| Rhodopsins are a kind of photoactive membrane chromoproteins, with a molecular weight of26kD containing248amino acid residues. Rhodopsins are the opsins comprised seven TM a-helix, and the chromophore in the protein is an all-trans retinal covalently bound via lysine-Schiff base to opsins. They were first discovered by analysis of the genome fragment of a marine gamma-proteobacterium from the SAR86clade, which has high sequence similarity to bR. The sequence codogenic protein was proteorhodopsin (PR). PR shares30%sequence identity to bR. Since then, PR belonging to different bacterial groups, have been found in many different marine environments, such as fresh water and marine environment. Subsequent screening of DNA from different oceans revealed a very large diversity of PR in bacteria belonging to divergent clades of the Alpha-proteobacteria, Gamma-proteobacteria classes and Bacteroides in the sea and so on. Proteorhodopsins (PRs) were distinguished for a-proteobacteria,(3-proteobacterial, flavobacteria and bacteroides according to PR host. The PRs protein can change the light absorption maximum from490nm (blue) to525nm (green), so PRs were also classified to a blue light-absorbing proteorhodopsin (BPR) and a green light-absorbing proteorhodopsin (GPR).Proteorhodopsins are bacterial light-dependent proton pumps and ubiquitous retinal-binding membrane proteins, which functions as photo-isomerization using retinal. Light excitation of PR initiates a photocycle, the retinal was caused isomerization when absorption of light energy, that triggers the translocation of a proton from the cytoplasmic to the extracellular side. The proton motive force is used to synthesize ATP, and then to drive chemiosmotic reactions, and power the rotary flagellar motor. The retinal was caused isomerization when absorption of light energy, induce a protonated Schiff base and Asp97, Glu108, Arg94, His75constitution changed. To urge de-protonated Asp97and protonated Glu108, the proton was translocated from the cytoplasmic to the extracellular side. Not only proteorhodopsin have been found in a variety of environments, but also have an important role in supplying light energy for microbial metabolism in the world. To compare with bR, the structure of PR and the light-dependent proton pumps have not be clearly known now, so the people should be extremely to study PR structure, especially three-dimensional structure.These bacteria which have proteorhodopsin were isolated from surface water and were unsuccessfully cultured. A memberane gene of blue-absorbing proteorhodopsin isolated from75-meter deep plankton from the Hawaiian Ocean Time (Hot75) Station was expressed in E. coli, and then uased for proton pumps and PR structure study and research.We use bioinformatics to find the amino acids of light-dependent proton pumps. Archaeal bacteriorhodopsin is a homologue of Proteorhodopsin, bR proton transfer amino acid were Arg85, E194and E204. The sequence comparison between PR and bR is demonstrated that most of the amino acid residues involved in the vectorial proton transfer from the cytoplasm to the extracellular medium are not conserved. There is only Arg85in bR corresponding to Arg94conservative in PR. The mutant proteins Arg94Lys, Arg94Leu, Arg94Glu and Arg94Ala did not show light-dependent proton pumps at all. Light excitation of PR triggers the translocation of a proton from the cytoplasmic to the extracellular side, but the PR Arg94mutants show no proton pumps. Otherwise, the Photochemical Reactions are diversity and a greatly retarded in proteorhodopsin Arg94mutant. The ground state of Photochemical Reactions in mutants recovers more than10-fold slower than in wild-type PR. The proton donor to the Schiff base for the proton transfer occurring during the transition of the M inter-mediate to later red-shifted states as evidenced by a greatly retarded M decay in PR mutant, and most accumulation is in the red-shifted M state without marked accumulation of M by the rise and decay of an O. In contrast to the natural mutant of PR Arg94has the same phenomenon. We also chose kytoplasm interspaceal acidity amino acid residue to catastrophe, such as D85,D89, E144, D213. These mutants have the functions as light-dependent proton pumps.We constructed expression vectors HotD97N no20for cutting20aas from the DHD of HotD97N with pET expression system respectively, and expressed them in E. coli C43(DE3) pLysS, the expression product (HotD97N no20) were induced by IPTG and appending retinal. The protein of PR was purified by Ni-NTA and gel-filtration, and was used to crystallize. Crystals were screened out at295K using sitting-drop vapor diffusion technique and Hampton research crystallization conditions kit. The mutant Hot D97N no20 crystals in six different crystallization conditions were screened out by using sitting-drop vapor diffusion technique. The condition:0.2mol/L Ammonium Acetate,0.1mol/L Bis-Tris pH6.5,45%v/v2-Methyl-2,4-pentanediol; anapoe-80; EDTA. The proteins were both at the concentration of20mg/mL. Data collection about Hot D97N no20crystals was collected on beamline Swiss Light Sourcesynchrotron radiation beamlines (SLS) at100K, which resulted in a complete3.3A resolution data set. The unit-cell parameters was a=163.47, b=169.39, c=69.58, with5-6PR molecules in the asymmetric unit. To solve the structure did not success due to the low sequence similarity with its homologs by using molecular replacement. The diffraction data were indexed, integrated and scaled with XDS, further conducted with the CCP4suite. We use COOT to build the structure and find part of the main chain, but we cannot find the hydro-amino acids. We collected diffraction data of Hot D97N no20crystal soaked with substrate Pt, Ta, Hg and KI, and anomalous signal was detected from the KI, Ta soaked crystal diffraction dataset. We collected diffraction data of Hot D97N no20selenomethionyl protein crystals, and anomalous signal was detected. But the results were not enough stronger to solve the phase of Hot D97N no20.To prepare selenomethionyl (Se-Met) protein crystals, anomalous signal was detected from the Se-Met crystal diffraction dataset to solve the phase of PR. The PR gene encoded a polypeptide of249amino acids, with11methionine amino acids. As to there is more anomalous signal in crystal diffraction, we can not find the phase, so we chose the six methionine mutated. Mutant HotD97N six M was constructed using two-step PCR, expressed in E. coli C43(DE3) pLysS. The expression product HotD97N six M was purified by Ni-NTA and gel-filtration and used to prepare Selenomethionyl protein crystals.Most of the amino acid residues involved in the vectorial proton transfer and those carboxylic acid residues involved in the proton release, the striking differences of Arg94were observed, which provides insight into proteorhodopsin diversity in mechanism and possibly physiological function. The mutantHotR94A was constructed using two-step PCR, expressed in E. coli C43(DE3) pLysS, and HotR94A was purified by Ni-NTA and gel-filtration. Otherwise, the difference of PR protein absorption spectra and photochemical reactions were due to amino acids at position105. Mutant BPR-HotQ105L no20was constructed using two-step PCR, expressed in E. coli C43(DE3) pLysS, and BPR-H otQ105L no20was purified by Ni-NTA and gel-filtration in the same way.The Hot R94A protein used are20mg/mL:0.2mol/L Magnesium chloride hexahydrate,0.1mol/L Hepes pH7.0,25%v/v Polyethylene glycol400, the crystals were grow quickly for three day. We try to sublimate crystals quality by using Additive Screen, Detergent Screen but the crystal was not changed. It diffracted to20A. The HotQ105L no20protein crystal condition is0.2mol/L Magnesium chloride hexahydrate,0.1mol/L HEPES sodium pH7.5,30%v/v Polyethylene glycol400, and crystal diffraction is also20A. |
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