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Experimental free-energy measurements of kinetic molecular states using fluctuation theorems

Nature Physics volume 8, pages 688–694 (2012)Cite this article

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Abstract

Recent advances in non-equilibrium statistical mechanics and single-molecule technologies have made it possible to use irreversible work measurements to extract free-energy differences associated with the mechanical (un)folding of molecules. To date, free-energy recovery has been focused on native (or equilibrium) molecular states, but free-energy measurements of kinetic states have remained unexplored. Kinetic states are metastable, finite-lifetime states that are generated dynamically, and play important roles in diverse physical processes. In biophysics, there are many examples in which these states determine the fate of molecular reactions, including protein binding, enzymatic reactions, as well as the formation of transient intermediate states during molecular-folding processes. Here we demonstrate that it is possible to obtain free energies of kinetic states by applying extended fluctuation relations, using optical tweezers to mechanically unfold and refold deoxyribonucleic acid (DNA) structures exhibiting intermediate and misfolded kinetic states.

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Figure 1: Schematic illustration of the configurational space, molecular free-energy landscapes and experimental set-up.
Figure 2: Hairpin I1, with an intermediate state.
Figure 3: Hairpin M1, with one misfolded state.
Figure 4: Hairpin I2, with two intermediate states.
Figure 5: Hairpin M2, with two misfolded states.

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Acknowledgements

A.A. is supported by grant AP2007-00995 (Spanish Research Council). A.M. acknowledges funding from Lundbeckfonden. I.J. is supported by a Novartis grant (CRG). F.R. is supported by grants FIS2010-19342, Icrea Academia 2008, and Human Frontier Science Program (RGP55-2008). We thank J. Horowitz and M. Palassini for a careful reading of the manuscript.

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Authors and Affiliations

  1. Small Biosystems Lab, Departament de Fı´sica Fonamental, Universitat de Barcelona, Avda. Diagonal 647, 08028 Barcelona, Spain

    Anna Alemany & Felix Ritort

  2. Department of Physics and Astronomy, University of Aarhus, DK-8000 Aarhus C, Ny Munkegade 120, Denmark

    Alessandro Mossa

  3. Centre de Regulació Genòmica (CRG), C/ Dr. Aiguader 88, 08003 Barcelona, Spain

    Ivan Junier

  4. CIBER-BBN de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, 28029 Madrid, C/ Sinesio Delgado 4, Spain

    Felix Ritort

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  1. Anna Alemany
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Contributions

I.J. and F.R. designed the experiment. A.A. made the measurements. A.A. and A.M. analysed the data. All authors wrote the paper.

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Correspondence to Felix Ritort.

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The authors declare no competing financial interests.

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Alemany, A., Mossa, A., Junier, I. et al. Experimental free-energy measurements of kinetic molecular states using fluctuation theorems. Nature Phys 8, 688–694 (2012). https://doi.org/10.1038/nphys2375

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