A eukaryotic protein, NOP-1, binds retinal to form an archaeal rhodopsin-like photochemically reactive pigment.
The nop-1 gene from Neurospora crassa is predicted to encode a seven-helix protein exhibiting conservation with the rhodopsins of the archaeon Halobacterium salinarum. In the work presented here we have expressed this gene heterologously in the yeast Pichia pastoris, obtaining a relatively high yield of 2.2 mg of NOP-1 protein/L of cell culture. The expressed protein is membrane-associated and forms with all-trans retinal a visible light-absorbing pigment with a 534 nm absorption maximum and approximately 100 nm half-bandwidth typical of retinylidene protein absorption spectra. Its lambda(max) indicates a protonated Schiff base linkage of the retinal. Laser flash kinetic spectroscopy demonstrates that the retinal-reconstituted pigment undergoes a photochemical reaction cycle with a near-UV-absorbing intermediate that is similar to the M intermediates produced by transient Schiff base deprotonation of the chromophore in the photocycles of bacteriorhodopsin and sensory rhodopsins I and II. The slow photocycle (seconds) and long-lived intermediates (M and O) are most similar to those of the phototaxis receptor sensory rhodopsin II. The results demonstrate a photochemically reactive member of the archaeal rhodopsin family in a eukaryotic cell.
Bieszke
J A
JA
Department of Microbiology and Molecular Genetics, The University of Texas Medical School, Houston, Texas 77030, USA.
Spudich
E N
EN
Scott
K L
KL
Borkovich
K A
KA
Spudich
J L
JL
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010777:Photochemistry; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010860:Pigments, Biological / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D019843:Archaeal Proteins; D002352:Carrier Proteins; D005656:Fungal Proteins; C401582:NOP-1 protein, Neurospora crassa; D010860:Pigments, Biological; D012243:Rhodopsin