Structural basis for reversible photobleaching of a green fluorescent protein homologue

Abstract

Fluorescent protein (FP) variants that can be reversibly converted between fluorescent and nonfluorescent states have proven to be a catalyst for innovation in the field of fluorescence microscopy. However, the structural basis of the process remains poorly understood. High-resolution structures of a FP derived from Clavularia in both the fluorescent and the light-induced nonfluorescent states reveal that the rapid and complete loss of fluorescence observed upon illumination with 450-nm light results from cistrans isomerization of the chromophore. The photoinduced change in configuration from the well ordered cis isomer to the highly nonplanar and disordered trans isomer is accompanied by a dramatic rearrangement of internal side chains. Taken together, the structures provide an explanation for the loss of fluorescence upon illumination, the slow light-independent recovery, and the rapid light-induced recovery of fluorescence. The fundamental mechanism appears to be common to all of the photoactivatable and reversibly photoswitchable FPs reported to date.crystallography ͉ fluorescence ͉ photoswitching ͉ protein structure G reen fluorescent protein (GFP) from the jellyfish Aequorea victoria (1) and more recently, cyan, yellow, and red fluorescent proteins (FPs), isolated from coral reef organisms (2), have become standard research tools in cellular biology. Structural studies have revealed a universally conserved fold for FPs: an 11-stranded ␤-barrel with a central and axial ␣-helix (3, 4). A sequence of three amino acid residues on this central ␣-helix (Ser-65-Tyr-66-Gly-67 in Aequorea GFP) undergoes a series of autocatalytic posttranslational modifications to form the chromophore. Despite overall structural similarity across FPs from a variety of species and a variety of hues, members of the GFP family are quite diverse with respect to their photophysical properties. For example, properties such as emission color, extinction coefficient, quantum yield, and fluorescence lifetime can vary dramatically among FP variants. These properties are determined by both the influence of the protein matrix on the chromophore environment and the particular covalent chemical structure of the chromophore (5).Another photophysical property common to all FPs is wavelength-dependent change in their f luorescence emission properties upon illumination. In most cases, these changes appear to result from irreversible covalent modification of the chromophore structure or its local protein environment. Examples of such permanent changes include light-induced transition from a f luorescent to a nonf luorescent state (photobleaching), transition from a nonf luorescent to a f luorescent state (photoactivation), or a change in excitation or emission wavelength maxima (photoconversion). Photobleaching, photoactivation, and photoconversion are well documented and can be advantageous for f luorescence imaging applications such as in vivo tracking of fusion protein movements (6 -9). A less well documented process, occasionally described as ''re...

Advances in fluorescent protein technology

“…4C). Similar behavior has been reported for a teal FP precursor, termed mTFP0.7 (Henderson et al, 2007), in which the dark and fluorescent states have been characterized by crystallography.…”

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

“…However, in all previously characterized RSFPs, the wavelength used for generating the fluorescence emission is identical to one of the wavelengths used for switching the fluorescence on or off. The result is a complex interlocking of switching and fluorescence readout [14][15][16][17][18][19][20][21][22] , impeding or even precluding many applications, including fluorescence nanoscopy (super-resolution microscopy). Hence, the identification of an RSFP in which the generation of fluorescence is disentangled from switching has long been pursued.…”

Single-molecule spectroscopy of fluorescent proteins

“…Although fluorescent proteins do not show the major structural rearrangements seen in many enzymes, the proteins are not static, and thermally-driven or photoinduced reorientations in the chromophore vicinity lead to a spread of photophysical properties [89]. Also the change between emitting and non-emitting states has been observed and linked to a structural reorientation in the chromophore environment [53,54].…”

“…The combination of these genetically encodable markers with advanced microscopic and spectroscopic techniques has enabled quantitative measurement of protein-protein interactions. Variants of VFPs exhibiting different colours and photophysical properties such as photoactivation [39][40][41][42][43][44][45][46][47][48][49][50][51][52], photobleaching [53][54][55], and phototoxicity [56,57] have provided new windows into the cell. However, many studies have established that VFPs exhibit intrinsically complex photophysical behaviour, and in order to effectively and correctly exploit their enormous power to enable visualization of dynamic biological processes, it is of paramount importance to comprehensively understand the intrinsic photophysical properties of these remarkable fluorophores.…”