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Note: Research in this publication was not performed at Janelia.
Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.