Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Abstract

A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics.Channelrhodopsins are involved in phototaxis and photophobia of unicellular green algae (1). They constitute a new class of light-gated ion channels containing the seven-transmembrane helix motif and the chromophore retinal as the light-sensitive cofactor, which are both common to the other retinal-containing proteins such as bacteriorhodopsin (bR) 4 or visual rhodopsin (2). In CrChR2 the chromophore retinal is bound to Lys-257 (3). Channelrhodopsin activation via lightinduced isomerization of retinal from all-trans to 13-cis is coupled to a transient cofactor deprotonation and a functional protein structural change. In channelrhodopsin, as well as in other retinal-containing photoreceptors, subtle changes in the chromophore vicinity trigger large scale protein conformational changes remote from the chromophore binding pocket. Rearrangement of helix B is hypothesized to constitute a key element in channel opening by allowing the entry of water molecules (4 -6). A continuous water wire is a prerequisite for the formation of the ion-conducting pathway. A hydrophilic pore between helices A, B, C, and G was suggested to serve as the ion permeation pathway based on the high resolution crystal structure of the C1C2 chimera (3 10).The inner gate was hypothesized to be involved in CrChR2 activation (formation of the open conducting channel state) together with the tilt of helix B (5). Evidence for light-induced movements of helix B comes from structural studies using electron crystallography and EPR spectroscopy (double electronelectron resonance) (11-13). Together these data suggest that in contrast to bR and sensory rhod...

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications

“…Double electron-electron resonance (DEER) spectroscopy showed that the intracellular end of helix 2, and of helix 6 to a lesser degree, move outward upon illumination (107–108). The results of time-resolved measurements of fluorescence anisotropy are consistent with an outward tilt of helix 2 (109). Projection maps obtained by cryo-electron microscopy suggested in addition a photoinduced movement of helix 7 (110).…”

Pitfalls in using fluorescence tagging of nanomaterials: tecto‐dendrimers in skin tissue as investigated by Cluster‐FLIM

“…The recorded solution fluorescence decay traces were fitted using a multiexponential model function: with n the total number of decay components, α i the amplitude, and τ i the fluorescence lifetime of the i th component . The analysis of the time‐resolved quenching data is based on the mean fluorescence lifetime τ m calculated by …”

“…with n the total number of decay components, ␣ i the amplitude, and i the fluorescence lifetime of the ith component. 29 The analysis of the time-resolved quenching data 30,31 is based on the mean fluorescence lifetime m calculated by…”

The redox-coupled proton-channel opening in cytochrome c oxidase

“…The resulting anisotropy decay curves reveal the rotational dynamics of the protein-bound probe and yield information on global and local protein dynamics as well as on the protein structure and conformational changes. 18,22,23,30 Since the probed rotational dynamics of the uorophore is affected by the motion of the protein segment to which it is covalently attached, we expect to detect nanosecond dynamics changes of helix 6 itself and of the immediate environment including structural constraints imposed by the surrounding helices (i.e., helices 7 and 8, Fig. 1A).…”