Role of the Cytoplasmic Domain in Anabaena Sensory Rhodopsin Photocycling: Vectoriality of Schiff Base Deprotonation

• Published in: Biophysical journal Vol. 91; no. 12; pp. 4519 - 4527
• Main Authors: Sineshchekov, Oleg A., Spudich, Elena N., Trivedi, Vishwa D., Spudich, John L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2006; Biophysical Society
• Subjects: Anabaena; Anabaena - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Proteins - physiology; Cell Membrane - physiology; Cells; Cytoplasm - metabolism; E coli; Escherichia coli; Escherichia coli - physiology; Escherichia coli - radiation effects; Light; Membranes; Models, Molecular; Mutation; Photobiophysics; Pigments; Protein Structure, Tertiary; Proteins; Protons; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Schiff Bases - metabolism; Sensory Rhodopsins - genetics; Sensory Rhodopsins - metabolism; Sensory Rhodopsins - physiology
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1529/biophysj.106.093641

Light-induced electric signals in intact E. coli cells generated by heterologously expressed full-length and C-terminally truncated versions of Anabaena sensory rhodopsin ( ASR) demonstrate that the charge movements within the membrane-embedded part of the molecule are stringently controlled by the cytoplasmic domain. In particular, truncation inverts the direction of proton movement during Schiff base deprotonation from outward to cytoplasmic. Truncation also alters faster charge movements that occur before Schiff base deprotonation. Asp 217 as previously shown by FTIR serves as a proton acceptor in the truncated ASR but not in the full-length version, and its mutation to Asn restores the natural outward direction of proton movement. Introduction of a potential negative charge (Ser 86 to Asp) on the cytoplasmic side favors a cytoplasmic direction of proton release from the Schiff base. In contrast, mutation of the counterion Asp 75 to Glu reverses the photocurrent to the outward direction in the truncated pigment, and in both truncated and full-length versions accelerates Schiff base deprotonation more than 10-fold. The communication between the cytoplasmic domain and the membrane-embedded photoactive site of ASR demonstrated here is likely to derive from the receptor’s use of a cytoplasmic protein for signal transduction, as has been suggested previously from binding studies.