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
