Insights into the spectral versatility of fluorescent proteins by single molecule spectroscopy

Understanding the photophysics of fluorescent proteins is essential for accurate interpretation of the biological and biochemical processes illuminated by the fluorescent proteins as well as for the development of biosensors based on fluorescent proteins.We used spectrally resolved single molecule spectroscopy to analyse aspects of fluorescent protein photophysics that are not accessible by conventional ensemble spectroscopy. We were able to identify and characterize different subensembles and spectral forms of a range of fluorescent proteins. We could follow transitions between the different spectral forms on the single molecule level and draw conclusions on the underlying molecular origins. Fluorescent proteins are excellent systems to analyze the interaction between a chromophore and its nanoenvironment since the local environment of the emitting chromophore is precisely defined by the protein that encapsulates the chromophore. We find that for the fluorescent proteins studied, the width of the distribution of the single molecule emission maximum positions is strictly correlated with the flexibility of the chromophore nanoenvironment. Further we analyzed the fluorescence resonance energy transfer coupling of different chromophores within one protein tetramer. We find that in a fraction of the tetramers the different chromophores are not effectively coupled. We propose an interruption of the energy transfer chain within the multichromophoric system by proteins lacking a chromophore.