipation coefficient is 5 × 10-7 kg s-1, which is much lower than the reported values. The entropic stiffness of single unfolded domains of protein measured using both methods is in the range of 10 mN m-1. We show that in a conventional deflection detection measurement, the phase of the bending signal can be a primary source of artefacts in the dissipation estimates. It is recognized that the measurement of cantilever displacement, which has negligibly small phase lag due to hydrodynamics of the cantilever at low excitation frequencies, is better suited for ensuring artefact-free measurement of viscoelasticity compared to the measurement of the cantilever bending. Further, it was possible to measure dissipation in molecular layers of water confined between the tip and the substrate using fibre interferometer based AFM with similar experimental parameters. It confirms that the dissipation coefficient of a single I278 is below the detection limit of AFM. The results shed light on the discrepancy observed in the measured diffusional dynamics of protein collapse measured using Force spectroscopic techniques and single-molecule optical techniques.