Functional characterization of sensory rhodopsin II from Halobacterium salinarum expressed in Escherichia coli

Abstract

Sensory rhodopsin II (SRII) from Halobacterium salinarum is heterologously expressed in Escherichia coli with a yield of 3-4 mg of purified SRII per liter cell culture. UV/ Vis absorption spectroscopy display bands characteristic for native SRII. The resonance Raman spectrum provides evidence for a strongly hydrogen-bonded Schiff base like in mammalian rhodopsin but unlike to the homologous pSRII from Natronobacterium pharaonis. Laser flash spectroscopy indicates that SRII in detergent as well as after reconstitution into polar lipids shows its typical photochemical properties with prolonged photocycle kinetics. The first functional heterologous expression of SRII from H. salinarum provides the basis for studies with its cognate transducer HtrII to investigate the molecular processes involved in phototransduction as well as in chemotransduction.

Resolving voltage-dependent structural changes of a membrane photoreceptor by surface-enhanced IR difference spectroscopy

“…However, a very different signature is apparent in the amide I range. This observation reveals structural changes in H. s. SR II clearly distinct from those in the N. p. receptor (26). The absence of a negative band at 1,544 cm Ϫ1 indicates that the dipole moment of the CAC stretch of retinal is close to parallel to the solid surface.…”

“…Each modification step was followed in situ by SEIRAS. His-tagged sensory rhodopsin II (SR II) from N. p. and from H. s. were expressed and purified as described (25,26). Binding of SR II via the C-terminal His-tag to the Ni-NTA modified gold surface was carried out by incubating a 6 M solution of protein in 0.05% (wt/vol) n-dodecyl-␤-D-maltopyranoside (DDM) (pH 5.8, 20°C, 4 M NaCl, 50 mM phosphate buffer, in the case of SRII from H. s.) or a 4.0 M solution of protein in 0.02% DDM (pH 8.0, 20°C, 0.5M NaCl, 10 mM bis-Tris-propane buffer, in the case of SRII from N. p.) atop the Ni-NTA modified gold surface.…”

“…Recombinant sensory rhodopsin II (25,26) was specifically adhered via the C-terminal His-tag to the Ni-NTA modified gold surface. The latter was created a top the Si hemiprism used for internal reflection spectroscopy.…”

Controlled In Meso Phase Crystallization – A Method for the Structural Investigation of Membrane Proteins

“…SRII was expressed in E. coli and purified in 4.0 M NaCl, 50 mM MES, pH 6, 0.05% DDM (n-dodecyl-ß-maltoside) as described previously [30].…”

Functional Expression of the Signaling Complex Sensory Rhodopsin II/Transducer II from Halobacterium salinarum in Escherichia coli†

“…Recently, HsSRII has been cloned, heterologously expressed in E. coli , and functionally characterized (16). Here we describe the characterization of both HsSRII and HsHtrII isolated and purified from E. coli membranes.…”