Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2.
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.
From the Freie Universität Berlin, Institut für Experimentalphysik, D-14195 Berlin, Germany and.
Pierre
P
Volz
From the Freie Universität Berlin, Institut für Experimentalphysik, D-14195 Berlin, Germany and.
Nils
N
Krause
From the Freie Universität Berlin, Institut für Experimentalphysik, D-14195 Berlin, Germany and.
Jens
J
Balke
From the Freie Universität Berlin, Institut für Experimentalphysik, D-14195 Berlin, Germany and.
Constantin
C
Schneider
From the Freie Universität Berlin, Institut für Experimentalphysik, D-14195 Berlin, Germany and.
Maria
M
Walter
the Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany.
Franziska
F
Schneider
From the Freie Universität Berlin, Institut für Experimentalphysik, D-14195 Berlin, Germany and.
Ramona
R
Schlesinger
From the Freie Universität Berlin, Institut für Experimentalphysik, D-14195 Berlin, Germany and ulrike.alexiev@fu-berlin.de.
Ulrike
U
Alexiev
D016825:Chlamydomonas reinhardtii / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D010940:Plant Proteins; D012243:Rhodopsin
channelrhodopsin; fluorescence; fluorescence anisotropy; fluorescence quenching; membrane protein; optogenetics; protein conformation