Predicted bacteriorhodopsin from Exiguobacterium sibiricum is a functional proton pump

Abstract

a b s t r a c tThe predicted Exigobacterium sibiricum bacterirhodopsin gene was amplified from an ancient Siberian permafrost sample. The protein bacteriorhodopsin from Exiguobacterium sibiricum (ESR) encoded by this gene was expressed in Escherichia coli membrane. ESR bound all-trans-retinal and displayed an absorbance maximum at 534 nm without dark adaptation. The ESR photocycle is characterized by fast formation of an M intermediate and the presence of a significant amount of an O intermediate. Proteoliposomes with ESR incorporated transport protons in an outward direction leading to medium acidification. Proton uptake at the cytoplasmic surface of these organelles precedes proton release and coincides with M decay/O rise of the ESR.

Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria

“…Both 3 10 -and π-structures have been implicated to possess functional roles other than simply structural in other membrane and soluble proteins (16)(17)(18). One possibility is that the novel structure facilitates the "unlatching" of the cytoplasmic site, similarly to what was proposed in bacteriorhodopsin photocycle (19).…”

“…occurrence of the π-and 3 10 -like structures, the overall register of the helix is unchanged. The structural element is stabilized by hydrogen bonds between the backbone and neighboring side chains (O 187 -Trp-154 and O 190 -Asn-224).…”

“…ESR with a C-terminal hexahistidine tag was expressed in E. coli strain Rosetta2(DE3)pLysS and purified as described (10). ESR was growing in a fermenter for 3 d at 30°C in 2× ZYM5052 autoinduction medium (31) with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol.…”

“…First, ESR's proton donor is a lysine side chain that is situated very close to the bulk solvent. Second, the α-helical structure in the middle of the helix F is replaced by 3 10 -and π-helix-like elements that are stabilized by the Trp-154 and Asn-224 side chains. This feature is characteristic for the proteorhodopsin family of proteins.…”

“…The E. sibiricum rhodopsin (ESR) expressed well in Escherichia coli in a functionally active form. The protein with a covalently linked alltrans retinal had maximum absorbance at 534 nm and was able to pump a proton upon illumination in a broad pH range of 4.5-8.5 (10,11).…”