Functional Analysis Of Aspartic 116/115 In The Light-driven Proton Pump Bacteriorhodopsin And Archearhodopsin-4

Microbial rhodopsin is a type of seven-transmembrane photoreceptor with retinal as the ligand,mainly including light-driven ion pumps,light-gated ion channels,sensors,and light-activated enzymes.Due to their diverse functionalities and high photo-electric conversion efficiency,microbial rhodopsins have a promising application prospect in solar energy storage,artificial retina,information storage and optogenetics.Bacteriorhodopsin(b R),as the representative of microbial photoreceptors,has been studied widely investigated,especially the relationship between structure and functions.Archaerorhodopsin-4(a R4)is a seventransmembrane photoreceptor found in Halobacterium species(H.sp.)XZ515,which has the similar trimeric structure and light-driven proton pumping function to b R.However,in addition to the retinal chromophore,a R4 also has a second chromophore bacterioruberin,embedding between the adjacent monomers.It exhibits a faster photocycle and reversed temporal order of proton release and uptake compared to b R.With the successful application of a R proteins in optogenetic studies,it is important to understand their properties,including the rapid photocycle dynamics,the relationship between the different proton temporal order and their corresponding structures,and the function of the carotenoid chromophore.Previous studies have shown that the aspartic acid 115(D115),a highly conserved charged residue on helix D of b R,regulates the conformations of helices C and D by forming the local interhelical hydrogen bonding network with T90 and L87,which has a great impact on the photocycle and proton transport.However,there was less attention on the corresponding residue D116 in a R4,and it remains unclear that whether the interaction between carotenoid chromophore and helix D influences its function.Here,we explored the functional similarity and difference of D115 and D116 during the photocycle of b R and a R4 through site-directed mutagenesis,UV-Vis absorption spectroscopy,flash-light induced photolysis spectroscopy,and p H titration measurements.Our results showed that D115 and D116 are both classified as key residues of proton transport channels,and D116 is more closely coupled with the retinal binding region.Meanwhile we also found that there is a regulatory effect between D116 and bacteriorhodopsin.In the absence of the second chromophore,helix D can lead to a weaker influence of D116 on the overall structure and function than D115 in b R.This study clarifies our current understanding of the functional role of helix D and the carotenoid chromophore in a R4.It will lay a foundation for studying other dualchromophore microbial rhodopsin and accelerate the development of new optogenetic tools.