title: 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.
authors: Laboratory of Physics of Living Matter, Institute of Physics of Biological Systems, School of Basic Sciences, École polytechnique fédérale de Lausanne, Lausanne, Switzerland.
authors: Livan
authors: L
authors: Alonso-Sarduy
authors: Laboratory of Statistical Biophysics, Institute of Theoretical Physics, School of Basic Sciences, École polytechnique fédérale de Lausanne, Lausanne, Switzerland.
authors: Paolo
authors: P
authors: De Los Rios
authors: Laboratory of Physics of Living Matter, Institute of Physics of Biological Systems, School of Basic Sciences, École polytechnique fédérale de Lausanne, Lausanne, Switzerland.
authors: Fabrizio
authors: F
authors: Benedetti
authors: Laboratory of Physics of Living Matter, Institute of Physics of Biological Systems, School of Basic Sciences, École polytechnique fédérale de Lausanne, Lausanne, Switzerland.
authors: Dusan
authors: D
authors: Vobornik
authors: Laboratory of Physics of Living Matter, Institute of Physics of Biological Systems, School of Basic Sciences, École polytechnique fédérale de Lausanne, Lausanne, Switzerland.
authors: Giovanni
authors: G
authors: Dietler
authors: Laboratory of Physics of Living Matter, Institute of Physics of Biological Systems, School of Basic Sciences, École polytechnique fédérale de Lausanne, Lausanne, Switzerland; Faculty of Biology and Medicine, Department of Fundamental Neurosciences, Lausanne University, Lausanne, Switzerland.
authors: Sandor
authors: S
authors: Kasas
authors: Laboratory of Physics of Living Matter, Institute of Physics of Biological Systems, School of Basic Sciences, École polytechnique fédérale de Lausanne, Lausanne, Switzerland; Istituto Superiore di Sanità, Rome, Italy.
authors: Giovanni
authors: G
authors: Longo
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000951:Antigens, Neoplasm / Q000737:chemistry*; D015374:Biosensing Techniques; D004250:DNA Topoisomerases, Type II / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry; D006801:Humans; D018625:Microscopy, Atomic Force; D036103:Nanotechnology; D011487:Protein Conformation
chemical_list: D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D004800:Enzymes, Immobilized; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II