@article {66028, title = {Structure-Function Dataset Reveals Environment Effects within a Fluorescent Protein Model System.}, journal = {Angew Chemie (International Edition English)}, year = {2021}, month = {2021 Feb 04}, abstract = {

Anisotropic environments can drastically alter the spectroscopy and photochemistry of molecules, leading to complex structure-function relationships. We examined this using fluorescent proteins as easy-to-modify model systems. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, including the determination of 43 crystal structures. Correlation and principal component analysis confirmed the complex relationship between structure and spectroscopy, but also allowed us to identify consistent trends and to relate these to the spatial organization. We find that changes in spectroscopic properties can come about through multiple underlying mechanisms, of which polarity, hydrogen bonding and presence of water molecules are key modulators. We anticipate that our findings and rich structure/spectroscopy dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

}, issn = {1521-3773}, doi = {10.1002/anie.202015201}, author = {De Zitter, Elke and Hugelier, Siewert and Duw{\'e}, Sam and Vandenberg, Wim and Tebo, Alison G and Van Meervelt, Luc and Dedecker, Peter} } @article {65762, title = {Integrated structure-function dataset reveals key mechanisms underlying photochromic fluorescent proteins}, journal = {bioRxiv}, year = {2020}, month = {09/2020}, pages = {2020.09.25.313528}, abstract = {

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.

}, doi = {10.1101/2020.09.25.313528}, author = {Zitter, Elke De and Hugelier, Siewert and Duw{\'e}, Sam and Vandenberg, Wim and Tebo, Alison G. and Meervelt, Luc Van and Dedecker, Peter} } @article {65761, title = {Orthogonal fluorescent chemogenetic reporters for multicolor imaging}, journal = {Nature Chemical Biology}, year = {2020}, month = {04/2020}, pages = {1{\textendash}9}, abstract = {

Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen-activating tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple protein{\textendash}protein interactions by live-cell fluorescence microscopy. The fluorescent chemogenetic reporters greenFAST and redFAST were engineered by protein engineering. They display orthogonal fluorogen recognition and spectral properties allowing efficient multicolor imaging of proteins in live cells and organisms.

}, issn = {1552-4450}, doi = {10.1038/s41589-020-0611-0}, author = {Tebo, Alison G. and Moeyaert, Benjamien and Thauvin, Marion and Carlon-Andres, Irene and B{\"o}ken, Dorothea and Volovitch, Michel and Padilla-Parra, Sergi and Dedecker, Peter and Vriz, Sophie and Gautier, Arnaud} }