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

Livan Alonso-Sarduy,Paolo De Los Rios,Dusan Vobornik,Giovanni Longo,Giovanni Dietler,Fabrizio Benedetti,Sandor Kasas,Etienne Dague

doi:10.1371/journal.pone.0103674

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.

Highlights

Monitoring protein activity is of paramount importance in several domains of biology and medicine, such as proteomics [1,2], investigation of biomolecular interactions [3,4] or drug development [5]
In absence of DNA, the basal ATPase rates of topoisomerase II (Topo II) are less than one order of magnitude lower than the DNA-stimulated rates and follow a standard Michaelis-Menten kinetic model [36]
These results indicate that cantilever fluctuations are the result of the combination of energy dissipation (ATP hydrolysis) associated to conformational changes in the molecule

Summary

Introduction

Monitoring protein activity is of paramount importance in several domains of biology and medicine, such as proteomics [1,2], investigation of biomolecular interactions [3,4] or drug development [5]. Human Topoisomerase type II (Topo II) is a interesting enzyme capable, through the hydrolysis of ATP, of managing tangled and supercoiled double-stranded DNA by changing its topology, facilitating numerous physiological processes such as gene expression, cell division, transcription or duplication [8]. Because of this it is often employed as target for anticancer drugs [9] and disparate techniques have been used to characterize its conformational changes [10,11,12]. This hampers their scalability, introduces greater complications in the assessment of the intrinsic kinetics of proteins and of how they are affected by different environmental conditions and ligands, e.g. drugs acting at the molecular level

Methods

Results

Conclusion