Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes.
Sensory rhodopsin II (SRII) is the primary light sensor in the photophobic reaction of the halobacterium Natronomonas pharaonis. Photoactivation of SRII results in a movement of helices F and G of this seven-helical transmembrane protein. This conformational change is conveyed to the transducer protein (HtrII). Global changes in the protein backbone have been monitored by IR difference spectroscopy by recording frequency shifts in the amide bands. Here we investigate local structural changes by judiciously inserting thiocyanides at different locations of SRII. These vibrational Stark probes absorb in a frequency range devoid of any protein vibrations and respond to local changes in the dielectric, electrostatics, and hydrogen bonding. As a proof of principle, we demonstrate the use of Stark probes to test the conformational changes occurring in SRII 12 ms after photoexcitation and later. Thus, a methodology is provided to trace local conformational changes in membrane proteins by a minimal invasive probe at the high temporal resolution inherent to IR spectroscopy.
Department of Physics, Experimental Molecular Biophysics, Freie Universität Berlin , 14195 Berlin, Germany.
Hendrik
H
Mohrmann
Max Planck Institute of Molecular Physiology , Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
Ines
I
Kube
Department of Physics, Experimental Molecular Biophysics, Freie Universität Berlin , 14195 Berlin, Germany.
Víctor A
VA
Lórenz-Fonfría
Max Planck Institute of Molecular Physiology , Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
Martin
M
Engelhard
Department of Physics, Experimental Molecular Biophysics, Freie Universität Berlin , 14195 Berlin, Germany.
Joachim
J
Heberle
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006217:Halobacterium / Q000378:metabolism; D006860:Hydrogen Bonding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D025603:Sensory Rhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D055672:Static Electricity
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D025603:Sensory Rhodopsins