Abstract
The covalent chemistry of reactants tethered within a single protein pore can be monitored by observing the time-dependence of ionic current flow through the pore, which responds to bond making and breaking in individual reactant molecules. Here we use this ‘nanoreactor’ approach to examine the reaction of a quinone with a thiol to form a substituted hydroquinone by reductive 1,4-Michael addition. Remarkably, a primary hydrogen–deuterium isotope effect is readily detected at the single-molecule level during prototropic rearrangement of an initial adduct. The observation of individual reaction intermediates allows the measurement of an isotope effect whether or not the step involved is rate limiting, which would not be the case in an ensemble measurement.
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References
Moffitt, J. R., Chemla, Y. R., Smith, S. B. & Bustamante, C. Recent advances in optical tweezers. Annu. Rev. Biochem. 77, 205–228 (2008).
Bustamante, C. In singulo biochemistry: when less is more. Ann. Rev. Biochem. 77, 45–50 (2008).
Benkovic, S. J., Hammes, G. G. & Hammes-Schiffer, S. Free-energy landscape of enzyme catalysis. Biochemistry 47, 3317–3321 (2008).
Liang, J. & Fernandez, J. M. Mechanochemistry: One bond at a time. ACS Nano 3, 1628–1645 (2009).
Puchner, E. M., Franzen, G., Gautel, M. & Gaub, H. E. Comparing proteins by their unfolding pattern. Biophys. J. 95, 426–434 (2008).
Zhuang, X. Single-molecule RNA science. Annu. Rev. Biophys. Biomol. 34, 399–414 (2005).
Comellas-Aragones, M. et al. A virus-based single-enzyme nanoreactor. Nature Nanotechnol. 2, 635–639 (2007).
Liu, S., Abbondanzieri, E. A., Rausch, J. W., Le Grice, S. F. & Zhuang, X. Slide into action: dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates. Science 322, 1092–1097 (2008).
Yanagida, T., Iwaki, M. & Ishii, Y. Single molecule measurements and molecular motors. Phil. Trans. R. Soc. B 363, 2123–2134 (2008).
Park, H., Toprak, E. & Selvin, P. R. Single-molecule fluorescence to study molecular motors. Q. Rev. Biophys. 40, 87–111 (2007).
Brandenburg, B. & Zhuang, X. Virus trafficking - learning from single-virus tracking. Nat. Rev. Microbiol. 5, 197–208 (2007).
Xie, X. S., Choi, P. J., Li, G. W., Lee, N. K. & Lia, G. Single-molecule approach to molecular biology in living bacterial cells. Ann. Rev. Biophys. 37, 417–444 (2008).
Ho, W. Single-molecule chemistry. J. Chem. Phys. 117, 11033–11061 (2002).
Rao, B. V., Kwon, K. Y., Liu, A. & Bartels, L. Measurement of a linear free energy relationship one molecule at a time. Proc. Natl Acad. Sci. USA 101, 17920–17923 (2004).
Maksymovych, P., Sorescu, D. C., Jordan, K. D. & Yates, J. T., Jr. Collective reactivity of molecular chains self-assembled on a surface. Science 322, 1664–1667 (2008).
Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H. & Gaub, H. E. How strong is a covalent bond? Science 283, 1727–1730 (1999).
Garcia-Manyes, S., Liang, J., Szoszkiewicz, R., Kuo, T. L. & Fernández, J. M. Force-activated reactivity switch in a bimolecular chemical reaction. Nature Chem. 1, 236–242 (2009).
Roeffaers, M. B. et al. Spatially resolved observation of crystal face dependent catalysis by single turnover counting. Nature 439, 572–575 (2006).
Xu, W., Kong, J. S., Yeh, Y. T. & Chen, P. Single-molecule nanocatalysis reveals heterogeneous reaction pathways and catalytic dynamics. Nature Mater. 7, 992–996 (2008).
Bayley, H., Luchian, T., Shin, S. H. & Steffensen, M. B. in Single Molecules and Nanotechnology (eds. Rigler, R. & Vogel, H.) 251–277 (Springer, 2008).
Shin, S. H., Luchian, T., Cheley, S., Braha, O. & Bayley, H. Kinetics of a reversible covalent-bond forming reaction observed at the single molecule level. Angew. Chem. Int. Ed. 41, 3707–3709 (2002).
Luchian, T., Shin, S. H. & Bayley, H. Kinetics of a three-step reaction observed at the single molecule level. Angew. Chem. Int. Ed. 42, 1926–1929 (2003).
Luchian, T., Shin, S. H. & Bayley, H. Single-molecule covalent chemistry with spatially separated reactants. Angew. Chem. Int. Ed. 42, 3766–3771 (2003).
Shin, S. H. & Bayley, H. Stepwise growth of a single polymer chain. J. Am. Chem. Soc. 127, 10462–10463 (2005).
Loudwig, S. & Bayley, H. Photoisomerization of an individual azobenzene molecule in water: an on-off switch triggered by light at a fixed wavelength. J. Am. Chem. Soc. 128, 12404–12405 (2006).
Shin, S. H., Steffensen, M. B., Claridge, T. D. W. & Bayley, H. Formation of a chiral center and pyramidal inversion at the single-molecule level. Angew. Chem. Int. Ed. 46, 7412–7416 (2007).
Wu, H. C. & Bayley, H. Single-molecule detection of nitrogen mustards by covalent reaction within a protein nanopore. J. Am. Chem. Soc. 130, 6813–6819 (2008).
Zhang, Z., Rajagopalan, P. T., Selzer, T., Benkovic, S. J. & Hammes, G. G. Single-molecule and transient kinetics investigation of the interaction of dihydrofolate reductase with NADPH and dihydrofolate. Proc. Natl Acad. Sci. USA 101, 2764–2769 (2004).
Finley, K. T. in The Chemistry of Quinonoid Compounds (eds. Patai, S. & Rappoport, Z.) 878–1144 (John Wiley & Sons, 1974).
Jameson, G. N., Zhang, J., Jameson, R. F. & Linert, W. Kinetic evidence that cysteine reacts with dopaminoquinone via reversible adduct formation to yield 5 cysteinyl dopamine: an important precursor of neuromelanin. Org. Biomol. Chem. 2, 777–782 (2004).
Ogata, Y., Sawaki, Y. & Goto, S. Kinetics of the reaction of p-benzoquinone with sodium thiosulfate. J. Am. Chem. Soc. 90, 3469–3472 (1968).
Ogata, Y., Sawaki, Y. & Isono, M. Kinetics of the addition of benzenesulfinic acid to p-benzoquinone. Tetrahedron 25, 2715–2721 (1969).
Ogata, Y., Sawaki, Y. & Isono, M. Kinetics of the addition of substituted benzenesulfinic acids to p-benzoquinone. Tetrahedron 26, 731–736 (1970).
Li, W. W., Heinze, J. & Haehnel, W. Site-specific binding of quinones to proteins through thiol addition and addition-elimination reactions. J. Am. Chem. Soc. 127, 6140–6141 (2005).
Li, W. W., Hellwig, P., Ritter, M. & Haehnel, W. De novo design, synthesis, and characterization of quinoproteins. Chem. Eur. J. 12, 7236–7245 (2006).
Riddles, P. W., Blakeley, R. L. & Zerner, B. Ellman's reagent: 5,5'-dithio(2-nitrobenzoic acid)- a reexamination. Anal. Biochem. 94, 75–81 (1979).
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This work was supported by the MRC.
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S.L. and W-W.L. performed the kinetic measurements and analysed the data. W-W.L. synthesized deuterated UQ (1). S.L. and E.M. made the PSH pore. S.L, W-W.L. and D.R. performed simulations using QuB. S.L., W-W.L. and H.B. planned the research and wrote the paper.
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Lu, S., Li, WW., Rotem, D. et al. A primary hydrogen–deuterium isotope effect observed at the single-molecule level. Nature Chem 2, 921–928 (2010). https://doi.org/10.1038/nchem.821
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DOI: https://doi.org/10.1038/nchem.821
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