Effect Of Y186F Mutation On Proton Transport And Energy Conversion In Photoreceptor Archaerhodopsin-4

Archaerhodopsin 4(aR4)is a seven transmembrane photoreceptor using retinal as a chromophore from Halobacterium species xz515.It functions as a proton pump similar to bacteriorhodopsin(bR)but with an opposite temporal order of proton uptake and release at neutral pH.Functions of much conservative aromatic residues have been one of the hotspots with this respect in the recent research.For example,Tyrosine 185(Y185),one of the aromatic residues within the retinal binding pocket on helix F of bR,may play an important role in stabilizing the pentagonal hydrogen-bond network on the extracellular side of the Schiff base(SB),and involved in the reprotonation of SB during the early stage of photocycle.It may also function as a micro-switch to modulate the cis-trans isomerization thermal equilibrium of the retinal chromophore and further affect the energy conversion in bR.Therefore,function of Tyrosine 186(Y186),a similar aromatic residue locating at the similar position within the retinal binding pocket as Y185 in bR is worthy to study deeply.Aiming on this hot topic,SOE-PCR is applied to clone the Y186F-ar4 gene,then the gene is expressed in Halobacterium salinarum L3 3 with the vector pXLNovR to form the recombinant protein Y186FL33-aR4.Y186FL33-aR4 is then characterized by comparing with the recombinant aR4(RCL33-aR4)through flash light induced kinetic absorption change measurements and UV-VIS spectroscopy.2D solid-state NMR,reinforced with Fourier transform infrared spectroscopy,dynamic light scattering,titration analysis and ATP formation rate measurements is employed to study the function of Y186.By comparing with Y185 in bR,modulation mechanism of Y186 on the cis-trans isomerization thermal equilibrium of the retinal chromophore,photocycle,and further on energy conversion in aR4 is discussed.4 nm blue shift of the absorption maximum in Y186FL33-aR4 is observed,which is similar to Y185FL33-bR.A fivefold longer lifetime of the M intermediate and a twofold longer lifetime of the O intermediate are observed in Y186FL33-aR4,by comparing with the recombinant aR4.In addition,the proton-pumping activity is reduced in Y186FL33-aR4 distinctly.However,on the other hand,the Y185F mutation of bR causes not only the similar affection,but also a missing O intermediate.Solid-state NMR and FTIR measurements of the RCL33-aR4 and Y186FL33-aR4 reconstituted with the[10,11,14,15-13C4]-all-trans-retinal show a different mediation mechanism of Y186 on the thermal equilibrium of the retinal chromophore and further on the photocycle in aR4.Furthermore,Y186F mutation affects the protein assembling status and causes a decrease of the ATP formation rate by about 37%.All the results indicate that Y186 may be one of the key residues within the retinal binding pocket to mediate the proton transport and energy conversion in aR4.