Influence Of Bacterioruberin On The Function Of The Key Residue F146 Of The Dual-chromophore Proton Pump AR4

Archaerhodopsin-4(aR4),a bacteriorhodopsin(b R)like light-driven proton pump,possesses the similar trimeric structure.aR4 contains not only an opsin molecule and a covalently bound retinal chromophore,but also a carotenoid chromophore bacterioruberin that is packed in the crevices between the adjacent trimer subunits of the protein trimer.In addition,aR4 displays an opposite temporal order of proton release and uptake to that of b R.After being excited by light,retinal changes from alltrans to 13-cis.The retinal thermal deactivation drives the conformational change of opsin,accompanied by the directional transfer of protons from intracellular to extracellular.Phenylalanine-146(F146)and Methionine-145(M145)are the only different residue in aR4 and b R retinal bonding pocket.In b R,M145 F mutation change the thermodynamic equilibrium of retinal in dark adaptation state,and has an important effect on the photocycle.Further studies on the role of F146 not only conducive to revealing the similarities and differences on the fnction of the key residues in the same position of the bonding pocket in the single and dual chromophore proteins,but also explore the effect of the second chromophore bacterioruberin on the role of key residues in the aR4 binding pocket.Therefore,this work successfully established a method to study the fuction role of key residues in dual chromophore proteins.Rely on this method with in situ UV-Vis absorption spectroscopy,solid-state NMR,flash light-induced transient absorption change measurements,p H titration and other technical means,we studied the function commonality and differences of F146 M and M145 F mutation in aR4 and b R.Our results shown that both F146 and M145 played a role in maintaining the cis-trans thermal equilibrium of retinal in the dark-adapted proteins.Both F146 M and M145 F mutations could cause changes to the retinal binding pocket and the hydrogen bond network with the proton translocation channel,thus participating in the regulation of proton transfer.In addition,it was identified for the first time that bacterioruberin not only can stabilize the trimeric structure,but also can modulate the function of F146,a key residue of the retinal binding pocket,removal of bacterioruberin will weaken the effect of F146 in aR4.