Single molecule biology: Coming of age - CiteSeerX
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www.rsc.org/molecularbiosystems | Molecular BioSystems
Single molecule biology: Coming of age Liming Ying DOI: 10.1039/b702845h
Cellular heterogeneity and stochastic fluctuation play key roles in biological processes. Single molecule approaches have the key advantage of avoiding ensemble averaging, allowing the observation of transient intermediates and heterogeneity (both static and dynamic). Thus they have revolutionised the way many biological questions are addressed. The challenge ahead is to develop integrated approaches such as the combination of single molecule imaging with single molecule manipulation to probe the dynamics of gene regulatory and cell signalling networks in living cells.
Introduction The key processes of life occur in the noisy, far from equilibrium cellular environment, which is characterised by heterogeneity and stochastic fluctuation. To unravel this complexity, biology requires new physical concepts and methodologies. One promising approach is measuring the properties and dynamics of biological molecules, macromolecular machines, and cellular properties at the single molecule level in both test-tubes and living cells. Around thirty years ago, an innovative method (using a glass micropipette to patch the cell membrane) to measure the electrical activity of a single ionic channel across a cell membrane was developed by Erwin Neher and Bert Sakmann.1 This patch-clamp technique has opened a new Biological Nanoscience Section, National Heart and Lung Institute, Imperial College London, London, UK SW7 2AZ. E-mail: [email protected]; Tel: 020 7594 3132
window for neurobiology and physiology research and the two scientists were subsequently awarded the Nobel Prize in 1991. The statistical analysis of the current flowing through the ionic channel provided unique and valuable information on the functional and dynamic states of these channels. This information had previously been buried or lost when one simply took measurements on the average current flowing through a large ensemble of channels. The patch-clamp technique was the first successful approach to taking measurements on single biological identities in living cells. The last decade has seen the development of several new tools for manipulating, visualising, and characterising single molecules and their interactions, and as a result many areas of biology are now undergoing transformation. Direct monitoring of conformations, movement, and interactions of individual biological molecules, molecular complexes and macromolecular machines as well as other biological events such as gene
Dr Liming Ying obtained his PhD in physical chemistry from Peking University in China. He was an AWU postgraduate fellow at the Pacific Northwest National Laboratory in Richland USA, working with Dr Sunney Xie in single molecule spectroscopy. After postdoctoral studies with Drs David Klenerman and Shankar Balasubramanian at the Department of Chemistry, University of Cambridge, he was awarded a BBSRC David Phillips Research Fellowship and continued his research at Cambridge. He recently joined Imperial College London. His research focuses on developing and applying novel single molecule approaches to address biological questions. Liming Ying This journal is ß The Royal Society of Chemistry 2007
expression has started to provide new temporal and spatial dynamic information not accessible with ensemble measurements. Single molecule biology is becoming a new paradigm of research in life sciences and an indispensable part of systems biology.
Why the single molecule approach in biology? Due to technical limitations and convenience, biological studies traditionally take averages over ensembles of cells or molecules. However, averaging is sometimes problematic as it may mask important individual variations in structural properties and dynamics at both the cellular and molecular levels. Single molecule approaches have the advantage of avoiding ensemble averaging, enabling observation of transient intermediates and heterogeneity (both static and dynamic). Hence they have revolutionised the way many biological questions are addressed. There are several fundamental and practical reasons behind the use of the single molecule approach. At the molecular level in test-tubes, one would like to ask mechanistic questions, such as how proteins fold and how macromolecular complexes and molecular motors work. In the living cell, one would like to address how proteins and molecular complexes interact with each other in the heterogeneous and fluctuating cellular environment. As we know, these molecular machines or motors cannot be synchronised in their functioning or operation, making detailed mechanistic investigation impossible with Mol. BioSyst., 2007, 3, 377–380 | 377
ensemble studies. Therefore it is necessary to examine molecular motors one by one, in analogy to the quality control procedures in the manufacturing of car engines. Protein folding is an emerging area demanding the single molecule approach.2 Molecular dynamics simulations of protein folding are classic single molecule theoretical ‘‘experiments,’’ providing atomic-resolution structural and dynamic information. However these unprecedented details are yet to be confirmed from single molecule measurements on individual proteins. Single molecule approaches can identify the distribution of microscopic pathways in protein folding and provide insights into protein misfolding and aggregation which are possible causes of a wide range of diseases, especially neurodegenerative disorders. It can also distinguish protein folding mechanisms.3 Biology is becoming increasingly quantitative as we enter the post-genomic era. It will be necessary to complement ensemble approaches with analyses of their individual units: single molecules in single cells to gain an integrative systematic level of understanding of complexity in biology. Cells are testtubes for the next level of experiments. In single cells, many proteins exist in low copy numbers. These proteins play important roles in the functioning of cells, including cell signalling and the regulation of gene expression. Without amplification procedures, their abundance is far below the sensitivity of conventional protein analysis methods. Therefore it is necessary to develop microfluidic devices in which manipulation, lysation, labelling, separation, and quantification of the protein contents of a single cell using single-molecule fluorescence counting can be carried out.4 On the other hand, ensemble kinetic measurements of reaction/signalling networks in a living cell is difficult partly because it involves low-copy-numbers of molecules and partly because individual processes in the network cannot be synchronised. Multi-colour single molecule imaging would allow several reactions/interactions to be observed and recorded simultaneously, thus allowing the measurements of reaction rate or binding constant in a single run. 378 | Mol. BioSyst., 2007, 3, 377–380
Current single molecule approaches Measurements on single molecules employ optical, electrical and mechanical detection. Most optical methods use single molecule fluorescence or fluorescence resonance energy transfer (FRET) via either one-photon or multi-photon excitation. A variety of single molecule fluorescence techniques have been developed over the last decade, notably singlepair FRET,5 two colour fluorescence coincidence6 and alternating laser excitation (ALEX).7 Single molecule fluorescence imaging, in particular, total-internal-reflection fluorescence (TIRF) microscopy (objective-type or prism-type)8 has drawn a great deal of interest in recent years. These techniques are now widely applied to study the structural heterogeneity and dynamics of protein and nucleic acids,9 the assembly of biomolecular complexes, the mechanism of enzymatic reactions,10 and finally protein–protein interactions in living cells.11 However, blinking and photobleaching of the fluorophores set a fundamental limit on the amount of fluorescence photons detectable and therefore observation time. For prolonged and multiplexed single molecule imaging, one may use quantum dots which offer better brightness and photostability. Engineered fluorescent proteins, especially those whose fluorescence can be photo-switched on and off, are finding increasing use in single molecule imaging in living cells. New optical approaches are continuing to emerge, offering optical resolution beyond the diffraction-limit, combined with single molecule sensitivity. Fluorescence switching is exploited most recently in stimulated emission depletion (STED) fluorescence microscopy,12 photo activated localisation microscopy (PALM)13 and stochastic optical reconstruction microscopy (STORM).14 These novel techniques are able to achieve a resolution of tens of nanometres. The most common electrical measurement on single molecules is the patch clamp for single ion channels on living cells. This has been extended to measure the ion current blockage when ssDNA passes through an engineered protein nanopore and nanofabricated solid-state nanopore.15 Individual bases can be
distinguished by the different current signature of each base as it passes and partly blocks the ion current. This method holds great promise for single molecule DNA or protein sequencing provided that the measurement bandwidth can be significantly improved in the future. In the mid-1990s the ability to directly measure interactive forces at the level of single biological macromolecules and/or their complexes was acquired. Single molecule force spectroscopy uses atomic force microscopy to stretch and record the force–extension curve of a biopolymer.16 This approach has been used to study the mechanical unfolding of the multi-unit proteins such as titin and the ligand receptor interaction. Apart from atomic force microscopy, other single molecule manipulation techniques include magnetic and optical tweezers,17 microfluidics/nanofluidics,18 and bioMEM/NEMs devices.19 These have been used to control biological reactions and functions and provide new routes to study biomolecular machinery. Statistical mechanics is an essential tool in understanding fluctuating biological machines and processes. For example, statistical analysis helps the biophysicist to overcome the noise in the single molecule signals to learn about the step size of molecular motors, their energy consumption, and the rate-limiting transitions in their enzymatic cycles. One can thus build and verify better models of how these enzymes function and compare with the well-established mechanism derived from ensemble observations. The ergodic hypothesis in statistical mechanics states that the average of a process parameter over time and the average over the statistical ensemble at a given time are the same. It assumes that it is as good to observe a process for a long time as sampling many independent realisations of the same process. For single molecule measurement on a homogenous sample, the average of a physical parameter over a sufficient long time trajectory from an individual molecule is equal to the statistical average of the same parameter from many single molecules at any time—the ensemble average. As heterogeneity and fluctuation are ubiquitous in biology, the significance of single molecule experiments lies in
This journal is ß The Royal Society of Chemistry 2007
their capability of capturing the static and dynamic heterogeneity of the biological system, and in underpinning microscopic insights of the fundamental mechanisms in the biological process. One marvellous example was given by Xie and his colleagues on the study of fluctuating single enzyme b-galactosidase molecules.20 It was observed that the memory effect, which in a dynamic system refers to the temporal dependency of a future prediction on the history, takes place at high substrate concentrations and it covers the time scale from a millisecond to a second due to the interconverting conformers with broadly distributed lifetimes. It was also found that the Michaelis–Menten equation which has been an essential tool for understanding enzyme kinetics since 1910s still holds even for a fluctuating single enzyme, but with a different microscopic interpretation: its apparent rate constant is in fact an average of the ever fluctuating conformations, as oppose to a well-defined one as conventionally thought. This might have considerable physiological consequences in a living cell where a low copy number of enzymes within a cellular compartment gives importance to the enzymatic fluctuations. How can one obtain equilibrium thermodynamic parameters from processes carried out far from equilibrium? In 1997 Jarzynski proved an equality which relates the irreversible work to the equilibrium free energy difference.21 This theorem was tested rigorously by mechanically stretching a single molecule of RNA reversibly and irreversibly between two conformations.22 The Jarzynski equality extends the thermodynamic analysis of single molecule manipulation data beyond the context of equilibrium experiments. Hence protein and RNA unfolding energy landscape can be extracted from single molecule pulling experiments.
complexes and their machinery. In order to fulfil these tasks, current single molecule imaging and manipulation techniques need to be integrated and improved. In the meantime chemists and protein biochemists need to develop optimised fluorophores, preferably inorganic or intrinsic such as fluorescent protein, to improve the photostability and brightness, for multiplexed single molecule detection and imaging. Gene expression influences most aspects of cellular behaviour, and its variation is responsible for the phenotypic differences in a population of cells. Because DNA, RNA, and proteins can be present and active at a few copies per cell, gene expression is an intrinsically stochastic process.23 Sunney Xie and coworkers24,25 have followed single protein expression events in vivo in an elegant set of studies. The work was done by two separate methods: in one study, the production of single molecules of a fluorescent, membrane-targeted fusion protein was visualised in live bacteria; while in the other, fluorescence generated by the enzyme b-galactosidase in a microfluidic system was used to track protein production in living cells. Their work has confirmed the stochasticity of gene expression at the single molecule level. Statistical analysis of the data
revealed that protein production occurs in stochastic bursts and that a variable number of expression events are included in each burst. The techniques described allow protein production to be monitored with unprecedented precision. The next step is to probe the expression of multiple genes simultaneously with the use of multiple reporters or monitor several components in gene regulatory networks with different colours synchronously, paving the way towards programming an individual cell. However, the challenge still remains for the study of gene expression in the eukaryotic cell at the single molecule level. Optical tweezers and fluorescence are two extensively employed single-molecule approaches. The combination of these two biophysical techniques in a single experiment offers a powerful tool for studying biomolecular systems, by allowing direct correlations to be made between nanoscale structural changes, reported by singlemolecule fluorescence, and biomechanical transitions, probed by piconewton forces generated with optical traps. Steven Block and coworkers26 have demonstrated the feasibility of this combination by simultaneously observing single-molecule fluorescence changes and force-induced strand separations in dye-labelled doublestranded DNA duplexes. This technique
Perspective for single molecule biology Looking into the future, the challenge ahead for single molecule biology is to probe the dynamics of gene expression and cellular communication at the single molecule level, which requires the dissection of the assembly, dynamics and mechanism of large biomolecular
Fig. 1 Schematic of the combination of single molecule manipulation with single molecule imaging. Fluorescence-tagged ligand molecules may be delivered to the living cell surface using a nanopipette with high temporal and spatial accuracy. The nanopipette is controlled by scanning ion-conductance microscopy. The motion of ligand bound receptors can then be tracked by single molecule imaging. (Courtesy of Dr Andreas Bruckbauer.)
This journal is ß The Royal Society of Chemistry 2007
Mol. BioSyst., 2007, 3, 377–380 | 379
has been improved recently by alternately modulating the optical trap and excitation beams to reduce the photobleaching of fluorescent dye.27 Trapping of individual biomolecules may be achieved by optical resonance28 or using the anti-Brownian electrokinetic (ABEL) scheme.29 Thus, observation times of single biomolecules could be greatly increased, allowing long trajectories of the conformational dynamics being taken without the scepticism of surface immobilisation approaches. Fluorescence technique may also unite with scanning ion-conductance microscopy (SICM),30 a special form of scanning probe microscopy which offers non-invasive imaging of living cells with high resolution and the capability of controlled delivery of single molecules onto the cell surface. A nanopipette may be used to deliver individual biomolecules through the tip of the pipette, which are then landed onto a specific site of the cell surface and bind to cell receptors, thus triggering a cascade of cell signalling events that could be followed by multicolour single molecule tracking, as illustrated in Fig. 1. This will allow the study of complex signalling networks with high spatial and temporal resolution at the single molecule level. A closely related technique, scanning electrochemical microscopy (SECM) with nanoelectrode, has been used to address and monitor local enzymatic activities on cell surfaces.31 Combined with single molecule FRET, proton-driven ATP synthesis by single membrane-bound F0F1-ATP synthases can be investigated.32 A growing number of integrated single molecule approaches and their applications in single living cell study are expected to arise in the coming years.
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Acknowledgements The author thanks David Klenerman, Xiaodong Zhang, Andreas Bruckbauer, Joe D. Piper, and Richard W. Clarke for critical reading of the manuscript. This work is supported by the BBSRC.
References 1 E. Neher and B. Sakmann, Single-channel currents recorded from membrane of denervated frog muscle-fibers, Nature, 1976, 260, 799–802. 2 X. Michalet, S. Weiss and M. Ja¨ger, Single-molecule fluorescence studies of
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protein folding and conformational dynamics, Chem. Rev., 2006, 106, 1785–1813. F. Huang, S. Sato, T. D. Sharpe, L. M. Ying and A. R. Fersht, Distinguishing between cooperative and unimodal downhill protein folding, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 123–127. B. Huang, H. K. Wu, D. Bhaya, A. Grossman, S. Granier, B. K. Kobilka and R. N. Zare, Counting low-copy number proteins in a single cell, Science, 2007, 315, 81–84. A. A. Deniz, M. Dahan, J. R. Grunwell, T. J. Ha, A. E. Faulhaber, D. S. Chemla, S. Weiss and P. G. Schultz, Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Forster distance dependence and subpopulations, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 3670–3675. H. T. Li, L. M. Ying, J. J. Green, S. Balasubramanian and D. Klenerman, Ultrasensitive coincidence fluorescence detection of single DNA molecules, Anal. Chem., 2003, 75, 1664–1670. A. N. Kapanidis, T. A. Laurence, N. K. Lee, E. Margeat, X. X. Kong and S. Weiss, Alternating-laser excitation of single molecules, Acc. Chem. Res., 2005, 38, 523–533. H. Schneckenburger, Total internal reflection fluorescence microscopy: technical innovations and novel applications, Curr. Opin. Biotechnol., 2005, 16, 13–18. T. Ha, Structural dynamics and processing of nucleic acids revealed by single-molecule spectroscopy, Biochemistry, 2004, 43, 4055–4063. W. Min, B. P. English, G. B. Luo, B. J. Cherayil, S. C. Kou and X. S. Xie, Fluctuating enzymes: lessons from singlemolecule studies, Acc. Chem. Res., 2005, 38, 923–931. Y. Sako and T. Yanagida, Single-molecule visualization in cell biology, Nat. Cell Biol., 2003, Suppl. S, SS1–SS5. T. A. Klar, S. Jakobs, M. Dyba, A. Egner and S. W. Hell, Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 8206–8210. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz and H. F. Hess, Imaging intracellular fluorescent proteins at nanometer resolution, Science, 2006, 313, 1642–1645. M. J. Rust, M. Bates and X. W. Zhuang, Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM), Nat. Methods, 2006, 3, 793–795. D. W. Deamer and D. Branton, Characterization of nucleic acids by nanopore analysis, Acc. Chem. Res., 2002, 35, 817–825. H. Clausen-Schaumann, M. Seitz, R. Krautbauer and H. E. Gaub, Force spectroscopy with single bio-molecules, Curr. Opin. Chem. Biol., 2000, 4, 524–530. C. Bustamante, S. B. Smith, J. Liphardt and D. Smith, Single-molecule studies of
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DNA mechanics, Curr. Opin. Struct. Biol., 2000, 10, 279–285. J. T. Mannion and H. G. Craighead, Nanofluidic structures for single biomolecule fluorescent detection, Biopolymers, 2007, 85, 131–143. H. Craighead, Future lab-on-a-chip technologies for interrogating individual molecules, Nature, 2006, 442, 387–393. B. P. English, W. Min, A. M. van Oijen, K. T. Lee, G. B. Luo, H. Y. Sun, B. J. Cherayil, S. C. Kou and X. S. Xie, Ever-fluctuating single enzyme molecules: Michaelis–Menten equation revisited, Nat. Chem. Biol., 2006, 2, 87–94. C. Jarzynski, Nonequilibrium equality for free energy differences, Phys. Rev. Lett., 1997, 14, 2690–2693. J. Liphardt, S. Dumont, S. B. Smith, I. Tinoco and C. Bustamante, Equilibrium information from nonequilibrium measurements in an experimental test of Jarzynski’s equality, Science, 2002, 296, 1832–1835. J. M. Raser and E. K. O’Shea, Noise in gene expression: Origins, consequences, a n d co n t r o l , S c i e n c e, 20 05, 30 9, 2010–2013. J. Yu, J. Xiao, X. J. Ren, K. Q. Lao and X. S. Xie, Probing gene expression in live cells, one protein molecule at a time, Science, 2006, 311, 1600–1603. L. Cai, N. Friedman and X. S. Xie, Stochastic protein expression in individual cells at the single molecule level, Nature, 2006, 440, 358–362. M. J. Lang, P. M. Fordyce, A. M. Engh, K. C. Neuman and S. M. Block, Simultaneous, coincident optical trapping and single-molecule fluorescence, Nat. Methods, 2004, 1, 133–139. R. R. Brau, P. B. Tarsa, J. M. Ferrer, P. Lee and M. J. Lang, Interlaced optical force-fluorescence measurements for single molecule biophysics, Biophys. J., 2006, 91, 1069–1077. H. T. Li, D. J. Zhou, H. Browne and D. Klenerman, Evidence for resonance optical trapping of individual fluorophore-labeled antibodies using single molecule fluorescence spectroscopy, J. Am. Chem. Soc., 2006, 128, 5711–5717. A. E. Cohen and W. E. Moerner, Suppressing Brownian motion of individual biomolecules in solution, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 4362–4365. L. M. Ying, A. Bruckbauer, D. J. Zhou, J. Gorelik, A. I. Shevchuk, M. Lab, Y. E. Korchev and D. Klenerman, The scanned nanopipette: a new tool for high resolution bioimaging and controlled deposition of biomolecules, Phys. Chem. Chem. Phys., 2005, 7, 2859–2866. P. Sun, F. O. Laforge and M. V. Mirkin, Scanning electrochemical microscopy in the 21st century, Phys. Chem. Chem. Phys., 2007, 9, 802–823. F.-M. Boldt, J. Heinze, M. Diez, J. Petersen and M. Bo¨rsch, Real-time pH microscopy down to the molecular level by combined scanning electrochemical microscopy/single-molecule fluorescence spectroscopy, Anal. Chem., 2004, 76, 3473–3481.
This journal is ß The Royal Society of Chemistry 2007
www.rsc.org/molecularbiosystems | Molecular BioSystems
Single molecule biology: Coming of age Liming Ying DOI: 10.1039/b702845h
Cellular heterogeneity and stochastic fluctuation play key roles in biological processes. Single molecule approaches have the key advantage of avoiding ensemble averaging, allowing the observation of transient intermediates and heterogeneity (both static and dynamic). Thus they have revolutionised the way many biological questions are addressed. The challenge ahead is to develop integrated approaches such as the combination of single molecule imaging with single molecule manipulation to probe the dynamics of gene regulatory and cell signalling networks in living cells.
Introduction The key processes of life occur in the noisy, far from equilibrium cellular environment, which is characterised by heterogeneity and stochastic fluctuation. To unravel this complexity, biology requires new physical concepts and methodologies. One promising approach is measuring the properties and dynamics of biological molecules, macromolecular machines, and cellular properties at the single molecule level in both test-tubes and living cells. Around thirty years ago, an innovative method (using a glass micropipette to patch the cell membrane) to measure the electrical activity of a single ionic channel across a cell membrane was developed by Erwin Neher and Bert Sakmann.1 This patch-clamp technique has opened a new Biological Nanoscience Section, National Heart and Lung Institute, Imperial College London, London, UK SW7 2AZ. E-mail: [email protected]; Tel: 020 7594 3132
window for neurobiology and physiology research and the two scientists were subsequently awarded the Nobel Prize in 1991. The statistical analysis of the current flowing through the ionic channel provided unique and valuable information on the functional and dynamic states of these channels. This information had previously been buried or lost when one simply took measurements on the average current flowing through a large ensemble of channels. The patch-clamp technique was the first successful approach to taking measurements on single biological identities in living cells. The last decade has seen the development of several new tools for manipulating, visualising, and characterising single molecules and their interactions, and as a result many areas of biology are now undergoing transformation. Direct monitoring of conformations, movement, and interactions of individual biological molecules, molecular complexes and macromolecular machines as well as other biological events such as gene
Dr Liming Ying obtained his PhD in physical chemistry from Peking University in China. He was an AWU postgraduate fellow at the Pacific Northwest National Laboratory in Richland USA, working with Dr Sunney Xie in single molecule spectroscopy. After postdoctoral studies with Drs David Klenerman and Shankar Balasubramanian at the Department of Chemistry, University of Cambridge, he was awarded a BBSRC David Phillips Research Fellowship and continued his research at Cambridge. He recently joined Imperial College London. His research focuses on developing and applying novel single molecule approaches to address biological questions. Liming Ying This journal is ß The Royal Society of Chemistry 2007
expression has started to provide new temporal and spatial dynamic information not accessible with ensemble measurements. Single molecule biology is becoming a new paradigm of research in life sciences and an indispensable part of systems biology.
Why the single molecule approach in biology? Due to technical limitations and convenience, biological studies traditionally take averages over ensembles of cells or molecules. However, averaging is sometimes problematic as it may mask important individual variations in structural properties and dynamics at both the cellular and molecular levels. Single molecule approaches have the advantage of avoiding ensemble averaging, enabling observation of transient intermediates and heterogeneity (both static and dynamic). Hence they have revolutionised the way many biological questions are addressed. There are several fundamental and practical reasons behind the use of the single molecule approach. At the molecular level in test-tubes, one would like to ask mechanistic questions, such as how proteins fold and how macromolecular complexes and molecular motors work. In the living cell, one would like to address how proteins and molecular complexes interact with each other in the heterogeneous and fluctuating cellular environment. As we know, these molecular machines or motors cannot be synchronised in their functioning or operation, making detailed mechanistic investigation impossible with Mol. BioSyst., 2007, 3, 377–380 | 377
ensemble studies. Therefore it is necessary to examine molecular motors one by one, in analogy to the quality control procedures in the manufacturing of car engines. Protein folding is an emerging area demanding the single molecule approach.2 Molecular dynamics simulations of protein folding are classic single molecule theoretical ‘‘experiments,’’ providing atomic-resolution structural and dynamic information. However these unprecedented details are yet to be confirmed from single molecule measurements on individual proteins. Single molecule approaches can identify the distribution of microscopic pathways in protein folding and provide insights into protein misfolding and aggregation which are possible causes of a wide range of diseases, especially neurodegenerative disorders. It can also distinguish protein folding mechanisms.3 Biology is becoming increasingly quantitative as we enter the post-genomic era. It will be necessary to complement ensemble approaches with analyses of their individual units: single molecules in single cells to gain an integrative systematic level of understanding of complexity in biology. Cells are testtubes for the next level of experiments. In single cells, many proteins exist in low copy numbers. These proteins play important roles in the functioning of cells, including cell signalling and the regulation of gene expression. Without amplification procedures, their abundance is far below the sensitivity of conventional protein analysis methods. Therefore it is necessary to develop microfluidic devices in which manipulation, lysation, labelling, separation, and quantification of the protein contents of a single cell using single-molecule fluorescence counting can be carried out.4 On the other hand, ensemble kinetic measurements of reaction/signalling networks in a living cell is difficult partly because it involves low-copy-numbers of molecules and partly because individual processes in the network cannot be synchronised. Multi-colour single molecule imaging would allow several reactions/interactions to be observed and recorded simultaneously, thus allowing the measurements of reaction rate or binding constant in a single run. 378 | Mol. BioSyst., 2007, 3, 377–380
Current single molecule approaches Measurements on single molecules employ optical, electrical and mechanical detection. Most optical methods use single molecule fluorescence or fluorescence resonance energy transfer (FRET) via either one-photon or multi-photon excitation. A variety of single molecule fluorescence techniques have been developed over the last decade, notably singlepair FRET,5 two colour fluorescence coincidence6 and alternating laser excitation (ALEX).7 Single molecule fluorescence imaging, in particular, total-internal-reflection fluorescence (TIRF) microscopy (objective-type or prism-type)8 has drawn a great deal of interest in recent years. These techniques are now widely applied to study the structural heterogeneity and dynamics of protein and nucleic acids,9 the assembly of biomolecular complexes, the mechanism of enzymatic reactions,10 and finally protein–protein interactions in living cells.11 However, blinking and photobleaching of the fluorophores set a fundamental limit on the amount of fluorescence photons detectable and therefore observation time. For prolonged and multiplexed single molecule imaging, one may use quantum dots which offer better brightness and photostability. Engineered fluorescent proteins, especially those whose fluorescence can be photo-switched on and off, are finding increasing use in single molecule imaging in living cells. New optical approaches are continuing to emerge, offering optical resolution beyond the diffraction-limit, combined with single molecule sensitivity. Fluorescence switching is exploited most recently in stimulated emission depletion (STED) fluorescence microscopy,12 photo activated localisation microscopy (PALM)13 and stochastic optical reconstruction microscopy (STORM).14 These novel techniques are able to achieve a resolution of tens of nanometres. The most common electrical measurement on single molecules is the patch clamp for single ion channels on living cells. This has been extended to measure the ion current blockage when ssDNA passes through an engineered protein nanopore and nanofabricated solid-state nanopore.15 Individual bases can be
distinguished by the different current signature of each base as it passes and partly blocks the ion current. This method holds great promise for single molecule DNA or protein sequencing provided that the measurement bandwidth can be significantly improved in the future. In the mid-1990s the ability to directly measure interactive forces at the level of single biological macromolecules and/or their complexes was acquired. Single molecule force spectroscopy uses atomic force microscopy to stretch and record the force–extension curve of a biopolymer.16 This approach has been used to study the mechanical unfolding of the multi-unit proteins such as titin and the ligand receptor interaction. Apart from atomic force microscopy, other single molecule manipulation techniques include magnetic and optical tweezers,17 microfluidics/nanofluidics,18 and bioMEM/NEMs devices.19 These have been used to control biological reactions and functions and provide new routes to study biomolecular machinery. Statistical mechanics is an essential tool in understanding fluctuating biological machines and processes. For example, statistical analysis helps the biophysicist to overcome the noise in the single molecule signals to learn about the step size of molecular motors, their energy consumption, and the rate-limiting transitions in their enzymatic cycles. One can thus build and verify better models of how these enzymes function and compare with the well-established mechanism derived from ensemble observations. The ergodic hypothesis in statistical mechanics states that the average of a process parameter over time and the average over the statistical ensemble at a given time are the same. It assumes that it is as good to observe a process for a long time as sampling many independent realisations of the same process. For single molecule measurement on a homogenous sample, the average of a physical parameter over a sufficient long time trajectory from an individual molecule is equal to the statistical average of the same parameter from many single molecules at any time—the ensemble average. As heterogeneity and fluctuation are ubiquitous in biology, the significance of single molecule experiments lies in
This journal is ß The Royal Society of Chemistry 2007
their capability of capturing the static and dynamic heterogeneity of the biological system, and in underpinning microscopic insights of the fundamental mechanisms in the biological process. One marvellous example was given by Xie and his colleagues on the study of fluctuating single enzyme b-galactosidase molecules.20 It was observed that the memory effect, which in a dynamic system refers to the temporal dependency of a future prediction on the history, takes place at high substrate concentrations and it covers the time scale from a millisecond to a second due to the interconverting conformers with broadly distributed lifetimes. It was also found that the Michaelis–Menten equation which has been an essential tool for understanding enzyme kinetics since 1910s still holds even for a fluctuating single enzyme, but with a different microscopic interpretation: its apparent rate constant is in fact an average of the ever fluctuating conformations, as oppose to a well-defined one as conventionally thought. This might have considerable physiological consequences in a living cell where a low copy number of enzymes within a cellular compartment gives importance to the enzymatic fluctuations. How can one obtain equilibrium thermodynamic parameters from processes carried out far from equilibrium? In 1997 Jarzynski proved an equality which relates the irreversible work to the equilibrium free energy difference.21 This theorem was tested rigorously by mechanically stretching a single molecule of RNA reversibly and irreversibly between two conformations.22 The Jarzynski equality extends the thermodynamic analysis of single molecule manipulation data beyond the context of equilibrium experiments. Hence protein and RNA unfolding energy landscape can be extracted from single molecule pulling experiments.
complexes and their machinery. In order to fulfil these tasks, current single molecule imaging and manipulation techniques need to be integrated and improved. In the meantime chemists and protein biochemists need to develop optimised fluorophores, preferably inorganic or intrinsic such as fluorescent protein, to improve the photostability and brightness, for multiplexed single molecule detection and imaging. Gene expression influences most aspects of cellular behaviour, and its variation is responsible for the phenotypic differences in a population of cells. Because DNA, RNA, and proteins can be present and active at a few copies per cell, gene expression is an intrinsically stochastic process.23 Sunney Xie and coworkers24,25 have followed single protein expression events in vivo in an elegant set of studies. The work was done by two separate methods: in one study, the production of single molecules of a fluorescent, membrane-targeted fusion protein was visualised in live bacteria; while in the other, fluorescence generated by the enzyme b-galactosidase in a microfluidic system was used to track protein production in living cells. Their work has confirmed the stochasticity of gene expression at the single molecule level. Statistical analysis of the data
revealed that protein production occurs in stochastic bursts and that a variable number of expression events are included in each burst. The techniques described allow protein production to be monitored with unprecedented precision. The next step is to probe the expression of multiple genes simultaneously with the use of multiple reporters or monitor several components in gene regulatory networks with different colours synchronously, paving the way towards programming an individual cell. However, the challenge still remains for the study of gene expression in the eukaryotic cell at the single molecule level. Optical tweezers and fluorescence are two extensively employed single-molecule approaches. The combination of these two biophysical techniques in a single experiment offers a powerful tool for studying biomolecular systems, by allowing direct correlations to be made between nanoscale structural changes, reported by singlemolecule fluorescence, and biomechanical transitions, probed by piconewton forces generated with optical traps. Steven Block and coworkers26 have demonstrated the feasibility of this combination by simultaneously observing single-molecule fluorescence changes and force-induced strand separations in dye-labelled doublestranded DNA duplexes. This technique
Perspective for single molecule biology Looking into the future, the challenge ahead for single molecule biology is to probe the dynamics of gene expression and cellular communication at the single molecule level, which requires the dissection of the assembly, dynamics and mechanism of large biomolecular
Fig. 1 Schematic of the combination of single molecule manipulation with single molecule imaging. Fluorescence-tagged ligand molecules may be delivered to the living cell surface using a nanopipette with high temporal and spatial accuracy. The nanopipette is controlled by scanning ion-conductance microscopy. The motion of ligand bound receptors can then be tracked by single molecule imaging. (Courtesy of Dr Andreas Bruckbauer.)
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has been improved recently by alternately modulating the optical trap and excitation beams to reduce the photobleaching of fluorescent dye.27 Trapping of individual biomolecules may be achieved by optical resonance28 or using the anti-Brownian electrokinetic (ABEL) scheme.29 Thus, observation times of single biomolecules could be greatly increased, allowing long trajectories of the conformational dynamics being taken without the scepticism of surface immobilisation approaches. Fluorescence technique may also unite with scanning ion-conductance microscopy (SICM),30 a special form of scanning probe microscopy which offers non-invasive imaging of living cells with high resolution and the capability of controlled delivery of single molecules onto the cell surface. A nanopipette may be used to deliver individual biomolecules through the tip of the pipette, which are then landed onto a specific site of the cell surface and bind to cell receptors, thus triggering a cascade of cell signalling events that could be followed by multicolour single molecule tracking, as illustrated in Fig. 1. This will allow the study of complex signalling networks with high spatial and temporal resolution at the single molecule level. A closely related technique, scanning electrochemical microscopy (SECM) with nanoelectrode, has been used to address and monitor local enzymatic activities on cell surfaces.31 Combined with single molecule FRET, proton-driven ATP synthesis by single membrane-bound F0F1-ATP synthases can be investigated.32 A growing number of integrated single molecule approaches and their applications in single living cell study are expected to arise in the coming years.
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Acknowledgements The author thanks David Klenerman, Xiaodong Zhang, Andreas Bruckbauer, Joe D. Piper, and Richard W. Clarke for critical reading of the manuscript. This work is supported by the BBSRC.
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