Direct Measurement of the Diffusion Dynamics of an Extended Poly-Ubiquitin Under Constant Force

Abstract

Force Spectroscopy is a technique in which single proteins are probed under mechanical perturbation. The force-length course measured in force spectroscopy is commonly described in terms of a diffusive process over a one dimensional potential of mean force, which reflects the end to end motion of the protein. Accordingly, the diffusion coefficient of a protein or polypeptide over its potential of mean force, D, is a basic property that relates the detected kinetics to the applied load. So far, D had been calculated from measurements taken in bulk, where the molecule is free to diffuse without any applied mechanical loads. The reported values indicate rapid dynamics that cannot explain the substantially slower timescales observed by a collapse trajectory of a single molecule under force. To this end we built a fast AFM apparatus with an improved characteristic time response of ∼100 μs. Using this novel setup we pulled on poly-ubiquitin, which has a distinct fingerprint, and then applied a force quench protocol between 250 and 100 pN to probe its recoiling dynamics. We fitted this data with a high force approximation analytical expression, which was verified using Brownian Dynamics, to measure the value of D for the recoiling traces. We report here for the first time an averaged value of D = 1374 ± 222 nm2/s, which is interestingly about five orders of magnitude smaller than the ones measured in bulk. The value of D measured here accounts for the observed slow recoiling timescales (∼1 ms) of a single poly-ubiquitin under mechanical load. Moreover, this value is significant when describing elastic systems where proteins are bound on both sides and still go through conformational changes.