Photofunctional proteins: how nature keeps the laboratories updated about light and life

Light is Life: this should always be the slogan of humanity, but it is presently particularly true. 2015 has in fact been proclaimed the International Year of Light and Light-based Technologies by the United Nations General Assembly. It is therefore especially significant that Photochemical & Photobiological Sciences (PPS) opens the year with a themed issue dedicated to photofunctional proteins, i.e. proteins whose functional properties are changed by illumination. In these biological macromolecules, light and proteins sum up the essentials of life: light is energy from the Sun and it is manifestly central to our daily lives on earth, through photosynthesis, heat and conversion into other useful forms of energy; proteins make up the molecular machinery that enables energy transformations and cellular activities, ensures structural integrity, plasticity and sense and responds to environmental stimuli, including light. Photoreceptors are naturally occurring photofunctional proteins, widely distributed among the three domains of life, whose activity can be switched on/regulated by light, thereby affecting cellular functions and biological photoresponses. During the last decade the properties of these chromophoric systems have been actively exploited and further engineered in the field of optogenetics, that is, artificial light-regulation of cell functions with high spatiotemporal resolution and minimal invasiveness. Conversely, the labeling of non-photofunctional proteins with photoactivable molecules can be used to trigger protein functions or conformational changes and study their dynamic properties at the molecular level. Last but not least, conventional and super-resolution microscopy are taking increasing advantage of naturally fluorescent and photochromic proteins, following the revolution introduced by the green fluorescent protein (GFP) that had led Shimomura, Chalfie and Tsien to win the Nobel Prize in Chemistry in 2008. In 2010 PPS published a first themed issue on photofunctional proteins. It is noteworthy that the 2014 Nobel Prize for chemistry was assigned to Betzig, Hell, and Moerner, for the development of super-resolution microscopy techniques. The increasing research effort in this area is strongly encouraging researchers to find, study and exploit novel and optimized fluorescent proteins.

The perspective of Polli et al. (DOI: 10.1039/c4pp00370e) is dedicated to a class of photoreceptors for which light activation is most evident. The authors present an interesting and comprehensive overview of the most recent experimental and computational studies on the fundamental mechanism for the activation of opsins by light. In particular, they focus on highly time-resolved pump-probe data, two-dimensional electronic spectroscopy and femtosecond stimulated Raman scattering to follow the early steps of the ultra fast 11-cis to all-trans photoisomerization of the chromophore. If it is true that the primary photochemical reactions of opsins and other photoreceptors are completed on the fs-to-ps time scale, LOV (light, oxygen and voltage) domains represent an exception, because in this case the photocycle proceeds via the formation of a long lived triplet state. In order to determine the contribution of radical species in the photocycle of LOV domains, Kutta and co-workers developed an ad hoc experimental setup for transient absorption spectra based on a streak camera (10.1039/c4pp00155a).

Equally innovative is the automated system developed by Richter and co-workers (10.1039/c4pp00361f) to study photoactivation/de-activation of photoreceptors as a function of light quality and quantity and in different microenvironments. To do this, the authors developed a fully programmable illumination device for a microplate reader.

A group of papers is devoted to novel photoreceptors. By means of infrared spectroscopy, Heilmann et al. (10.1039/c4pp00246f) have uncovered sites of light-induced conformational changes in UVR8, a plant UV receptor that employs a group of tryptophans as chromophores. The contributions of Rockwell (10.1039/c4pp00336e) and Pennacchietti (10.1039/c4pp00337c) deal instead with innovative photosensory proteins found in cyanobacteria, the so-called cyanobacteriochromes that, similar to canonical phytochromes, rely on the photoisomerization of a bilin chromophore, but can tune their response over a wide range of the visible spectrum. These two papers investigate important photochemical features of these “multicolored” photoswitchable proteins, their potential for fluorescence applications in vivo and the effects of hydration on the photocycle dynamics. Also considering hydration as a key determinant for the dynamics of light-induced phenomena in proteins, Malferrari and co-authors (10.1039/c4pp00245h) focus on the effect of dehydration on the conformational relaxations which stabilize the charge-separated state in a bacterial photosynthetic reaction center.

Turning to other applications of photofunctional or photo-functionalized proteins, Torra et al. (10.1039/c4pp00338a) report on the photosensitizing properties of a mutated flavin-binding fluorescent protein from Pseudomonas putida, whereas the paper from Decaneto and co-workers (10.1039/c4pp00297k) supports the idea of light-triggered activation of hydrolytic enzymes using caged compounds. In this contribution the first caged substrate peptide for matrix metalloproteinases is developed and investigated.

From natural photofunctional proteins at their best, to the best of lab applications: the second perspective of this themed issue, written by Hense et al. (10.1039/c4pp00212a), is dedicated to red fluorescent proteins and their broad range of applications in microscopic imaging. Red light is less toxic and penetrates better in living tissue than green light, therefore continuing efforts are being devoted to the design, development and testing of novel red-emitting GFP variants.

We wish to take the opportunity to thank all authors for their valuable contributions and we hope you all find this second issue on photofunctional proteins an up-to-date and enjoyable read.

 

Aba Losi, Stefania Abbruzzetti

Guest editors

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