Phenomena that can be observed for a large number of molecules may not be understood if it is not possible to observe the events on the single-molecule level. We measured the fluorescence lifetimes of individual tetramethylrhodamine molecules, linked to an 18-mer deoxyribonucleotide sequence specific for M13 DNA, by time-resolved, singlephoton counting in a confocal fluorescence microscope during Brownian motion in solution. When many molecules were observed, a biexponential fluorescence decay was observed with equal amplitudes. However, on the single-molecule level, the fraction of one of the amplitudes spanned from 0 to unity for a collection of single-molecule detections. Further analysis by fluorescence correlation spectroscopy made on many molecules revealed a process that obeys a stretched exponential relaxation law. These facts, combined with previous evidence of the quenching effect of guanosine on rhodamines, indicate that the tetramethylrhodamine molecule senses conformational transitions as it associates and dissociates to a guanosine-rich area. Thus, our results reveal conformational transitions in a single molecule in solution under conditions that are relevant for biological processes.A new era of molecular analysis was begun by the introduction of extremely sensitive methods to detect and characterize species at the single-molecule level by spectroscopic means. Several techniques to trace individual fluorescent particles in solution have been reported over the last 12 years (1-9). Single-molecule detection (SMD) in solids and on surfaces has also been accomplished (10-14); however, many features important for chemical and biological systems can only be realized in the liquid phase, as is pointed out by reports regarding applications in DNA sequencing (15, 16) or detection and selection of rare events in diagnostics and biotechnology (17). Another important application for the SMD technique is the study of reaction dynamics at the singlemolecule level.Wang and Wolynes (18) have recently reported how measurements of single molecules can make it possible to gain information about phenomena that cannot be easily understood when a large number of molecules are observed simultaneously. A complex behavior (e.g., a distribution of a physical entity) will not provide information on whether all molecules share a common distribution or whether each molecule gives its own specific contribution to the distribution seen for many molecules. Measurements of single molecules can be decidedly important in obtaining such information.Previously (22), but we used it as a sensitive instrument -to provide precise statistical characteristics of the system. Single-Molecule Measurements. To achieve SMD, we measured repeatedly for short periods (t). The ratio of the measurement time to the characteristic passage time of a molecule through the VE is defined as T, the relative measuring time. By selecting only those measurements that experienced a large time integral of the detected fluorescence, we could effectively...

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Immobilized single horseradish peroxidase enzymes were observed by confocal fluorescence spectroscopy during catalysis of the oxidation reaction of the nonfluorescent dihydrorhodamine 6G substrate into the highly fluorescent product rhodamine 6G. By extracting only the non-Markovian behavior of the spectroscopic two-state process of enzyme-product complex formation and release, memory landscapes were generated for single-enzyme molecules. The memory landscapes can be used to discriminate between different origins of stretched exponential kinetics that are found in the first-order correlation analysis. Memory landscapes of single-enzyme data shows oscillations that are expected in a single-enzyme system that possesses a set of transient states. Alternative origins of the oscillations may not, however, be ruled out. The data and analysis indicate that substrate interaction with the enzyme selects a set of conformational substates for which the enzyme is active. Non-ergodic properties of a process of a molecule (12, 13) as well as non-exponential state transition probabilities for a single process (5) are both indicators of a complex behavior. Enlarged dynamic models on the single molecule level are then required as compared with models derived from standard chemical kinetics of an ensemble of molecules.Catalysis of the oxidation of the dihydrorhodamine 6G into rhodamine 6G by the enzyme horseradish peroxidase on the single enzyme level has recently been observed at room temperature (5). Horseradish peroxidase is a 44-kDa heme protein (14,15) and is an effective catalyst of the decomposition of hydrogen peroxide (H 2 O 2 ) in the presence of hydrogen donors (14, 15). The reaction is monitored by existing experimental methods (8) based on confocal fluorescence spectroscopy (16,17). We used the nonfluorescent substrate dihydrorhodamine 6G, which after oxidation yields the highly fluorescing rhodamine 6G fluorophore. Hence, direct observation of successive single substrate turnovers into product is made possible by means of fluorescence microscopy if a single enzyme molecule is observed. The enzyme, the substrate, and the enzyme-substrate complex are nonfluorescent. However, the enzyme-product complex (EP) (18) is fluorescent and is formed as the result of the substrate being oxidized while still bound to the enzyme; the enzyme-substrate complex transforms into a fluorescent EP. For each catalytic cycle, a new substrate is bound to the enzyme and is turned over into a product (EP), after which the product dissociates from the enzyme. Then, another substrate attaches to the enzyme, is turned over into a product (EP), and so on. The average binding time of the product (lifetime of EP) was determined in ref. 5 to be approximately 50 ms. The observable state from a spectroscopic viewpoint is the EP. All other states of the enzyme are nonfluorescent. It is assumed that the spectroscopic properties of the EP are unaffected by the oxidation state of the enzyme (e.g., 4 ϩ or 5 ϩ oxidized state) because we directly monitor the...

Measurement of f luorescent lifetimes of dyetagged DNA molecules reveal the existence of different conformations. Conformational f luctuations observed by f luorescence correlation spectroscopy give rise to a relaxation behavior that is described by ''stretched'' exponentials and indicates the presence of a distribution of transition rates between two conformations. Whether this is an inhomogeneous distribution, where each molecule contributes with its own reaction rate to the overall distribution, or a homogeneous distribution, where the reaction rate of each molecule is time-dependent, is not yet known. We used a tetramethylrhodamine-linked 217-bp DNA oligonucleotide as a probe for conformational f luctuations. Fluorescence f luctuations from single DNA molecules attached to a streptavidin-coated surface directly show the transitions between two conformational states. The conformational f luctuations typical for single molecules are similar to those seen in single ion channels in cell membranes.Fluorescence correlation spectroscopy (FCS) allows the analysis of single-molecule events and their time correlations in solution (1-6). Since the introduction of confocal excitation in extremely small volume elements (2), single molecules can be detected almost background-free in solution (3, 4). FCS has opened the possibility to analyze the behavior of single molecules in relation to their ensemble averages. In particular information can be obtained from the analysis of single molecules that cannot be obtained from the ensemble average alone.As has been pointed out by Wang and Wolynes (7), properties found for the molecular ensemble such as the distribution of states can also be a property of a single molecule (the homogenous case). Alternatively, the ensemble behavior can be caused by a collection of individual molecules, each representing a different state (the inhomogeneous case). Such situations are likely to be found in biological systems as has been put forward recently by Frauenfelder (8).We have earlier been able to demonstrate the existence of different conformational states in single molecules of M13 phage DNA (9) from the analysis of the exited state of tetramethylrhodamine (TMR) linked to the DNA by a 6-atom carbon linker and serving as a sensor for different conformational states of the DNA molecule. The redox potential between aromatic dye molecules and purine as well as pyrimidine bases (10) leads in the case of rhodamine dyes to an electron transfer from guanine to the dye that competes with the photon emission from the excited singlet state of TMR (11,12). The electron transfer is characteristic for guanine and indicates that in one conformation electron transfer takes place but not in the other. The time range of the observed conformational transitions is in the millisecond region, pointing to the involvement of intercalative processes (13,14). The rates as measured in a molecular ensemble by FCS exhibited a nonexponential behavior that could be best represented by a ''stretch'' parameter (␤ ...

Conformational fluctuations in single nucleic acid molecules have recently been observed through excited state lifetime measurements. Immobilisation of the sample molecule has also enabled direct observation of the fluorescence intensity fluctuations generated as the molecule switches between two conformations. As a probe for conformational fluctuations we use tetramethylrhodamine linked to a 217-bp DNA oligonucleotide. The measurements on this and similar systems reveal the existence of a distribution of reaction rates between the conformations. Here we report 37 detected single-molecule conformational fluctuations collected with the same immobilisation method as described earlier. Within the time window of observation the reaction rates differ between the molecules, but stay constant within a single molecule. The distribution of the relaxation rates between the molecules correspond to the distribution seen in a bulk measurement on a similar system. We therefore conclude that within the observation time window the single DNA molecules behave in a non-ergodic way. q