Real-time monitoring of protein conformational changes using a nano-mechanical sensor

Abstract

Proteins can switch between different conformations in response to stimuli, such as pH or temperature variations, or to the binding of ligands. Such plasticity and its kinetics can have a crucial functional role, and their characterization has taken center stage in protein research. As an example, Topoisomerases are particularly interesting enzymes capable of managing tangled and supercoiled double-stranded DNA, thus facilitating many physiological processes. In this work, we describe the use of a cantilever-based nanomotion sensor to characterize the dynamics of human topoisomerase II (Topo II) enzymes and their response to different kinds of ligands, such as ATP, which enhance the conformational dynamics. The sensitivity and time resolution of this sensor allow determining quantitatively the correlation between the ATP concentration and the rate of Topo II conformational changes. Furthermore, we show how to rationalize the experimental results in a comprehensive model that takes into account both the physics of the cantilever and the dynamics of the ATPase cycle of the enzyme, shedding light on the kinetics of the process. Finally, we study the effect of aclarubicin, an anticancer drug, demonstrating that it affects directly the Topo II molecule inhibiting its conformational changes. These results pave the way to a new way of studying the intrinsic dynamics of proteins and of protein complexes allowing new applications ranging from fundamental proteomics to drug discovery and development and possibly to clinical practice.

Figure 1. The experimental setup and the experiment using high Topo II concentration (107.6 nM).

Panel a: Schematic illustration of the experimental setup: C) cantilever, L) laser beam, D) photodiode. Topo II indicates the molecules adsorbed to both sides of the cantilever. Panel b: The cantilever deflections as a function of ATP concentration. Different media were flowed through the analysis chamber: buffer (with no ATP), ATP-enriched medium, containing in order 0.2 µM, 2.0 µM, 0.02 mM, 0.2 mM and 2.0 mM ATP, and then again the no-ATP buffer. Panel c: Corresponding variance values.

Figure 2. The model analysis.

Power spectral density plot of the deflection data for different ATP concentrations (2.0 µM – black squares, 0.2 mM – black circles and 2.0 mM – black triangles) for a 107.7 nM Topo II preparation concentration. The white squares indicate the buffer data that were used as baseline. The data for the 2.0 mM concentration were shifted horizontally (by a multiplicative factor of 3) to enhance readability. For each curve the corresponding fit from the model is shown.

Figure 3. The Aclarubicin experiment.

The cantilever deflections as different media were flowed through the analysis chamber: buffer, an ATP-enriched solution (20 µM), and finally a solution containing both ATP (20 µM) and Aclarubicin (100 µM). The variance values were calculated from five independent experiments.