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The ability to detect emission from a single fluorophore presents a powerful tool to probe the dynamic properties of protein molecules during their interactions with ligands. Here, different classes of experiments are reviewed and a distinction is drawn between experiments that monitor signals from a large number of proteins, one molecule at a time, from those that follow a single protein molecule over many individual cycles. The latter approach is potentially capable of resolving dynamic heterogeneity, such as that displayed by enzymes that fluctuate between high and low activity states. Other factors that need to be considered are the origin of the fluctuations in the emission signal and the extent to which this represents the properties of the protein under investigation, as opposed to the probe itself. Most fluorophores show fluctuations in their emission rates, termed flickering, blinking or intermittency, which may occur on a similar time-scale as the event under investigation.

Blinking fluorophores: what do they tell us about protein dynamics?