Covalent chemistry can be observed at the single-molecule level by using engineered protein pores as “nanoreactors”. By recording the ionic current driven through single engineered alpha-hemolysin (αHL) pores in a transmembrane potential, individual bond-making and bond-breaking steps that occur within the pore and perturb the current are monitored with sub-millisecond time-resolution. Recently, a variety of covalent reactions of small molecules have been observed by this approach including irreversible light-activated chemistry, multiple turnovers of reversible reactions, the turnover of normally irreversible reactions in a twocompartment system and a step-by- step polymerization. These single-molecule experiments are revealing information about fundamental chemical processes that cannot be extracted from ensemble measurements. Further, the approach can be used to examine the effects of the local environment on chemistry and catalysis, and to construct sensors for reactive molecules based on covalent chemistry rather than non-covalent binding interactions. Alternative approaches to small molecule covalent chemistry at the single-molecule level are described in the review, as well as the problems and present limitations of the nanoreactor approach.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Abbondanzieri, E. A., Greenleaf, W. J., Shaevitz, J. W., Landick, R., and Block, S. M. (2005). Direct observation of base-pair stepping by RNA polymerase. Nature 438, 460–465.
Baker, G. A., Baker, S. N., Pandey, S., and Bright, F. V. (2005). An analytical view of ionic liquids. The Analyst 130, 800–808.
Bard, A. J., and Fan, F.-R. F. (1996). Electrochemical detection of single molecules. Acc Chem Res 29, 572–578.
Bayley, H., and Cremer, P. S. (2001). Stochastic sensors inspired by biology. Nature 413, 226–230.
Bayley, H., and Jayasinghe, L. (2004). Functional engineered channels and pores. Mol Membrane Biol 21, 209–220.
Beckstein, O., and Sansom, M. S. (2004). The influence of geometry, surface character, and flexibility on the permeation of ions and water through biological pores. Phys Biol 1, 42–52.
Bezrukov, S. M., and Kasianowicz, J. J. (1993). Current noise reveals protonation kinetics and number of ionizable sites in an open protein ion channel. Phys Rev Lett 70, 2352–2355.
Blatz, A. L., and Magleby, K. L. (1986). Correcting single channel data for missed events. Biophys J 49, 967–980.
Braha, O., Walker, B., Cheley, S., Kasianowicz, J. J., Song, L., Gouaux, J. E., and Bayley, H. (1997). Designed protein pores as components for biosensors. Chem Biol 4, 497–505.
Braslavsky, I., Hebert, B., Kartalov, E., and Quake, S. R. (2003). Sequence information can be obtained from single DNA molecules. Proc Natl Acad Sci USA 100, 3960–3964.
Collinson, M. M., and Wightman, R. M. (1995). Observation of individual chemical reactions in solution. Science 268, 1883–1885.
Conti, M., Falini, G., and Samori, B. (2000). How strong is the coordination bond between a histidine tag and Ni-nitrilotriacetate? An experiment of mechanochemistry on single molecules. Angew Chem Int Ed Engl 39, 215–218.
Cymes, G. D., Ni, Y., and Grosman, C. (2005). Probing ion-channel pores one proton at a time. Nature 438, 975–980.
Düllmann, C. E., et alet al. (2002). Chemical investigation of hassium (element 108). Nature 418, 859–862.
Eckel, R., Ros, R., Decker, B., Mattay, J., and Anselmetti, D. (2005). Supramolecular chemistry at the single molecule level. Angew Chem Int Ed Engl 44, 484–488.
Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H., and Gaub, H. E. (1999). How strong is a covalent bond? Science 283, 1727–1730.
Gronheid, R., Stefan, A., Cotlet, M., Hofkens, J., Qu, J., Mullen, K., van der Auweraer, M., Verhoeven, J. W., and De Schryver, F. C. (2003). Reversible intramolecular electron transfer at the single-molecule level. Angew Chem Int Ed Engl 42, 4209–4214.
Gu, L.-Q., and Bayley, H. (2000). Interaction of the non-covalent molecular adapter, β-cyclodextrin, with the staphylococcal α-hemolysin pore. Biophys J 79, 1967–1975.
Gu, L.-Q., Braha, O., Conlan, S., Cheley, S., and Bayley, H. (1999). Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature 398, 686–690.
Gu, L.-Q., Cheley, S., and Bayley, H. (2001). Capture of a single molecule in a nanocavity. Science 291, 636–640.
Gu, L.-Q., Cheley, S., and Bayley, H. (2003). Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore. Proc Natl Acad Sci USA 100, 15498–15503.
Helveg, S., Lopez-Cartes, C., Sehested, J., Hansen, P. L., Clausen, B. S., Rostrup-Nielsen, J. R., Abild-Pedersen, F., and Norskov, J. K. (2004). Atomic-scale imaging of carbon nanofibre growth. Nature 427, 426–429.
Henzl, J., Mehlhorn, M., Gawronski, H., Rieder, K. H., and Morgenstern, K. (2006). Reversible cis-trans isomerization of a single azobenzene molecule. Angew Chem Int Ed Engl 45, 603–606.
Herschbach, D. (2000). Fifty years in physical chemistry: Homage to mentors, methods, and molecules. Annu Rev Phys Chem 51, 1–39.
Hille, B. (2001). Ion Channels of Excitable Membranes, 3rd edition, (Sunderland, MA, USA: Sinauer).
Hladky, S. B., and Haydon, D. A. (1970). Discreteness of conductance change in biomolecular lipid membranes in the presence of certain antibiotics. Nature 225, 451–453.
Ho, W. (2002). Single-molecule chemistry. J Chem Phys 117, 11033–11061.
Howorka, S., and Bayley, H. (2002). Probing distance and electrical potential within a protein pore with tethered DNA. Biophys J 83, 3202–3210.
Howorka, S., Movileanu, L., Lu, X., Magnon, M., Cheley, S., Braha, O., and Bayley, H. (2000). A protein pore with a single polymer chain tethered within the lumen. J Am Chem Soc 122, 2411–2416.
Jaikaran, D. C. J., and Woolley, G. A. (1995). Characterization of thermal isomerization at the single molecule level. J Phys Chem 99, 13352–13355.
Johansson, L., Gafvelin, G., and Arner, E. S. (2005). Selenocysteine in proteins-properties and biotechnological use. Biochim Biophys Acta 1726, 1–13.
Kang, X., Gu, L.-Q., Cheley, S., and Bayley, H. (2005). Single protein pores containing molecular adapters at high temperatures. Angew Chem Int Ed Engl 44, 1495–1499.
Kasianowicz, J. J., and Bezrukov, S. M. (1995). Protonation dynamics of the α-toxin channel from spectral analysis of pH-dependent current fluctuations. Biophys J 69, 94–105.
Kersey, F. R., Yount, W. C., and Craig, S. L. (2006). Single-molecule forces spectroscopy of bimolecular reactions: System homology in the mechanical activation of ligand substitution reactions. J Am Chem Soc 128, 3886–3887.
Kong, C. Y., and Muthukumar, M. (2005). Simulations of stochastic sensing of proteins. J Am Chem Soc 127, 18252–18261.
Lawrence, D. S. (2005). The preparation and in vivo applications of caged peptides and proteins. Curr Opin Chem Biol 9, 570–575.
Lee, A. I., and Brody, J. P. (2005). Single-molecule enzymology of chymotrypsin using water-in-oil emulsion. Biophys J 88, 4303–4311.
Lee, H. J., and Ho, W. (1999). Single-bond formation and characterization with a scanning tunneling microscope. Science 286, 1719–1722.
Lindström, U. (2002). Stereoselective organic reactions in water. Chem Rev 102, 2751–2772.
Loudwig, S., and Bayley, H. (2005). In Light-Activated Proteins: An Overview Dynamic Studies in Biology: Phototriggers, Photoswitches and Caged Biomolecules, M. Goeldner, and R. Givens, eds. (Weinheim, Germany: Wiley-VCH Verlag), pp. 253–304.
Lu, H. P., Xun, L., and Xie, X. S. (1998). Single-molecule enzymatic dynamics. Science 282, 1877–1882.
Luchian, T., Shin, S.-H., and Bayley, H. (2003a). Kinetics of a three-step reaction observed at the single molecule level. Angew Chem Int Ed 42, 1926–1929.
Luchian, T., Shin, S.-H., and Bayley, H. (2003b). Single-molecule covalent chemistry with spatially separated reactants. Angew Chem Int Ed 42, 3766–3771.
Mathé, J., Aksimentiev, A., Nelson, D. R., Schulten, K., and Meller, A. (2005). Orientation discrimination of single-stranded DNA inside the alpha-hemolysin membrane channel. Proc Natl Acad Sci U S A 102, 12377–12382.
Mayer, M., Kriebel, J. K., Tosteson, M. T., and Whitesides, G. M. (2003). Microfabricated teflon membranes for low-noise recordings of ion channels in planar lipid bilayers. Biophys J 85, 2684–2695.
Min, W., English, B. P., Luo, G., Cherayil, B. J., Kou, S. C., and Xie, X. S. (2005). Fluctuating enzymes: Lessons from single-molecule studies. Acc Chem Res 38, 923–931.
Mindell, J. A., Zhan, H., Huynh, P. D., Collier, R. J., and Finkelstein, A. (1994). Reaction of diphtheria toxin channels with sulfhydryl reagents: Observation of chemical reactions at the single molecule level. Proc Natl Acad Sci USA 91, 5272–5276.
Moczydlowski, E. (1986). In Single-Channel Enzymology Ion Channel Reconstitution, C. Miller, ed. (New York: Plenum Press), pp. 75–113.
Morrill, J. A., and MacKinnon, R. (1999). Isolation of a single carboxyl-carboxylate proton binding site in the pore of a cyclic nucleotide-gated channel. J Gen Physiol 114, 71–83.
Movileanu, L., and Bayley, H. (2001). Partitioning of a polymer into a nanoscopic protein pore obeys a simple scaling law. Proc Natl Acad Sci USA 98, 10137–10141.
Movileanu, L., Cheley, S., and Bayley, H. (2003). Partitioning of individual flexible polymers into a nanoscopic protein pore. Biophys J 85, 897–910.
Movileanu, L., Cheley, S., Howorka, S., Braha, O., and Bayley, H. (2001). Location of a constriction in the lumen of a transmembrane pore by targeted covalent attachment of polymer molecules. J Gen Physiol 117, 239–251.
Movileanu, L., Howorka, S., Braha, O., and Bayley, H. (2000). Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore. Nature Biotechnology 18, 1091–1095.
Muir, T. (2003). Semisynthesis of proteins by expressed protein ligation. Ann Rev Biochem 72, 249–289.
Musyanovych, A., Mailander, V., and Landfester, K. (2005). Miniemulsion droplets as single molecule nanoreactors for polymerase chain reaction. Biomacromolecules 6, 1824–1828.
Nitzan, A., and Ratner, M. A. (2003). Electron transport in molecular wire junctions. Science 300, 1384–1389.
Noskov, S. Y., Im, W., and Roux, B. (2004). Ion permeation through the alpha-hemolysin channel: Theoretical studies based on Brownian dynamics and Poisson-Nernst-Planck electrodiffusion theory. Biophys J 87, 2299–2309.
Otero, R., Rosei, F., and Besenbacher, F. (2006). Scanning tunneling microscopy manipulation of complex organic molecules on solid surfaces. Annu Rev Phys Chem 57, 497–525.
Paula, S., Akeson, M., and Deamer, D. (1999). Water transport by the bacterial channel α-hemolysin. Biochim Biophys Acta 1418, 117–126.
Pietrobon, D., Prod’hom, B., and Hess, P. (1988). Conformation changes associated with ion permeation in L-type calcium channels. Nature 333, 373–376.
Pietrobon, D., Prod’hom, B., and Hess, P. (1989). Interactions of protons with single open L-type calcium channels: pH Dependence of proton-induced current fluctuations with Cs+, K+, and Na+ as permeant ions. J Gen Physiol 94, 1–21.
Prod’hom, B., Pietrobon, D., and Hess, P. (1987). Direct measurement of proton transfer rates to a group controlling the dihydropyridine-sensitive Ca2+ channel. Nature 329, 243–246.
Prod’hom, B., Pietrobon, D., and Hess, P. (1989). Interactions of protons with single open L-type calcium channels: Location of protonation site and dependence of proton-induced current fluctuations on concentration and species of permeant ion. J Gen Physiol 94, 23–42.
Raushel, F. M., Thoden, J. B., and Holden, H. M. (2003). Enzymes with molecular tunnels. Acc Chem Res 36, 539–548.
Raviv, U., Perkin, S., Laurat, P., and Klein, J. (2004). Fluidity of water confined down to subnanometer films. Langmuir 20, 5322–5332.
Richard, E. A., and Miller, C. (1990). Steady-state coupling of ion-channel conformations to a transmembrane ion gradient. Science 247, 1208.
Roeffaers, M. B., Sels, B. F., Uji, I. H., De Schryver, F. C., Jacobs, P. A., De Vos, D. E., and Hofkens, J. (2006). Spatially resolved observation of crystal-face-dependent catalysis by single turnover counting. Nature 439, 572–575.
Rondelez, Y., Tresset, G., Tabata, K. V., Arata, H., Fujita, H., Takeuchi, S., and Noji, H. (2005). Microfabricated arrays of femtoliter chambers allow single molecule enzymology. Nat Biotechnol 23, 361–365.
Root, M. J., and MacKinnon, R. (1994). Two identical noninteracting sites in an ion channel revealed by proton transfer. Science 265, 1852–1856.
Sakata, T., Yan, Y., and Marriott, G. (2005). Optical switching of dipolar interactions on proteins. Proc Natl Acad Sci U S A 102, 4759–4764.
Schadel, M. (2006). Chemistry of superheavy elements. Angew Chem Int Ed Engl 45, 368–401.
Selzer, Y., and Allara, D. L. (2006). Single-molecule electrical junctions. Annu Rev Phys Chem 57, 593–623.
Shapovalov, G., and Lester, H. A. (2004). Gating transitions in bacterial ion channels measured at 3 microseconds resolution. J Gen Physiol 124, 151–161.
Shilov, I. Y., and Kurnikova, M. G. (2003). Energetics and dynamics of a cyclic oligosaccharide molecule in a confined protein pore environment. A molecular dynamics study. J Phys Chem B 107, 7189–7201.
Shin, S.-H., and Bayley, H. (2005). Stepwise growth of a single polymer chain. J Am Chem Soc 127, 10462–10463.
Shin, S.-H., Luchian, T., Cheley, S., Braha, O., and Bayley, H. (2002). Kinetics of a reversible covalent–bond forming reaction observed at the single molecule level. Angew Chem Int Ed 41, 3707–3709.
Spuches, A. M., Kruszyna, H. G., Rich, A. M., and Wilcox, D. E. (2005). Thermodynamics of the As(III)-thiol interaction: Arsenite and monomethylarsenite complexes with glutathione, dihydrolipoic acid, and other thiol ligands. Inorganic Chemistry 44, 2964–2972.
Stauffer, D. A., and Karlin, A. (1994). The electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates. Biochemistry 33, 6840–6849.
Wang, L., and Schultz, P. G. (2004). Expanding the genetic code. Angew Chem Int Ed Engl 44, 34–66.
Woolley, G. A., Jaikaran, A. S. I., Zhang, Z., and Peng, S. (1995). Design of regulated ion channels using measurements of cis-trans isomerization in single molecules. J Am Chem Soc 117, 4448–4454.
Woolley, G. A., Zunic, V., Karanicolas, J., Jaikaran, A. S. I., and Starostin, A. V. (1997). Voltage-dependent behavior of a ball-and-chain gramicidin channel. Biophys J 73, 2465–2475.
Yasuda, S., Nakamura, T., Matsumoto, M., and Shigekawa, H. (2003). Phase switching of a single isomeric molecule and associated characteristic rectification. J Am Chem Soc 125, 16430–16433.
Zangi, R., and Mark, A. E. (2003a). Bilayer ice and alternate liquid phases of confined water. J Chem Phys 119, 1694–1700.
Zangi, R., and Mark, A. E. (2003b). Monolayer ice. Phys Rev Lett 91, 025502–025501 to 025502–025504.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Bayley, H., Luchian, T., Shin, SH., Steffensen, M.B. (2008). Single-Molecule Covalent Chemistry in a Protein Nanoreactor. In: Rigler, R., Vogel, H. (eds) Single Molecules and Nanotechnology. Springer Series in Biophysics, vol 12. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-540-73924-1_10
Download citation
DOI: https://doi.org/10.1007/978-3-540-73924-1_10
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-540-73923-4
Online ISBN: 978-3-540-73924-1
eBook Packages: Physics and AstronomyPhysics and Astronomy (R0)