Molecular Dynamic Simulations Of The Structure-Function Relationship Of The Light-Driven Proton Pumps With Single Or Dual Chromophore

The cytoplasmic membrane is composed of liposomes,proteins,and carbohydrates,which have multiple functions and are the basis of life.The dynamics of membrane structure provide an important temporal correlation for the realization of membrane protein function through the protein-protein and protein-lipid interactions.More importantly,the completeness of the membrane protein dynamic function has a direct correspondence with the original dynamic conformational changes of its complete structure in the native plasma membrane environment.Microbial light-driven proton pumps are a class of seven-transmembrane light energy conversion systems containing retinal chromophores.Their efficient proton pumping ability and photocycling properties,make them highly application value in light energy conversion,optoelectronic materials,and optogenetics.In particular,the successful application of Archaerhodopsin 3(a R3)as an optogenetic tool has set off an upsurge in the study of such proteins.The successful resolution of numerous microbial light-driven proton pumping structures provides the possibility for the in-depth understanding of its activation mechanism and structure-based protein-directed engineering.However,the differences in oligomeric forms,lipid composition,and ligand assembly caused by different reconstitution strategies to the perturbation of plasma membrane environment destroy the completeness of protein structures,resulting in inconsistent results of photocycling kinetics,and some conclusions are even contradictory.This greatly affects the application research of light-driven proton pumps,especially the construction of novel optogenetic tools based on their structures.To further explore the relationship between the structural completeness and functional completeness of proton pump proteins,the single-chromophore proton pump Bacteriorhodopsin(b R)containing only retinal and the dual-chromophore proton pump Archaerhodopsin 4(a R4)containing both retinal and carotenoid bacterioruberin were selected as the research object.Using long-term all-atoms molecular dynamics(MD)simulations that more closely approximate the timescales of membrane protein’s native dynamic conformational changes,supplemented by automated fragmentation quantum mechanics/molecular mechanics method(AF-QM/MM),to systematically elucidated the influence of oligomeric structure,specific phospholipids,and second chromophores on the photocycling kinetics,proton uptake and release at the atomic level,as well as revealed the dynamic couplings of key residues in the retinal binding pocket with photocycling intermediates.This main work includes:(1)At the atomic level,we systematically and thoroughly studied the influence of the oligomeric structure on the overall dynamic conformational changes,the retinal cistrans thermal equilibrium,the key residues involved in proton transport,as well as water dynamics of b R and a R4.Also,we elucidated the molecular mechanism by which oligomeric structure regulate b R and a R4 normal photocycling and proton pumping function,and the reason for reversal of proton release and uptake temporal sequence in the a R4 monomer.(2)We provided unambiguous structural information about the specific interactions of non-bilayer archaeal lipids S-TGA-1 and PGP-Me in b R,and in-depth systematically studied the effects of archaeal lipids-b R interactions on the dynamic conformation,key domains involved in proton release and uptake,as well as water dynamics of b R.For the first time,we revealed the underlying molecular mechanism of how non-bilayer archaeal lipids archaeal lipids-b R specific interactions affect b R normal photocycling and proton transport.(3)At the atomic level,we systematically and thoroughly investigated the influence of the second chromophore bacterioruberin on the overall dynamic conformational changes,the retinal cis-trans thermal equilibrium,and the key residues involved in proton transport,as well as water dynamics of a R4.And we revealed the underlying molecular mechanism by which bacterioruberin regulates a R4 normal photocycling and proton pumping function.(4)Combined with solid-state NMR chemical shifts determination,automated fragmentation quantum mechanics/molecular mechanics(AF-QM/MM)approach to optimize the conformation of b R retinal binding pocket to obtain more accurate allatom MD calculation results.We systematically and thoroughly studied the effects of the highly conserved aromatic residues tyrosine185(Y185)in the microbial rhodopsins on the retinal cis-trans thermal equilibrium,the pentagonal hydrogen bond network,and the F42 gate at the atomic level,and revealed that the dynamic coupling of Y185 with the b R photocycling dynamic behaviors.The work could not only help to ensure the accuracy of the study on the activation mechanism of the light-driven proton pumps,and provide more theoretical guidelines for the rational design of the proteins.