Effects Of E194 / E195 And K129 / K130 On The Photocycle And Proton Pump In Photoreceptor BR And AR4

Bacteriorhodopsin?bR?and Archaerhodopsin-4?aR4?are both light-driven proton pump proteins found on the cell membranes of halophilic archaea,but their proton release and uptake are in reverse order,and Carotenoids are present in aR4 as the second chromophore.The difference in structure and function between the two proteins is very helpful to further study the differences in the proton transport mechanism of proton pump proteins.The mechanism of proton pump proton transport has been a research hotspot.Despite extensive research on bR,the proton transport mechanism,especially the proton release mechanism,is still unclear.As a key amino acid residue of the proton release complex,E194/E195 is an important part of the hydrogen bond network involved in proton transport.At the same time,K129/K130 is the only lysine residue on the outer side of bR/aR4,which may participate in maintaining the hydrogen of the interface and have a certain effect on proton transport.This article focuses on the goal of the influence of the two interface residues E194/E195 and K129/K130 on the structure and function of the photoreceptor protein bR/aR4.The UV-visible spectrum,dynamic light scattering,kinetic spectrum,proton pump function detection,pH titration and other methods were used to characterize the mutants under the conditions of the native membrane environment.To study the effects of E194/E195 and K129/K130 residue mutations on protein structural characteristics,photocycle,and proton pump function,explore the similarities and differences between the proton transport mechanisms in the two proton pump proteins,and lay the foundation for the determination of aR4.Through a comparative study,it is found that the proton transport mechanism in aR4 is not the same with bR.The mutation of E195 in the RC^MPK409-aR4 system causes the loss of the function of the proton pump.The RC^L33-bR and RC^L33-aR4 protein pumps are not lost,but the protons release process becomes slower.This is related to the structural differences of the proteins.The disappearance of the E195Q^MPK409-aR4and E195L^MPK409-aR4 proton pumps indicates that salinixanthin not only stabilizes the protein structure,but also has a modulation effect on the proton release process.The mutation of E194/E195 destroys the coupling relationship with D86/D86.Protonation of D85/D86 cannot drive the release of protons outside the cell,and the increase of pKa is not conducive to the transfer of protons from O state to PRC.Therefore,the decay of the kinetic intermediate state M state and O state is greatly prolonged,the protons cannot be released in the M state,and the O state is released to the outside of the cell,so the proton release process becomes slower.As the only lysine residue on the outside of the cell,K129 plays a very important role in maintaining the hydrogen bonding network on the extracellular interface and the proton transmission channel.Our research shows that both the K129E and K129L mutations cause the ability of bR to pump protons from the intracellular to the extracellular,the M-state generation is difficult,and the O-state signal becomes weak or even disappears,indicating that K129 residues participate in proton transmission.In conclusion,K129,as a positively charged amino acid residue on the outside of the cell,plays an important role in stabilizing the charge of the proton releasing group and the hydrogen bonding network of the proton transmission channel,which directly affects the photocycling dynamics and proton pump function of bR.K130 is also the only lysine on the outer side of the aR4 protein.Through a comparative study of K130E^L33-aR4 and K129E^L33-bR,it is found that the effect on the protein is not the same.The M state decays faster,and the proton pump decays faster,indicating that although they are conservative residues at the same position,due to the structural differences of the bR and aR4 proteins themselves,their effects on the function of the proton pump also differ.In general,E194/E195 and K129/K130,two polar residues with opposite charges,play an important role in the photocycle and proton transport of proton pump proteins.The study can further reveal the protons of proton pump proteins Transmission mechanism.