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We present the first demonstration that f luorescence resonance energy transfer can be used to track the motion of a single molecule undergoing conformational changes. As a model system, the conformational changes of individual three-helix junction RNA molecules induced by the binding of ribosomal protein S15 or Mg 2؉ ions were studied by changes in single-molecule f luorescence. The transition from an open to a folded configuration was monitored by the change of f luorescence resonance energy transfer between two different dye molecules attached to the ends of two helices in the RNA junction. Averaged behavior of RNA molecules closely resembles that of unlabeled molecules in solution determined by other bulk assays, proving that this approach is viable and suggesting new opportunities for studying protein-nucleic acids interactions. Surprisingly, we observed an anomalously broad distribution of RNA conformations at intermediate ion concentrations that may be attributed to foldability differences among RNA molecules. In addition, an experimental scheme was developed where the real-time response of single molecules can be followed under changing environments. As a demonstration, we repeatedly changed Mg 2؉ concentration in the buffer while monitoring single RNA molecules and showed that individual RNA molecules can measure the instantaneous Mg 2؉ concentration with 20-ms time resolution, making it the world's smallest Mg 2؉ meter.The study of individual molecules allows one to look beyond ensemble-averaged properties. For example, one can measure the distributions of physical properties and observe the detailed time trajectories of molecular conformations. The study of single molecules has also led to the discovery that identical DNA molecules under identical conditions follow a multitude of paths as they extend in elongational flows (1). This ''molecular individualism'' was shown to be a consequence of thermal variations in the initial random polymer coil before extension and of nonequilibrium statistical mechanics (2). It is possible that complex processes such as protein folding and enzyme activity will also show a rich set of kinetic paths that can be fully characterized only at the single-molecule level. Thus, it is important to develop techniques that extend our ability to observe such molecular processes at this level of sensitivity.Fluorescence resonance energy transfer (FRET) (3, 4) provides a powerful way of observing the behavior of molecules. Donor and acceptor dyes attached to two sites of a biological molecule can be used to measure the distance between the two dyes. Donor fluorescence emission is strongly quenched in a distance-dependent manner by the acceptor, whereas the acceptor emission increases because of the energy transfer. Thus, measurement of a change in fluorescence from the two dyes can be used as an indicator of a change in the conformation of the host molecule. Because the two fluorophores are on different parts of the molecule, intramolecular motion can be measured in the mol...

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The roles of specific and nonspecific interactions in the regulation of protein kinase C by lipid have been examined. Binding and activity measurements reveal two mechanisms by which protein kinase C interacts with membranes: (1) a specific binding to the activating lipid phosphatidylserine and (2) a nonspecific binding to nonactivating, acidic lipids. The specific interaction with phosphatidylserine is relatively insensitive to ionic strength, surface charge, and the presence of nonactivating lipids. The two second messengers of the kinase, diacylglycerol and Ca2+, increase markedly the affinity of the kinase for phosphatidylserine. In contrast, the nonspecific interaction is sensitive to ionic strength and surface charge, and is unaffected by diacylglycerol. These results suggest that electrostatic interactions promote the binding of protein kinase C to membranes but the cooperative and selective binding of phosphatidylserine is the dominant driving force in a productive protein-lipid interaction.