Different Effects Of W86A And W86F Mutations On The Bacteriorhodopsin Photocycle

Bacteriorhodopsin(bR)is a light-driven photoreceptor,each active unit is made up three monomers and each monomer contains one opsin molecule and one retinal(Ret)chromophore covalently bonded to each other,functioning as a proton pump to transport protons from the cytoplasmic side to the extracellular side directionally to convert light energy into chemical energy through formed proton concentration gradient.Photo-isomerization of the Ret chromophore from the all-trans to the 13-cis configuration triggers a cyclic conformation changes that includes a series of stable photo-intermediates to transfer a proton vectorial across the cell membrane.Among the 248 amino acid residues in the bR,there were 8 Tryptophan(W)residues distributed at different positions in the 7 transmembrane helices.Tryptophan 86(W86),one of the key aromatic amino acid residues in the Ret binding pocket,is highly conservative in all microbial rhodopsin family proteins.However,the mechanism of the coupling of W86 with the bR photocycle has not been well revealed.In this study,in situ solid-state NMR correlation experiments,combined with lightinduced transient absorption change measurements,pH titration,DLS,ATP testing and site-specific mutation,were employed to elucidate the functional roles of W86 during the bR photocycle.Our studies indicate that both W86 F and W86 A mutations shift the dark-adapted isomerization thermal equilibrium of Ret towards a cis dominant direction with the all-trans isomer completely disappear in W86 F.In addition,both photocycle kinetics,proton pumping capability and ATP production rate of bR are weakened differently by the mutations.Two mutations with different perturbations on W86 also had different effects on the stability and trimerization of the protein.Different perturbation mechanisms to the Ret polyene chain and the surrounding residues within the binding pocket may attribute to this.By carrying out two mutations on W86 site and analyzing the differences in protein structure and function caused by mutations,we can better understand the regulatory mechanism and value of W86 on photocycle and proton pump functions.We also can provide reference for the structure and function oriented transformation of protein like bR.On this basis,this study also conducted a preliminary study on the structural and functional effects of W87 mutation on aR4,another microbial rhodopsin protein.