RESUMEN
Multidimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterized by its arrival time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multidimensional TCSPC makes a fluorescence lifetime technique with multiwavelength capability, near-ideal counting efficiency, and the capability to resolve multiexponential decay functions. We show that the same technique and the same hardware can be used for precision fluorescence decay analysis and fluorescence correlation spectroscopy (FCS) in selected spots of a sample.
Asunto(s)
Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Línea Celular , Núcleo Celular/química , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/análisis , Humanos , Péptidos y Proteínas de Señalización Intracelular , Microscopía Confocal/métodos , Fotones , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Recombinantes de Fusión/análisis , Piel/ultraestructuraRESUMEN
Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state.