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Research Progresses in Single Molecule Enzymology
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 44, Issue 9, September 2016 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2016, 44(9), 1437–1446.
REVIEW
Research Progresses in Single Molecule Enzymology XU Yan, SUN Le-Le, GAO Yan-Jing, QIN Wei-Wei, PENG Tian-Huan, LI Di* Division of Physical biological & Bioimaging Center, ShanghaiInstitute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China
Abstract: The advent of single molecule imaging technologies in 1990s made the dynamics of single molecule enzyme catalysis be successfully probed in real time in vitro. Ever since then, single molecule enzymology entered the golden age of rapid developing. Individual features of single enzyme molecule hidden in the overall average were discovered, and many new catalytic mechanisms were proposed. Single molecule enzymology sheds light on the dynamic interactions between enzyme and substrate or product, deepening the understanding of biochemical reactions. Key Words:
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Single molecule; Enzyme; Ribozyme; Fluorescence resonance energy transfer; Fluorescence microscopy; Review
Introduction
As an important constituent part of biology, enzyme could catalyze a series of biochemical reactions, making it an indispensable element for organisms to maintain normal physiological requirements. Although enzyme has been studied for a long time, new questions in understanding how enzyme functions in real time arise. How does individual enzyme dynamically change during the catalytic process? Is there any difference among individuals of enzyme? How does enzyme interact with its substrate? Nevertheless, traditional measurements often recorded the overall average, and the results represented overall similarities of countless molecules, and could not reflect the individual characteristics of single enzyme. It is an impossible mission for typical traditional ensemble measurements to probe specific features of single enzyme functions. The development of technology has expanded the horizons of thinking as well as exploring. In the early 1990s, researchers set out to participate in the development of single molecule imaging techniques at room temperature[1‒7]. The continuing improvements of imaging, detection as well as single-molecule manipulation offered suitable opportunities for the observation of biological macromolecules functions in single molecule level at that time.
The application of single molecule fluorescent microscopy provided an excellent avenue to monitor the dynamic motions of individual molecule, and addressed the dynamic features of enzyme from deeper depth. The present single molecule study uncovered the dynamic feature of enzyme-activity of single enzyme fluctuates during catalysis. Afterwards, this phenomenon has brought new concerns that what cause enzyme fluctuate? As known to all, enzyme is constituted of amino acids chains (polypeptide chain) and only when the polypeptide chain folds into the specific configuration, could enzyme functions normally as biocatalyst to generate product. However, in the presence of thermal motion, polypeptide chains have to fluctuate to maintain the specific configuration, and the fluctuation of polypeptide chains inevitably induces conformational fluctuation of enzyme[8]. That is why enzyme could fluctuate in different conformations, each with specific activity. Meanwhile, the energy landscape determines the thermodynamically and kinetically accessible conformations, their relative ratios and interconversion rates. Therefore, enzyme turns over many times before converting to a low active state, which gives the explanation for memory effect. Studying enzyme catalysis at single molecule level by using fluorescence microcopy becomes an important research field. The general study procedures are shown as follows. Firstly,
________________________ Received 18 March 2016; accepted 18 April 2016 *Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (Nos. 21227804, 21373260, 31371015), the Youth Innovation Promotion Association, Chinese Academy of Sciences, and the Open Project of State Key Laboratory of Oral Diseases of China (No. SKLOD2015OF05). Copyright © 2016, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(16)60957-X
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enzymes are loosely fixed onto the quartz or glass interface. Secondly, enzyme is immersed in the reaction buffer which contains the substrate, then the enzyme starts to catalyze the substrate to generate product. During the catalytic process, the interactions between enzyme and substrate or product would lead to the fluorescent change which could be recorded by fluorescent microscopy. Thirdly, fluorescent intensity trajectories of single enzyme are obtained and analyzed to build suitable catalytic model. The study is based on fluorescent change. However, owing to the limited experimental conditions, several rules should be considered to carry out a single molecule study: (1) the fluorescent active state of enzyme can be changed during the reaction; (2) the enzyme could catalyze fluorogenic substrate to generate fluorescent product; (3) the substrate could be easily labeled with flurophore and its fluorescent state may change after the reaction; (4) the enzyme or enzyme and substrate could be labeled with fluorescence resonance energy transfer (FRET) pairs to study the conformational change; (5) the intensity of fluorophore could be dramatically changed due to conformational change of enzyme. Researchers could independently record the fluorescent signals generated by the catalytic process of enzymes through single molecule fluorescent microscopy, then build mathematical catalytic model through statistical analysis of intensity trajectories. Therefore, single molecule enzymology could obtain the dynamical fluctuation of individual enzyme and provide statistical and theoretical support for understanding a specific catalytic system. In this review, we make a brief introduction of research progress in single molecule study on enzyme and ribozyme. We hope it will help the readers make a better understanding of single molecule enzymology.
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Research progresses in single molecule enzymology
2.1 Research progress in single molecule protein enzyme The advent of DNA double-helix structure model has opened a new chapter of interpreting structures of biological macromolecules. Essential to all life, enzyme is an indispensable element for organisms to maintain normal physiological functions. Although crystal structures of different types of enzyme have been resolved using X-ray crystallography and other physical techniques, we are still confused with some problems. How does enzyme work in real time during catalysis? What is the relationship between structure and function of enzyme? While through observation of enzyme catalysis from single molecule perspective, these questions have been gradually uncovered. The pioneer work of single molecule study of enzyme was conducted by Lu et al[9] through real-time observation of catalytic process of
cholesterol oxidase (COx) in 1998. As shown in Fig.1A, the active center of COx involves a flavin adenine dinucleotide (FAD), the oxidized form of FAD is naturally fluorescent while the reduced form is not fluorescent active. When COx catalyzing its substrate, FAD is reduced to the reduced form (FADH2) followed by the re-oxidization to FAD, and then a new catalytic cycle starts. The emission intensity is recorded in real time through an inverted fluorescence microscopy. Figure 1B shows a typical intensity trajectory of single COx. It is obvious that the fluorescent intensity stochastically fluctuate, and each on-off represents a complete catalytic turnover. The authors separated the whole reaction into two parts to simplify the analytic procedures, and analyzed a series of on-time and off-time using mathematics. The authors made product generation process rate limiting using a cholesterol derivative to evaluate the rate constant variance of individual enzyme. The result is illustrated in Fig.1C. The distribution of on-time was monotonic exponential decay and the distribution of rate constant among individuals of enzyme was broad. This discovery indicated that static disorder was present in enzyme catalysis[10]. Previous bulk measurements proposed that enzyme catalysis was a Markovian process, the catalytic rate was constant and there was no cross influence between adjacent two reactions. However, as shown in Fig.1D, there was an obvious diagonal feature which indicates the memory effect between the adjacent on-time pair. Moreover, when quantitatively analyzing the difference using autocorrelation function (r), r(m) decayed when the two-turnover pair was separated by increasing numbers of turnovers (Fig.1E). This discovery indicated that the reaction velocity and configuration of enzyme fluctuated during catalysis, and verified that there was no specific rate constant for enzyme from single molecule perspective. In the subsequent study, Yang et al[11] again confirmed that the conformational fluctuation of enzyme was a common phenomenon when using electron transfer as probe to correspond to distance variances. In general, probing conformational change of enzyme is a difficult task for ensemble averaging. However, individual features of single enzyme could be recorded and analyzed to reveal new insights in catalytic mechanism through single molecule techniques. The experimental designs and analytical methods developed by this single molecule assay made solid foundations for researching on different types of enzyme. In addition to the above mentioned COx whose fluorescent active state could be altered during catalysis, there was another kind of enzyme which catalyzed fluorogenic substrates to generate fluorescent products, this feature made it suitable candidate for single molecule study. Different from previous kind of enzyme, the second type had no fluorescent active state, and the observed fluorescent transformation was caused by the fluorophore generation reaction catalyzed by enzyme. It was noted that the substrate-product could not
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reversibly interconvert, every intensity burst indicated that a new substrate was converted to a product. Although different from the origin of intensity change, these two kinds of enzymes shared one same point that the intensity change represented the interactions between enzyme and substrate or product. Therefore, same or similar single-molecule techniques and analytical methods could be applied to conduct single molecule experiments. Taking horseradish peroxidase (HRP) for example, in 1999, Rigler et al[12] confined biotinylated HRP onto streptavidin-modified glass and studied the process of HRP catalyzing dihydrorhodamine 6G with combination use of fluorescence microscopy and FCS. In analogue to COx, the authors also discovered the activity of HRP fluctuated during catalytic process (Fig.2A) and considered the fluctuation of enzyme activity was caused by the thermodynamics induced conformational transformation in a time scale. Lu et al[13] modified maleimide-activated HRP onto sulphydryl- functioned cover glass, and developed a new method which combined the anisotropy and lifetime measurements with single molecule fluorescence intensity assay and studied the catalytic behaviors of HRP in single turnover level. As shown in Fig.2B, they confirmed the
configurations of HRP still switched between loosed and contracted state even in the product releasing process. They estimated that about 80% of products could diffuse directly from loosed active center of HRP, while the rest 20% of products would be spilled out from condensed active center. Lu et al[14] further proposed a new strategy to manipulate the conformation and activity of enzyme through home-built single molecule magnetic tweezers-total internal reflection microscopy. The experimental scheme is illustrated in Fig.2C. The authors modified one side of HRP onto the surface of cover slips, while connected the opposite side to magnetic beads. Therefore, the conformation manipulation of HRP was achieved by alteration of external force, and the dynamic catalytic activity was recorded by real-time monitoring single HRP catalyzing Amplex Red to generate fluorescent products. The authors discovered that the deformation of HRP molecules could not totally deprive activity of HRP under external force. Enzyme molecules maintained conformational fluctuation even under external force interference. Although it was stretched by external pulling forces, they were capable of refolding to the substrate-binding accessible conformational subset state due to conformational fluctuation. Therefore, the binding of
Fig.1 (A) Scheme of cholesterol oxidase (COx) catalysis. When COx catalyzing its substrate to generate product, the flavin adenine dinucleotide (FAD) containing in active site is reduced to reduced flavin adenine dinucleotide (FADH2), then re-oxidized by O2, corresponding changes of its fluorescent state. (B) Single molecule COx fluorescence emission intensity changes with time. (C) On-time distribution of single COx with 2 mM substrate. (D) The 2D conditional probability distribution of on-time separated by certain numbers of turnovers. (E) The autocorrelation function of on-time. (F) Proposed simple model of enzyme catalysis. (A)‒(F) were reproduced with permission from Ref [9], copyright 1998 The American Association for the Advancement of Science
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Fig.2 (A) Scheme for study of horseradish peroxidase (HRP) activity fluctuation. a, Immobilization scheme of HRP; b,Correlation analysis of fluorescence intensity changes of single HRP catalysis. (B) Product release process study of HRP. a, Scheme for immobilization of HRP; b, Correlation plots of the lifetime, anisotropy, and intensity on the single HRP-catalyzed Amplex Red fluorogenic assay; c,Theory model of two kinds of product releasing processes.(C) Conformational manipulation of HRP through single molecule magnetic microscopy. a, The conceptual scheme of the experimental system; b, The catalytic rates changes under external force; c, Conceptual scheme of conformational fluctuation of single enzyme protein when being deformed by external force. (A) was reproduced with permission from Ref [12], copyright 1999 Elsevier Limited. (B) was reproduced with permission from Ref [13], copyright 2014 American Chemical Society. (C) was reproduced with permission from Ref [14], copyright 2015 National Academy of Sciences
substrates and enzyme molecules induced partially unfolded or deformed enzymes refold to active and native conformation which promoted the generation of products. The immobilization and modification of the enzyme might also influence the flexibility and individual state of enzyme, leading to artificial static disorder. To seek a solution to this problem, different strategies were proposed to confine single enzyme inside nano-containers to keep its native state. Cornelissen et al[15] proposed to trap single HRP through virus vesicle, and achieved the single molecule assay with native enzyme (Fig.3A). During the assay, the substrates diffused into the cavity of the capsulds freely, and then were catalyzed by the trapped HRP to generate fluorescent products. Although the authors confirmed the catalytic process using a simple diffusion model, they did not study the reaction mechanism. Meanwhile, this method could not be adapted to all researches because the stability of virus vesicle would change with varied pH values. Afterwards, simultaneous
single molecule observation of individual enzymes was achieved by Walt et al[16] using an array of glass optical fiber bundles to trap single HRP. As depicted in Fig.3B, when trapped in the small chambers, the rates of HRP catalyzing product formation decreased 10 times compared with that in bulk solution. The authors attributed this phenomenon to a two-step mechanism of product formation. Briefly, HRP firstly catalyzed fluorogenic substrate molecules to generate radical intermediates, and then two radical molecules generated a product in an enzyme-independent dismutation process. The radical intermediates could be consumed when interacting with the wall of the chamber, therefore yielded less products compared with solution assays. Haran et al[17] described another approach to trap single enzyme in liposome for single molecule research. As shown in Fig.3C, after the confinement of HRP through liposomes, the fluorogenic substrates could penetrate into the vesicles and subsequently be catalyzed by HRP to generate charged products. However,
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the products were unable to diffuse out of liposomes and then accumulated in vesicles because the liposomes were uncharged. The accumulation of products led to an interesting phenomenon that the catalytic reaction would be stalled when the concentration of product was sufficiently high inside the liposome. The bulk and single molecule measurements confirmed that the enzyme was noncompetitively inhibited by the products, moreover, the distribution of the initial reaction rates and the number of products required for completely inhibition was broad and dynamically varied. Furthermore, correlation analysis revealed a high correlation of the two parameters, indicating that the active site of HRP was in synchronous motion with the allosteric inhibition site. However, as the products accumulated in the liposome, it was impossible for this assay to identify individual turnover catalyzed by enzyme. Single molecule study of enzyme was not limited to HRP. Enzymes as lactate dehydrogenase[10,18],
alkaline phosphate[19,20] and β-galactosidase[21], which generated fluorescent products were also studied through similar single molecule procedures. In addition, fluorescent labeling the substrate and tracking the fluorescent variation before and after catalytic reaction is also a frequently used single molecule research strategy. Iversen et al[22] proposed to study the Ras activation by Son of Sevenless (SOS) through confining guanosine triphosphatase Ras onto the membrane interface. The experimental scheme is depicted in Fig.4A. Single SOS molecule was labeled with atto-647, and Ras was fixed onto supported bilayer loading with atto-488 labeled GTP. Single SOS molecule catalyzed GTP to activate Ras, the generated GDP-atto-488 was released into solution, while the unlabeled GTP rebound to Ras. Therefore, the fluorescence intensity of atto-488 decreased with catalysis process. The authors observed the distribution of catalytic rates was broad, indicating the stochastic fluctuation
Fig.3 (A) A virus-based study of HRP catalysis. a, Experiment scheme for HRP capsuled in virus capsid, substrates diffuse into the cavity of the virus capsid and are catalyzed by HRP; b, Autocorrelation analysis of HRP catalysis. (B) Study of HRP catalysis revealing a two-step radical dismutation reaction mechanism. a, Scheme for glass optical fiber bundle confined single HRP catalysis study; b, Averaged catalysis turnover rate for single HRP detection; c, Averaged catalysis turnover rate in bulk measurement. (C) Allosteric inhibition study of product generated by HRP. a, Scheme for HRP trapped by liposome; b, A typical time trace of fluorescence intensity from HRP catalyzing product formation inside single liposome vesicle as a function of time; c, Simple model for noncompetition inhibition and the correlation function between initial reaction rate of individual HRP and the number of product molecules needed for completely inhibition. (A) was reproduced with permission from Ref [15], copyright 2007 Nature Publishing Group. (B) was reproduced with permission from Ref [16], copyright 2010 American Chemical Society. (C) was reproduced with permission from Ref [17], copyright 2012 National Academy of Sciences
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Fig.4 (A) Ras activation by Son of Senvenless: a, Experimental design, Ras loaded with fluorescent labeled GTP is trapped on to supported bilayer, after catalyzed by SOS, the resulted fluorescent GDP is released to solution; b, Wide-field epifluorescence image of fluorescently loaded Ras before injection of SOS (left) shows no initial dark corrals. Injection of a pulse of SOS was followed by continuous flow of nonfluorescent nucleotide (right) leads to enzymatic turnover in a subset of corrals. (B) Specific interaction of anaphase-promoting complex (APC) and substrate. a, Experimental design, fluorescent labeled substrate is fixed onto the interface, meanwhile ubiquitin and APC are also fluorescently labeled, and the interaction of APC and substrate are monitored by fluorescence microscopy; b, Processive affinity amplification enhances the specificity of APC. (C) Substrate degradation by the proteasome: a, Construction of substrate with different ubiquitin configurations; b, Single molecule study strategy, the proteasome is trapped on the coverslips, the substrate is conjugated with different ubiquitin conformers, and when the substrate interacting with proteasome, fluorescent spot could be recorded. (A) was reproduced with permission from Ref [22], copyright 2014 The American Association for the Advancement of Science. (B) was reproduced with permission from Ref [23], copyright 2015 The American Association for the Advancement of Science. (C) was reproduced with permission from Ref [24], copyright 2015 The American Association for the Advancement of Science
of various states. An important discovery was that the RasGTP modulated the fluctuation into highly active states, revealing a mechanism that the dynamical spectrum of rate sampled by a small number of enzymes determined the functional output. Krischner et al[23] also investigated the interaction of ubiquitin and anaphase-promoting complex (APC)-substrate by modifying the substrate, the APC and ubiquitin with different fluorophores and confined the substrates on the interface (Fig.4B). The authors discovered that APC-mediated ubiquitylation involved a highly processive reaction of conjugating several ubiquitin molecules on substrate, leading to increased specific affinity of substrate and APC in subsequent reactions. The authors postulated these cycles of positive feedback endowed substrates containing shot recognition motifs with high specificity in the complex cellular environments. Krischner et al[24] further studied the process of proteasome recognizing substrates, as shown in Fig.4C. The authors modified proteasome on the surface of coverslip and designed the substrates conjugated with multiple configuration of ubiquitin chains (each ubiquitin was fluorescently labeled). Bright fluorescent spot was observed while proteasome catalyzing the substrates, and disappeared if
the substrate was completely degraded. The authors discovered that the level of ubiquitylation influenced the interactions of proteasome and substrate. Meanwhile, the chain structure of ubiquitin affected substrate translocation into the channel of proteasome. The two factors determined how susceptibility of proteasome degrading the substrates in great extent. Generally, although catalytic mechanisms might be different, almost all enzymes share one similarity that the enzyme fluctuates during catalytic process. The fluctuation of enzyme is not important in a solution containing countless numbers of enzyme molecules and the overall average represents the overall characters of enzyme through ensemble assays. However, this is different in cell. As only one copy or several copies of enzyme involved in one cell, any fluctuation may cause stochastic result. Meanwhile, most of single molecule researches were carried out in a nonequilibrium steady state in vitro, single molecule is more common in cell, and there are huge differences between the in vitro and in vivo environments. Therefore, studying single molecule behaviors of enzyme in vivo may provide accurate information of enzyme functions.
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2.2
Single ribozyme catalysis research
RNA plays a vital role in series of biological processes such as gene slicing and translation, etc. As the activity of ribozyme directly reflects the extent of its native configuration, the enzyme-independent ribozyme is considered ideal model for studying the relationship between structures and functions. Single molecule study of ribozyme has long been explored. Early in 2000, Zhang et al[25] probed ribozyme catalysis and folding using single molecule FRET. They designed a tether strand with Cy5 and biotin dual modification (Fig.5A). The tether strand could hybridize with the extension part of ribozyme, fixing the ribozyme onto the streptavidin modified interface. The substrate strand was further modified with Cy3, when substrate-ribozyme interactions happened, the generated FRET could be measured and the FRET efficiency directly reflected the distance variance between donor and acceptor. The authors firstly verified that the modification and fixation of ribozyme did not affect the catalytic activity and ribozyme folding. Then the helix P1 formed by ribozyme and substrate hybridization was verified reversibly docking or undocking on
the active center of ribozyme. There arose a question whether a tertiary contact of P1 and active center could be formed during transition. A previous study implied that the 2’hydroxyl. of residue U-3 on substrate made tertiary contact[26,27] with the ribozyme core in the docked state, the authors modified the hydroxyl with methoxy, then discovered that the tertiary contact occurred after P1 docking on the active center. Afterwards, ribozyme folding was explored, the authors confirmed that there were at least three pathways when ribozyme folded into native configuration, and each pathway contained at least one intermediate which was rate limiting. Among all three pathways, a substrate-dependent fast folding pathway was overlook in bulk averaging. The single molecule method established by this assay provided a useful approach to study ribozyme catalysis as well as folding. Based on this assay, Zhang et al[28] further calculated the interrelation between structure dynamics and the functions subsequently (Fig.5B). The authors also confirmed that the complex conformational transformations even existed in the simplest hairpin ribozyme. When in the docked state of ribozyme, there were four active configures with individual specific stability.
Fig.5 (A) Single-molecule study of ribozyme catalysis and folding. a, Structural model of ribozyme and experimental scheme; b, The model of P1 interaction with the active center of ribozyme; c, Time histogram of folding into native structure. (B) Study of structural dynamics and function. a, Single-molecule and bulk solution measurements of enzymatic activities; b, The undocking kinetics is complex, suggesting four docked states of distinct stabilities; c, Docked states have a strong memory effect. (C) Study of modification showing impacts on catalysis. a, Exemplary single-molecule fluorescence resonance energy transfer (FRET) time trajectories of the major subpopulations of the wild type (WT) and variant hairpin ribozyme-noncleavable substrate analog complexes; b, Experimental and simulated cleavage time courses of WT and variant ribozymes. (D) Molecular Crowding Accelerates Ribozyme Docking and Catalysis. a, Polyethylene glycol (PEG) stabilizes the docked conformation of the hairpin ribozyme; b, PEG helps the ribozyme fold in near physiological conditions; c, PEG accelerates ribozyme cleavage. (A) was reproduced with permission from Ref [25], copyright 2000 The American Association for the Advancement of Science. (B) was reproduced with permission from Ref [28], copyright 2002 The American Association for the Advancement of Science. (C) was reproduced with permission from Ref [29], copyrights 2004 National Academy of Sciences. (D) was reproduced with permission from Ref [30], copyright 2014 The American Chemical Society
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Meanwhile, ribozyme hardly converted to other configurations during a period of time when it was in one configuration, and this interesting phenomenon suggested that the memory effect was also applicable to ribozyme catalysis. However, these configurations interconverted when slicing the substrate, and ribozyme catalysis was also heterogeneous which was in analogy to protein enzyme. Then, Rueda et al[29] probed how modification influenced catalysis, as shown in Fig.5C. They designed several modifications on the nucleotides which were in different distances away from the active center of ribozyme. They indicated that the modified variants exhibited heterogeneous docking and undocking rates, and impacted the internal chemistry-slicing process. The authors considered that the coupled molecular motions could form a network, which would communicate remote parts with reaction center. This interesting phenomenon indicated that the ribozymes had similar functions to protein, opening up new study direction of ribozyme. Moreover, Paudel et al[30] developed an assay mimicking the crowing cell environments in single ribozyme study (Fig.5D). They verified increasing polyethylene glycol (PEG) concentration could promote ribozyme folding into active formation, reduced Mg2+-induced folding, and improved the slicing activity through diminishing heterogeneous catalysis. These results suggested the ribozyme could evolve to adapt to unfamiliar environments. In summary, single ribozyme catalytic researches still focus on exploring the interactions between structures and functions. Due to easily labeling and fixing, by using single molecule FRET measurements, the dynamic conformational transformation could be directly calculated during ribozymesubstrate interactions. The existing results also suggested the fact that ribozymes have homologous features to protein enzymes. Ribozymes have many configurations and interconvert during catalysis. Ribozymes catalysis have memory effect, the nucleotides of ribozymes can form a molecular motion network to connect the active center with their remote parts and ribozymes evolve to make better function while in new environments.
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Perspectives in single molecule catalysis
during detection? And how to establish new analytical models to analyze the data? (2) When monitoring the catalysis process of enzyme in real-time, some important phenomena may be ignored due to the limited resolution of techniques. Therefore, in order to improve the temporal and spatial resolution of single molecule detection to dig out the potential information hiding in the catalytic process, it is necessary to investigate the way of combined use of super-resolution microscopy techniques and other imaging techniques, or establish new single molecule techniques. (3) The present single molecule studies are mainly performed based on fluorescent microscopy. However, many important enzymes have neither fluorescent-active state nor fluorogenic substrate to generate fluorescent product, these situations restrict single molecule study in a limited area. It is worth exploring new detection methods which do not rely on fluorescence change or developing new universal probes or fluorophore labeling methods that could be adopted by different biological reactions. (4) The present single molecule study mainly focuses on the fundamental research. However, it is narrow and limited, if the techniques and methods are only restricted to single molecule filed. Therefore, it is worth thinking how to use the techniques, methods as well as theories to instruct other fields and find real application. The field of single molecule enzymology has accomplished a lot in the last two decades. Plenty of new phenomena were discovered and many theories of biological macromolecules were established. Nevertheless, all the achievements are faded into the past, and the increasing matured single molecule techniques and methods will undoubtedly bring more wonderful findings in the future.
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Cite this article as: Chin J Anal Chem, 2016, 44(9), 1437–1446.
REVIEW
Research Progresses in Single Molecule Enzymology XU Yan, SUN Le-Le, GAO Yan-Jing, QIN Wei-Wei, PENG Tian-Huan, LI Di* Division of Physical biological & Bioimaging Center, ShanghaiInstitute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China
Abstract: The advent of single molecule imaging technologies in 1990s made the dynamics of single molecule enzyme catalysis be successfully probed in real time in vitro. Ever since then, single molecule enzymology entered the golden age of rapid developing. Individual features of single enzyme molecule hidden in the overall average were discovered, and many new catalytic mechanisms were proposed. Single molecule enzymology sheds light on the dynamic interactions between enzyme and substrate or product, deepening the understanding of biochemical reactions. Key Words:
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Single molecule; Enzyme; Ribozyme; Fluorescence resonance energy transfer; Fluorescence microscopy; Review
Introduction
As an important constituent part of biology, enzyme could catalyze a series of biochemical reactions, making it an indispensable element for organisms to maintain normal physiological requirements. Although enzyme has been studied for a long time, new questions in understanding how enzyme functions in real time arise. How does individual enzyme dynamically change during the catalytic process? Is there any difference among individuals of enzyme? How does enzyme interact with its substrate? Nevertheless, traditional measurements often recorded the overall average, and the results represented overall similarities of countless molecules, and could not reflect the individual characteristics of single enzyme. It is an impossible mission for typical traditional ensemble measurements to probe specific features of single enzyme functions. The development of technology has expanded the horizons of thinking as well as exploring. In the early 1990s, researchers set out to participate in the development of single molecule imaging techniques at room temperature[1‒7]. The continuing improvements of imaging, detection as well as single-molecule manipulation offered suitable opportunities for the observation of biological macromolecules functions in single molecule level at that time.
The application of single molecule fluorescent microscopy provided an excellent avenue to monitor the dynamic motions of individual molecule, and addressed the dynamic features of enzyme from deeper depth. The present single molecule study uncovered the dynamic feature of enzyme-activity of single enzyme fluctuates during catalysis. Afterwards, this phenomenon has brought new concerns that what cause enzyme fluctuate? As known to all, enzyme is constituted of amino acids chains (polypeptide chain) and only when the polypeptide chain folds into the specific configuration, could enzyme functions normally as biocatalyst to generate product. However, in the presence of thermal motion, polypeptide chains have to fluctuate to maintain the specific configuration, and the fluctuation of polypeptide chains inevitably induces conformational fluctuation of enzyme[8]. That is why enzyme could fluctuate in different conformations, each with specific activity. Meanwhile, the energy landscape determines the thermodynamically and kinetically accessible conformations, their relative ratios and interconversion rates. Therefore, enzyme turns over many times before converting to a low active state, which gives the explanation for memory effect. Studying enzyme catalysis at single molecule level by using fluorescence microcopy becomes an important research field. The general study procedures are shown as follows. Firstly,
________________________ Received 18 March 2016; accepted 18 April 2016 *Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (Nos. 21227804, 21373260, 31371015), the Youth Innovation Promotion Association, Chinese Academy of Sciences, and the Open Project of State Key Laboratory of Oral Diseases of China (No. SKLOD2015OF05). Copyright © 2016, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(16)60957-X
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enzymes are loosely fixed onto the quartz or glass interface. Secondly, enzyme is immersed in the reaction buffer which contains the substrate, then the enzyme starts to catalyze the substrate to generate product. During the catalytic process, the interactions between enzyme and substrate or product would lead to the fluorescent change which could be recorded by fluorescent microscopy. Thirdly, fluorescent intensity trajectories of single enzyme are obtained and analyzed to build suitable catalytic model. The study is based on fluorescent change. However, owing to the limited experimental conditions, several rules should be considered to carry out a single molecule study: (1) the fluorescent active state of enzyme can be changed during the reaction; (2) the enzyme could catalyze fluorogenic substrate to generate fluorescent product; (3) the substrate could be easily labeled with flurophore and its fluorescent state may change after the reaction; (4) the enzyme or enzyme and substrate could be labeled with fluorescence resonance energy transfer (FRET) pairs to study the conformational change; (5) the intensity of fluorophore could be dramatically changed due to conformational change of enzyme. Researchers could independently record the fluorescent signals generated by the catalytic process of enzymes through single molecule fluorescent microscopy, then build mathematical catalytic model through statistical analysis of intensity trajectories. Therefore, single molecule enzymology could obtain the dynamical fluctuation of individual enzyme and provide statistical and theoretical support for understanding a specific catalytic system. In this review, we make a brief introduction of research progress in single molecule study on enzyme and ribozyme. We hope it will help the readers make a better understanding of single molecule enzymology.
2
Research progresses in single molecule enzymology
2.1 Research progress in single molecule protein enzyme The advent of DNA double-helix structure model has opened a new chapter of interpreting structures of biological macromolecules. Essential to all life, enzyme is an indispensable element for organisms to maintain normal physiological functions. Although crystal structures of different types of enzyme have been resolved using X-ray crystallography and other physical techniques, we are still confused with some problems. How does enzyme work in real time during catalysis? What is the relationship between structure and function of enzyme? While through observation of enzyme catalysis from single molecule perspective, these questions have been gradually uncovered. The pioneer work of single molecule study of enzyme was conducted by Lu et al[9] through real-time observation of catalytic process of
cholesterol oxidase (COx) in 1998. As shown in Fig.1A, the active center of COx involves a flavin adenine dinucleotide (FAD), the oxidized form of FAD is naturally fluorescent while the reduced form is not fluorescent active. When COx catalyzing its substrate, FAD is reduced to the reduced form (FADH2) followed by the re-oxidization to FAD, and then a new catalytic cycle starts. The emission intensity is recorded in real time through an inverted fluorescence microscopy. Figure 1B shows a typical intensity trajectory of single COx. It is obvious that the fluorescent intensity stochastically fluctuate, and each on-off represents a complete catalytic turnover. The authors separated the whole reaction into two parts to simplify the analytic procedures, and analyzed a series of on-time and off-time using mathematics. The authors made product generation process rate limiting using a cholesterol derivative to evaluate the rate constant variance of individual enzyme. The result is illustrated in Fig.1C. The distribution of on-time was monotonic exponential decay and the distribution of rate constant among individuals of enzyme was broad. This discovery indicated that static disorder was present in enzyme catalysis[10]. Previous bulk measurements proposed that enzyme catalysis was a Markovian process, the catalytic rate was constant and there was no cross influence between adjacent two reactions. However, as shown in Fig.1D, there was an obvious diagonal feature which indicates the memory effect between the adjacent on-time pair. Moreover, when quantitatively analyzing the difference using autocorrelation function (r), r(m) decayed when the two-turnover pair was separated by increasing numbers of turnovers (Fig.1E). This discovery indicated that the reaction velocity and configuration of enzyme fluctuated during catalysis, and verified that there was no specific rate constant for enzyme from single molecule perspective. In the subsequent study, Yang et al[11] again confirmed that the conformational fluctuation of enzyme was a common phenomenon when using electron transfer as probe to correspond to distance variances. In general, probing conformational change of enzyme is a difficult task for ensemble averaging. However, individual features of single enzyme could be recorded and analyzed to reveal new insights in catalytic mechanism through single molecule techniques. The experimental designs and analytical methods developed by this single molecule assay made solid foundations for researching on different types of enzyme. In addition to the above mentioned COx whose fluorescent active state could be altered during catalysis, there was another kind of enzyme which catalyzed fluorogenic substrates to generate fluorescent products, this feature made it suitable candidate for single molecule study. Different from previous kind of enzyme, the second type had no fluorescent active state, and the observed fluorescent transformation was caused by the fluorophore generation reaction catalyzed by enzyme. It was noted that the substrate-product could not
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reversibly interconvert, every intensity burst indicated that a new substrate was converted to a product. Although different from the origin of intensity change, these two kinds of enzymes shared one same point that the intensity change represented the interactions between enzyme and substrate or product. Therefore, same or similar single-molecule techniques and analytical methods could be applied to conduct single molecule experiments. Taking horseradish peroxidase (HRP) for example, in 1999, Rigler et al[12] confined biotinylated HRP onto streptavidin-modified glass and studied the process of HRP catalyzing dihydrorhodamine 6G with combination use of fluorescence microscopy and FCS. In analogue to COx, the authors also discovered the activity of HRP fluctuated during catalytic process (Fig.2A) and considered the fluctuation of enzyme activity was caused by the thermodynamics induced conformational transformation in a time scale. Lu et al[13] modified maleimide-activated HRP onto sulphydryl- functioned cover glass, and developed a new method which combined the anisotropy and lifetime measurements with single molecule fluorescence intensity assay and studied the catalytic behaviors of HRP in single turnover level. As shown in Fig.2B, they confirmed the
configurations of HRP still switched between loosed and contracted state even in the product releasing process. They estimated that about 80% of products could diffuse directly from loosed active center of HRP, while the rest 20% of products would be spilled out from condensed active center. Lu et al[14] further proposed a new strategy to manipulate the conformation and activity of enzyme through home-built single molecule magnetic tweezers-total internal reflection microscopy. The experimental scheme is illustrated in Fig.2C. The authors modified one side of HRP onto the surface of cover slips, while connected the opposite side to magnetic beads. Therefore, the conformation manipulation of HRP was achieved by alteration of external force, and the dynamic catalytic activity was recorded by real-time monitoring single HRP catalyzing Amplex Red to generate fluorescent products. The authors discovered that the deformation of HRP molecules could not totally deprive activity of HRP under external force. Enzyme molecules maintained conformational fluctuation even under external force interference. Although it was stretched by external pulling forces, they were capable of refolding to the substrate-binding accessible conformational subset state due to conformational fluctuation. Therefore, the binding of
Fig.1 (A) Scheme of cholesterol oxidase (COx) catalysis. When COx catalyzing its substrate to generate product, the flavin adenine dinucleotide (FAD) containing in active site is reduced to reduced flavin adenine dinucleotide (FADH2), then re-oxidized by O2, corresponding changes of its fluorescent state. (B) Single molecule COx fluorescence emission intensity changes with time. (C) On-time distribution of single COx with 2 mM substrate. (D) The 2D conditional probability distribution of on-time separated by certain numbers of turnovers. (E) The autocorrelation function of on-time. (F) Proposed simple model of enzyme catalysis. (A)‒(F) were reproduced with permission from Ref [9], copyright 1998 The American Association for the Advancement of Science
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Fig.2 (A) Scheme for study of horseradish peroxidase (HRP) activity fluctuation. a, Immobilization scheme of HRP; b,Correlation analysis of fluorescence intensity changes of single HRP catalysis. (B) Product release process study of HRP. a, Scheme for immobilization of HRP; b, Correlation plots of the lifetime, anisotropy, and intensity on the single HRP-catalyzed Amplex Red fluorogenic assay; c,Theory model of two kinds of product releasing processes.(C) Conformational manipulation of HRP through single molecule magnetic microscopy. a, The conceptual scheme of the experimental system; b, The catalytic rates changes under external force; c, Conceptual scheme of conformational fluctuation of single enzyme protein when being deformed by external force. (A) was reproduced with permission from Ref [12], copyright 1999 Elsevier Limited. (B) was reproduced with permission from Ref [13], copyright 2014 American Chemical Society. (C) was reproduced with permission from Ref [14], copyright 2015 National Academy of Sciences
substrates and enzyme molecules induced partially unfolded or deformed enzymes refold to active and native conformation which promoted the generation of products. The immobilization and modification of the enzyme might also influence the flexibility and individual state of enzyme, leading to artificial static disorder. To seek a solution to this problem, different strategies were proposed to confine single enzyme inside nano-containers to keep its native state. Cornelissen et al[15] proposed to trap single HRP through virus vesicle, and achieved the single molecule assay with native enzyme (Fig.3A). During the assay, the substrates diffused into the cavity of the capsulds freely, and then were catalyzed by the trapped HRP to generate fluorescent products. Although the authors confirmed the catalytic process using a simple diffusion model, they did not study the reaction mechanism. Meanwhile, this method could not be adapted to all researches because the stability of virus vesicle would change with varied pH values. Afterwards, simultaneous
single molecule observation of individual enzymes was achieved by Walt et al[16] using an array of glass optical fiber bundles to trap single HRP. As depicted in Fig.3B, when trapped in the small chambers, the rates of HRP catalyzing product formation decreased 10 times compared with that in bulk solution. The authors attributed this phenomenon to a two-step mechanism of product formation. Briefly, HRP firstly catalyzed fluorogenic substrate molecules to generate radical intermediates, and then two radical molecules generated a product in an enzyme-independent dismutation process. The radical intermediates could be consumed when interacting with the wall of the chamber, therefore yielded less products compared with solution assays. Haran et al[17] described another approach to trap single enzyme in liposome for single molecule research. As shown in Fig.3C, after the confinement of HRP through liposomes, the fluorogenic substrates could penetrate into the vesicles and subsequently be catalyzed by HRP to generate charged products. However,
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the products were unable to diffuse out of liposomes and then accumulated in vesicles because the liposomes were uncharged. The accumulation of products led to an interesting phenomenon that the catalytic reaction would be stalled when the concentration of product was sufficiently high inside the liposome. The bulk and single molecule measurements confirmed that the enzyme was noncompetitively inhibited by the products, moreover, the distribution of the initial reaction rates and the number of products required for completely inhibition was broad and dynamically varied. Furthermore, correlation analysis revealed a high correlation of the two parameters, indicating that the active site of HRP was in synchronous motion with the allosteric inhibition site. However, as the products accumulated in the liposome, it was impossible for this assay to identify individual turnover catalyzed by enzyme. Single molecule study of enzyme was not limited to HRP. Enzymes as lactate dehydrogenase[10,18],
alkaline phosphate[19,20] and β-galactosidase[21], which generated fluorescent products were also studied through similar single molecule procedures. In addition, fluorescent labeling the substrate and tracking the fluorescent variation before and after catalytic reaction is also a frequently used single molecule research strategy. Iversen et al[22] proposed to study the Ras activation by Son of Sevenless (SOS) through confining guanosine triphosphatase Ras onto the membrane interface. The experimental scheme is depicted in Fig.4A. Single SOS molecule was labeled with atto-647, and Ras was fixed onto supported bilayer loading with atto-488 labeled GTP. Single SOS molecule catalyzed GTP to activate Ras, the generated GDP-atto-488 was released into solution, while the unlabeled GTP rebound to Ras. Therefore, the fluorescence intensity of atto-488 decreased with catalysis process. The authors observed the distribution of catalytic rates was broad, indicating the stochastic fluctuation
Fig.3 (A) A virus-based study of HRP catalysis. a, Experiment scheme for HRP capsuled in virus capsid, substrates diffuse into the cavity of the virus capsid and are catalyzed by HRP; b, Autocorrelation analysis of HRP catalysis. (B) Study of HRP catalysis revealing a two-step radical dismutation reaction mechanism. a, Scheme for glass optical fiber bundle confined single HRP catalysis study; b, Averaged catalysis turnover rate for single HRP detection; c, Averaged catalysis turnover rate in bulk measurement. (C) Allosteric inhibition study of product generated by HRP. a, Scheme for HRP trapped by liposome; b, A typical time trace of fluorescence intensity from HRP catalyzing product formation inside single liposome vesicle as a function of time; c, Simple model for noncompetition inhibition and the correlation function between initial reaction rate of individual HRP and the number of product molecules needed for completely inhibition. (A) was reproduced with permission from Ref [15], copyright 2007 Nature Publishing Group. (B) was reproduced with permission from Ref [16], copyright 2010 American Chemical Society. (C) was reproduced with permission from Ref [17], copyright 2012 National Academy of Sciences
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Fig.4 (A) Ras activation by Son of Senvenless: a, Experimental design, Ras loaded with fluorescent labeled GTP is trapped on to supported bilayer, after catalyzed by SOS, the resulted fluorescent GDP is released to solution; b, Wide-field epifluorescence image of fluorescently loaded Ras before injection of SOS (left) shows no initial dark corrals. Injection of a pulse of SOS was followed by continuous flow of nonfluorescent nucleotide (right) leads to enzymatic turnover in a subset of corrals. (B) Specific interaction of anaphase-promoting complex (APC) and substrate. a, Experimental design, fluorescent labeled substrate is fixed onto the interface, meanwhile ubiquitin and APC are also fluorescently labeled, and the interaction of APC and substrate are monitored by fluorescence microscopy; b, Processive affinity amplification enhances the specificity of APC. (C) Substrate degradation by the proteasome: a, Construction of substrate with different ubiquitin configurations; b, Single molecule study strategy, the proteasome is trapped on the coverslips, the substrate is conjugated with different ubiquitin conformers, and when the substrate interacting with proteasome, fluorescent spot could be recorded. (A) was reproduced with permission from Ref [22], copyright 2014 The American Association for the Advancement of Science. (B) was reproduced with permission from Ref [23], copyright 2015 The American Association for the Advancement of Science. (C) was reproduced with permission from Ref [24], copyright 2015 The American Association for the Advancement of Science
of various states. An important discovery was that the RasGTP modulated the fluctuation into highly active states, revealing a mechanism that the dynamical spectrum of rate sampled by a small number of enzymes determined the functional output. Krischner et al[23] also investigated the interaction of ubiquitin and anaphase-promoting complex (APC)-substrate by modifying the substrate, the APC and ubiquitin with different fluorophores and confined the substrates on the interface (Fig.4B). The authors discovered that APC-mediated ubiquitylation involved a highly processive reaction of conjugating several ubiquitin molecules on substrate, leading to increased specific affinity of substrate and APC in subsequent reactions. The authors postulated these cycles of positive feedback endowed substrates containing shot recognition motifs with high specificity in the complex cellular environments. Krischner et al[24] further studied the process of proteasome recognizing substrates, as shown in Fig.4C. The authors modified proteasome on the surface of coverslip and designed the substrates conjugated with multiple configuration of ubiquitin chains (each ubiquitin was fluorescently labeled). Bright fluorescent spot was observed while proteasome catalyzing the substrates, and disappeared if
the substrate was completely degraded. The authors discovered that the level of ubiquitylation influenced the interactions of proteasome and substrate. Meanwhile, the chain structure of ubiquitin affected substrate translocation into the channel of proteasome. The two factors determined how susceptibility of proteasome degrading the substrates in great extent. Generally, although catalytic mechanisms might be different, almost all enzymes share one similarity that the enzyme fluctuates during catalytic process. The fluctuation of enzyme is not important in a solution containing countless numbers of enzyme molecules and the overall average represents the overall characters of enzyme through ensemble assays. However, this is different in cell. As only one copy or several copies of enzyme involved in one cell, any fluctuation may cause stochastic result. Meanwhile, most of single molecule researches were carried out in a nonequilibrium steady state in vitro, single molecule is more common in cell, and there are huge differences between the in vitro and in vivo environments. Therefore, studying single molecule behaviors of enzyme in vivo may provide accurate information of enzyme functions.
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2.2
Single ribozyme catalysis research
RNA plays a vital role in series of biological processes such as gene slicing and translation, etc. As the activity of ribozyme directly reflects the extent of its native configuration, the enzyme-independent ribozyme is considered ideal model for studying the relationship between structures and functions. Single molecule study of ribozyme has long been explored. Early in 2000, Zhang et al[25] probed ribozyme catalysis and folding using single molecule FRET. They designed a tether strand with Cy5 and biotin dual modification (Fig.5A). The tether strand could hybridize with the extension part of ribozyme, fixing the ribozyme onto the streptavidin modified interface. The substrate strand was further modified with Cy3, when substrate-ribozyme interactions happened, the generated FRET could be measured and the FRET efficiency directly reflected the distance variance between donor and acceptor. The authors firstly verified that the modification and fixation of ribozyme did not affect the catalytic activity and ribozyme folding. Then the helix P1 formed by ribozyme and substrate hybridization was verified reversibly docking or undocking on
the active center of ribozyme. There arose a question whether a tertiary contact of P1 and active center could be formed during transition. A previous study implied that the 2’hydroxyl. of residue U-3 on substrate made tertiary contact[26,27] with the ribozyme core in the docked state, the authors modified the hydroxyl with methoxy, then discovered that the tertiary contact occurred after P1 docking on the active center. Afterwards, ribozyme folding was explored, the authors confirmed that there were at least three pathways when ribozyme folded into native configuration, and each pathway contained at least one intermediate which was rate limiting. Among all three pathways, a substrate-dependent fast folding pathway was overlook in bulk averaging. The single molecule method established by this assay provided a useful approach to study ribozyme catalysis as well as folding. Based on this assay, Zhang et al[28] further calculated the interrelation between structure dynamics and the functions subsequently (Fig.5B). The authors also confirmed that the complex conformational transformations even existed in the simplest hairpin ribozyme. When in the docked state of ribozyme, there were four active configures with individual specific stability.
Fig.5 (A) Single-molecule study of ribozyme catalysis and folding. a, Structural model of ribozyme and experimental scheme; b, The model of P1 interaction with the active center of ribozyme; c, Time histogram of folding into native structure. (B) Study of structural dynamics and function. a, Single-molecule and bulk solution measurements of enzymatic activities; b, The undocking kinetics is complex, suggesting four docked states of distinct stabilities; c, Docked states have a strong memory effect. (C) Study of modification showing impacts on catalysis. a, Exemplary single-molecule fluorescence resonance energy transfer (FRET) time trajectories of the major subpopulations of the wild type (WT) and variant hairpin ribozyme-noncleavable substrate analog complexes; b, Experimental and simulated cleavage time courses of WT and variant ribozymes. (D) Molecular Crowding Accelerates Ribozyme Docking and Catalysis. a, Polyethylene glycol (PEG) stabilizes the docked conformation of the hairpin ribozyme; b, PEG helps the ribozyme fold in near physiological conditions; c, PEG accelerates ribozyme cleavage. (A) was reproduced with permission from Ref [25], copyright 2000 The American Association for the Advancement of Science. (B) was reproduced with permission from Ref [28], copyright 2002 The American Association for the Advancement of Science. (C) was reproduced with permission from Ref [29], copyrights 2004 National Academy of Sciences. (D) was reproduced with permission from Ref [30], copyright 2014 The American Chemical Society
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Meanwhile, ribozyme hardly converted to other configurations during a period of time when it was in one configuration, and this interesting phenomenon suggested that the memory effect was also applicable to ribozyme catalysis. However, these configurations interconverted when slicing the substrate, and ribozyme catalysis was also heterogeneous which was in analogy to protein enzyme. Then, Rueda et al[29] probed how modification influenced catalysis, as shown in Fig.5C. They designed several modifications on the nucleotides which were in different distances away from the active center of ribozyme. They indicated that the modified variants exhibited heterogeneous docking and undocking rates, and impacted the internal chemistry-slicing process. The authors considered that the coupled molecular motions could form a network, which would communicate remote parts with reaction center. This interesting phenomenon indicated that the ribozymes had similar functions to protein, opening up new study direction of ribozyme. Moreover, Paudel et al[30] developed an assay mimicking the crowing cell environments in single ribozyme study (Fig.5D). They verified increasing polyethylene glycol (PEG) concentration could promote ribozyme folding into active formation, reduced Mg2+-induced folding, and improved the slicing activity through diminishing heterogeneous catalysis. These results suggested the ribozyme could evolve to adapt to unfamiliar environments. In summary, single ribozyme catalytic researches still focus on exploring the interactions between structures and functions. Due to easily labeling and fixing, by using single molecule FRET measurements, the dynamic conformational transformation could be directly calculated during ribozymesubstrate interactions. The existing results also suggested the fact that ribozymes have homologous features to protein enzymes. Ribozymes have many configurations and interconvert during catalysis. Ribozymes catalysis have memory effect, the nucleotides of ribozymes can form a molecular motion network to connect the active center with their remote parts and ribozymes evolve to make better function while in new environments.
3
Perspectives in single molecule catalysis
during detection? And how to establish new analytical models to analyze the data? (2) When monitoring the catalysis process of enzyme in real-time, some important phenomena may be ignored due to the limited resolution of techniques. Therefore, in order to improve the temporal and spatial resolution of single molecule detection to dig out the potential information hiding in the catalytic process, it is necessary to investigate the way of combined use of super-resolution microscopy techniques and other imaging techniques, or establish new single molecule techniques. (3) The present single molecule studies are mainly performed based on fluorescent microscopy. However, many important enzymes have neither fluorescent-active state nor fluorogenic substrate to generate fluorescent product, these situations restrict single molecule study in a limited area. It is worth exploring new detection methods which do not rely on fluorescence change or developing new universal probes or fluorophore labeling methods that could be adopted by different biological reactions. (4) The present single molecule study mainly focuses on the fundamental research. However, it is narrow and limited, if the techniques and methods are only restricted to single molecule filed. Therefore, it is worth thinking how to use the techniques, methods as well as theories to instruct other fields and find real application. The field of single molecule enzymology has accomplished a lot in the last two decades. Plenty of new phenomena were discovered and many theories of biological macromolecules were established. Nevertheless, all the achievements are faded into the past, and the increasing matured single molecule techniques and methods will undoubtedly bring more wonderful findings in the future.
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