Novel expression and characterization of a light driven proton pump archaerhodopsin 4 in a Halobacterium salinarum...

Archaerhodopsin 4 (AR4), a new member of the microbial rhodopsin family, is isolated from Halobacterium species xz515 in a Tibetan salt lake. AR4 functions as a proton pump similar to bacteriorhodopsin (BR) but with an opposite temporal order of proton uptake and release at neutral pH. However, further studies to elucidate the mechanism of the proton pump and photocycle of AR4 have been inhibited due to the difficulty of establishing a suitable system in which to express recombinant AR4 mutants. In this paper, we report a reliable method for expressing recombinant AR4 in Halobacterium salinarum L33 with a high yield of up to 20mg/l. Experimental results show that the recombinant AR4 retains the light-driven proton pump characteristics and photo-cycling kinetics, similar to that in the native membrane. The functional role of bacterioruberin in AR4 and the trimeric packing of AR4 in its native and recombinant forms are investigated through light-induced kinetic measurements, two-dimensional solid-state NMR experiments, dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). Such approaches provide new insights into structure-function relationships of AR4, and form a basis for other archaeal rhodopsins.