FTIR spectroscopy of the K photointermediate of Neurospora rhodopsin: structural changes of the retinal, protein, and water molecules after photoisomerization.
Neurospora rhodopsin (NR, also known as NOP-1) is the first rhodopsin of the haloarchaeal type found in eucaryotes. NR demonstrates a very high degree of conservation of the amino acids that constitute the proton-conducting pathway in bacteriorhodopsin (BR), a light-driven proton pump of archaea. Nevertheless, NR does not appear to pump protons, suggesting the absence of the reprotonation switch that is necessary for the active transport. The photocycle of NR is much slower than that of BR, similar to the case of pharaonis phoborhodopsin (ppR), an archaeal photosensory protein. The functional and photochemical differences between NR and BR should be explained in the structural context. In this paper, we studied the structural changes of NR following retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy and compared the obtained spectra with those for BR. For the spectroscopic analysis, we established the light-adaptation procedure for NR reconstituted into 1,2-dimyristoyl-sn-glycero- 3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC/DMPA) liposomes, which takes approximately 2 orders of magnitudes longer than in BR. The structure of the retinal chromophore and the hydrogen-bonding strength of the Schiff base in NR are similar to those in BR. Unique spectral features are observed for the S-H stretching vibrations of cysteine and amide-I vibrations for NR before and after retinal isomerization. In NR, there are no spectral changes assignable to the amide bands of alpha helices. The most prominent difference between NR and BR was seen for the water O-D stretching vibrations (measured in D(2)O). Unlike for haloarchaeal rhodopsins such as BR and ppR, no O-D stretches of water under strong hydrogen-bonded conditions (<2400 cm(-1)) were observed in the NR(K) minus NR difference spectra. This suggests a unique hydrogen-bonded network of the Schiff base region, which may be responsible for the lack of the reprotonation switch in NR.
Furutani
Yuji
Y
Department of Materials Science and Engineering, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
Bezerra
Arandi G
AG
Waschuk
Stephen
S
Sumii
Masayo
M
Brown
Leonid S
LS
Kandori
Hideki
H
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry; D001436:Bacteriorhodopsins / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D005615:Freezing; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D007536:Isomerism; D008027:Light; D008969:Molecular Sequence Data; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012172:Retinaldehyde / Q000737:chemistry*; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D012545:Schiff Bases / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D014867:Water / Q000737:chemistry*
D019843:Archaeal Proteins; D002352:Carrier Proteins; D005656:Fungal Proteins; C401582:NOP-1 protein, Neurospora crassa; D020283:Photoreceptors, Microbial; D011994:Recombinant Proteins; D012545:Schiff Bases; D014867:Water; D001436:Bacteriorhodopsins; D012243:Rhodopsin; D003545:Cysteine; D012172:Retinaldehyde