Deciphering Structural Photophysics of Fluorescent Proteins by Kinetic Crystallography

Abstract

Because they enable labeling of biological samples in a genetically-encoded manner, Fluorescent Proteins (FPs) have revolutionized life sciences. Photo-transformable fluorescent proteins (PTFPs), in particular, recently attracted wide interest, as their fluorescence state can be actively modulated by light, a property central to the emergence of super-resolution microscopy. PTFPs, however, exhibit highly complex photophysical behaviours that are still poorly understood, hampering the rational engineering of variants with improved performances. We show that kinetic crystallography combined with in crystallo optical spectroscopy, modeling approaches and single-molecule measurements constitutes a powerful tool to decipher processes such as photoactivation, photoconversion, photoswitching, photoblinking and photobleaching. Besides potential applications for the design of enhanced PTFPs, these investigations provide fundamental insight into photoactivated protein dynamics.

Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

“…Shifting the occupancy of chromophore states with different photochromism through binding by a modified Enhancer further intensifies the idea of it serving as a contrast mechanism, as was mentioned above. In any case, a detailed characterization of both species at the molecular level should provide useful information to guide future developments [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ].…”

“…These sub-diffraction techniques rely heavily on the performance of the fluorophores [ 14 ]. Consequently, plenty of effort has been devoted to creating more optimized photoresponsive FPs, as well as to achieving a fundamental understanding of the mechanisms through which this “smart” behavior is defined [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ].…”

Fluorogenic Protein‐Based Strategies for Detection, Actuation, and Sensing

“…[6][7][8][9] Furthermore, they often exhibit complicated photophysics that impedes quantitative analysis. [10,11] Despite efforts to engineer monomeric variants, many fluorescent proteins retain their tendency to oligomerize, which renders imaging in crowded environments particularly difficult. Finally, fluorescent proteins are comprised of a rigid and rather large (%26 kDa) β barrel that can impede the proper secretion, [12] folding, [13] and/or function of many proteins.…”

“…The production of hydrogen peroxide during the maturation of GFP‐like proteins can complicate in vivo experiments, as hydrogen peroxide is a known mediator of cell survival, growth, differentiation, and is implicated in diseases . Furthermore, they often exhibit complicated photophysics that impedes quantitative analysis . Despite efforts to engineer monomeric variants, many fluorescent proteins retain their tendency to oligomerize, which renders imaging in crowded environments particularly difficult.…”

Reversible photobleaching of photoconvertible SAASoti-FP

“…While the cisconfiguration is stabilized by the E144-H194-E212 Hbonded triad, it is replaced by the E144-R66-E212 triad in the case of the trans-configuration [4]. Accordingly, either H194 or R66 stabilizes the chromophore by πstacking or π-cation interactions, respectively [6]. As there exist FPs with trans-chromophore in their 'on' state [7] it should be emphasized that it is not the configuration (cis-or trans-) but its protonation state that is responsible for the fluorescence.…”