Intramolecular photo-switching and intermolecular energy transfer as primary photoevents in photoreceptive processes: The case of Euglena gracilis Raffaella Mercatelli a , Franco Quercioli a , Laura Barsanti b , Valter Evangelista b , Primo Coltelli c , Vincenzo Passarelli b , Anna Maria Frassanito b , Paolo Gualtieri b, * a Istituto Sistemi Complessi, CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy b Istituto di Biofisica, CNR, Via Moruzzi 1, 56124 Pisa, Italy c ISTI, CNR, Via Moruzzi 1, 56124 Pisa, Italy article info Article history: Received 23 April 2009 Available online xxxx Keywords: Euglena gracilis Photoreception Photoreceptor Primary photoevent Intramolecular photo-switch Intermolecular energy transfer Rhodopsin-like proteins FLIM FRET abstract In this paper we report the results of measurements performed by FLIM on the photoreceptor of Euglena gracilis. This organelle consists of optically bistable proteins, characterized by two thermally stable isomeric forms: A 498, non fluorescent and B 462 , fluorescent. Our data indicate that the primary photoevent of Euglena photoreception upon photon absorption con- sists of two contemporaneous different phenomena: an intramolecular photo-switch (i.e., A 498 becomes B 462 ), and a intermolecular and unidirectional Forster-type energy transfer. During the FRET process, the fluorescent B 462 form acts as donor for the non-fluorescent A 498 form of the protein nearby, which acts as acceptor. We hypothesize that in nature these phenomena follow each other with a domino progression along the orderly organized and closely packed proteins in the photoreceptor layer(s), modulating the isomeric composition of the photoreceptive protein pool. This mechanism guarantees that few photons are sufficient to produce a signal detectable by the cell. Ó 2009 Elsevier Inc. All rights reserved. Introduction The photoreceptor of the unicellular alga Euglena gracilis can be defined as a prototypic eye according to the Darwin’s definition as reported by Gehring [1]. This ‘‘eye” is formed within a single cell by the assembly of photoreceptive and pigmented molecules into two distinct organelles, i.e., the photoreceptor and the eyespot, within that cell. The photoreceptor is detectable by light microscopy as a distinct organelle about of 1–2 lm 3 in close association with the eyespot, a red-orange pigmented organelle about of 3–4 lm 3 , which acts as a screen device [2]. Euglena photoreceptor has a three-dimensional organization consisting of up to 50 closely stacked membrane lamellae. About 10 6 photoreceptive proteins (about 2 mM) assembled in a hexago- nal lattice span the lamellae [3]. The proteins are characterized by optical bistability, i.e., they possess two isomeric forms A and B, which interconvert along a photocycling path photochemically but not thermically [4]. A is the non-fluorescent form, while B is the fluorescent form, energetically lower, which can be considered the signaling state of the protein. The absorption spectrum of A has a band centered at 498 nm; the absorption spectrum of B has a band centered at 462 nm. Three well defined regions are present in the difference spectrum between the two isomers: a positive UV region below 400 nm, a negative region from 400 to 484 nm, and a positive region from 484 to 600 nm. The wavelengths in the positive part of the spec- trum cause the transition of A 498 to B 462 , while the wavelengths in the negative part cause the opposite transition [5]. The fluores- cence spectrum of B 462 shows a band centered at 550 nm [6]. The extraction of retinal from the Euglena photoreceptor [7], to- gether with the matching between the spectral properties and structural peculiarities of Euglena photoreceptor protein and those of proteorhodopsin [8,9], a membrane protein used by marine bac- terio-plankton as light-driven proton pump, allowed us to say that this organelle is a three-dimensional assemblage of one photore- ceptive protein belonging to the superfamily of rhodopsin-like proteins. In this paper we investigated the primary events of Euglena photoreception by means of fluorescence lifetime imaging micros- copy (FLIM) using a two-photon confocal microscope. Our results show that upon absorption of a photon that causes the transition of A 498 to B 462 , the proteins of the photoreceptor undergo two contemporaneous different phenomena: an intramolecular switch, 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.05.034 * Corresponding author. Fax: +39 050 315 2760. E-mail address: paolo.gualtieri@pi.ibf.cnr.it (P. Gualtieri). Biochemical and Biophysical Research Communications xxx (2009) xxx–xxx Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc ARTICLE IN PRESS Please cite this article in press as: R. Mercatelli et al., Intramolecular photo-switching and intermolecular energy transfer as primary photoevents in photo- receptive processes: The case of Euglena gracilis, Biochem. Biophys. Res. Commun. (2009), doi:10.1016/j.bbrc.2009.05.034