Effects Of M145/F146 On The Photocycle Of Photoreceptor Bacteriorhodopsin And Archearhodopsin-4

Bacteriarhodopsin(bR),a light-driven proton pump found in the purple membrane of Halobacterium salinarum,was the first microbial rhodopsin photoreceptor to be discovered and has served as an ideal model for the study of other microbial rhodopsins.Photo-isomerization of the retinal chromophore from the all-trans to the13-cis configuration triggers a cyclic reaction that includes a series of K-,L-,M-,Nand O-like photo-intermediates to transfer a proton vectorially across the cell membrane.Archaerhodopsin-4(aR4),a proton pump of unknown crystallographic structure found in the claret membrane of Halobacterium species XZ515,is also a retinal-based photoreceptor sharing 59% sequence similarity with bR,and exhibits a similar trimeric form with it.aR4 employs a similar light capture and proton translocation mechanism as bR,however,it displays an opposite proton release and uptake temporal order to that of bR under physiological conditions.Methionine 145(M145)is a key residue within the retinal binding pocket in bR and is very important to mediate its photocycle.On the other hand,Phenylalanine 146(F146)is the residue within the retinal binding pocket in aR4 locating at the same position as M145 in bR,and is the only different residue within the retinal binding pocket in the two proteins.So it is worthy to take a deep study of the functional role of F146 during aR4 photocycle by comparing with the role of M145 in bR.In this study,we exploited in situ two-dimensional(2D)magic-angle spinning(MAS)solid-state NMR(ssNMR)to address the molecular mechanisms of the functions of F146 during the aR4 photocycle in the native membrane environment.Light-induced transient absorption change measurements,dynamic light scattering(DLS)measurements,titration analysis,ATP formation rate measurement and site-directed mutagenesis were combined to elucidate the functional roles of F146 during the aR4 photocycle by comparing with the functional roles of M145 during the bR photocycle.A shifted Ret cis-trans isomerization thermal equilibrium,adeficient photocycle,and unfavorable trimeric assembly dynamics and energy conversion were observed in the M145F-R,indicating that the influences of M145 F mutation to the bR photo-intermediates and photocycle are much more severe than that of F146 M to the aR4 photo-intermediates and photocycle.A possible explanation is that the steric effect of M145 F mutation to the bR retinal binding pocket is bigger than that of F146 M to the aR4 retinal binding pocket due to the packing rigidity in the two proteins are different.