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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2019, Vol. 39 Issue (1): 21-30    DOI: 10.13523/j.cb.20190103
    
Effect of Mutation of M145F / F146M on the Photocycle of Photoreceptors Bacteriorhodopsin and Archaerhodopsin 4
Ya-nan YANG,Chao SUN,Hao-lin CUI,Xin ZHAO()
Shanghai Key Laboratory of Magnetic Resonance, School of Physics and Materials Science,East China Normal University, Shanghai 200062, China
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Abstract  

Both archaerhodopsin 4 (aR4) and bacteriorhodopsin (bR) belong to the Halobacterium family, share 59% homology and function as proton pumps to transfer protons from the cytoplasmic side to the extracellular side to convert light energy to chemical energy through ATP synthesis. Although aR4 has a similar photocycle as bR, it has an opposite temporal order of proton uptake and release at neutral pH. Methionine-145 (M145), one of the key residues locating within the retinal binding pocket in bR, has significant influence on the bR photocycle. Phenylalanine-146 (F146) is the corresponding residue within the retinal binding pocket in aR4, and is the only different binding residue between the two proteins. Effects of M145F and F146M mutations on the photocycle of bR and aR4 were studied by UV-VIS spectroscopy, light-induced kinetic change spectroscopy, proton pumping analysis and low temperature transmission FTIR spectroscopy. Loss of L state and decrease of the proton pumping capability in M145F mutation were observed in the bR photocycle, whereas neither a significant affect to the photocycle nor any a change to proton release and uptake order by F146M mutation were observed in aR4, which clear indicated that the function roles of the two residues are not exactly the same in the two proton pumps.

Key words： Archaerhodopsin 4      M145F/F146M single point mutation      Intermediate state and photocycle      Proton pump      Low temperature transmission FTIR spectroscopy     
Received: 15 March 2018      Published: 28 February 2019
ZTFLH:  Q93  
Corresponding Authors: Xin ZHAO     E-mail: xzhao@phy.ecnu.edu.cn
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Ya-nan YANG
Chao SUN
Hao-lin CUI
Xin ZHAO
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Ya-nan YANG,Chao SUN,Hao-lin CUI,Xin ZHAO. Effect of Mutation of M145F / F146M on the Photocycle of Photoreceptors Bacteriorhodopsin and Archaerhodopsin 4. China Biotechnology, 2019, 39(1): 21-30.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190103     OR     https://manu60.magtech.com.cn/biotech/Y2019/V39/I1/21

Gene Primer name Primer sequence(5'- 3')
bop P1 5'-CGGGATCCGACGTGAAGATGGGG-3'
bop P2 5'-GCCAAGCTTCTAGATCAGTCGCTG -3'
M145FL33-bop P3 5'- CACCGCAGCGTTCCTGTACATC -3'
P4 5'-GATGTACAGGAACGCTGCGGTG-3'
F146ML33-aR4 P5 5'-CGATGCTGTTCGTGCTGTACTACCTC-3'
P6 5'-GAGGTAGTACAGCACGAACAGCATCG-3'
Table 1 Primers used for amplification
Fig.1 Amino acid sequence alignments of bR and aR4
Fig.2 SDS-PAGE of the recombinant of bR and aR4
Fig.3 UV-VIS absorption spectra of the recombinant of bR and aR4
Fig.4 Light-induced transient absorption changes of the M, O states and recovery towards the ground state at 410nm,660nm and 570nm, respectively for the recombinant bR and aR4 (a) RCL33-aR4 (b) F146ML33-aR4 (c) RCL33-bR (d) M145FL33-bR
Protein M-decay (ms) O-rise (ms) O-decay (ms) Recovery to G state (ms)
1st 2nd 3rd
RCL33-aR4 2.41
100%		 0.35
100%		 3.39
100%		 0.49
79.2%		 2.85
17.0%		 36.60
3.8%
F146ML33-aR4 2.73
100%		 0.27
100%		 2.88
100%		 142.8
16.0%		 8.10
19.3%		 1.19
64.6%
RCL33-bR 5.14
100%		 1.32
100%		 3.22
100%		 3.63
39.4%		 22.14
0.6%		 0.43
59.9%
M145FL33-bR 4.64
100%		 1.76
100%		 2.37
100%		 3.29
58.9%		 6.95
30.8%		 0.31
10.4%
Table 2 Time constants and amplitudes resulting from multi-exponential fitting to the data traces depicted in the Figure 4
Fig.5 Retinal binding pocket of bR (green) and aR4(orange)
Fig.6 Proton pumping curves of recombinant bR and aR4 (a) RCL33-aR4 (b) F146ML33-aR4 (c) RCL33-bR (d) M145FL33-bR
Protein RCL33-aR4 F146ML33-aR4 RCL33-bR M145FL33-bR
Recovery time (ms) 1.38 2.09 5.93 2.17
Table 3 Time constants resulting from exponential fitting to the data traces depicted in the Figure 6
Fig.7 Low temperature transmission infrared spectra of photo-intermediate states of RCL33-aR4 and M145FL33-bR The spectra record the intermediate states of RCL33-aR4(blue) and M145FL33-bR(red) (a) The spectra of K state (b) The spectra of L state (c) The spectra of M state (d) The spectra of N state
Fig.8 Low temperature transmission infrared spectra of photo-intermediate states of RCL33-bR and F146ML33-aR4 The spectra record the intermediate states of RCL33-bR(blue) and F146ML33-aR4(red) (a) The spectra of K state (b) The spectra of L state (c) The spectra of M state (d) The spectra of N state
[1]   Wang Y Z, Ma D W, Zhao Y C , et al. Light-driven proton pumps of archaerhodopsin and bacteriorhodopsin and polymer-matrix composite materials of those functional proteins. Acta Polymerica Sinica, 2012,12(7):698-713.
doi: 10.3724/SP.J.1105.2012.12051
[2]   Wang J, El-Sayed M A . Time-resolved Fourier transform infrared spectroscopy of the polarizable proton continua and the proton pump mechanism of bacteriorhodopsin. Biophysical Journal, 2001,80(2):961-971.
doi: 10.1016/S0006-3495(01)76075-4
[3]   Ernst O P, Lodowski D T, Elstner M , et al. Microbial and animal rhodopsins: structures, functions, and molecular mechanisms. Chemical Reviews, 2014,114(1):126-163.
doi: 10.1021/cr4003769 pmid: 3979449
[4]   Mukohata Y, Sugiyama Y, Ihara K , et al. An Australian halobacterium contains a novel proton pump retinal protein: archaerhodopsin. Biochemical and Biophysical Research Communications, 1988,151(3):1339-1345.
doi: 10.1016/S0006-291X(88)80509-6 pmid: 2833260
[5]   唐琳, 孙庆安, 李庆国 , 等. 西藏嗜盐菌xz515紫红膜中类菌紫质分子的AFM成像. 科学通报, 2001,46(14):1176-1179.
doi: 10.3321/j.issn:0023-074X.2001.14.009
[5]   Tang L, Sun Q A, Li Q G , et al. AFM imaging of bacteriorhodopsin-like protein in the claret membrane of Halobacterium species xz515. Chinese Science Bulletin, 2001,46(14):1176-1179.
doi: 10.3321/j.issn:0023-074X.2001.14.009
[6]   Braiman M S, Mogi T, Marti T , et al. Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212. Biochemistry, 1988,27(23):8516-8520.
doi: 10.1021/bi00423a002 pmid: 2851326
[7]   Bousche O, Braiman M, W He Y , et al. Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that ASP-96 deprotonates during the M-N transition. Journal of Biological Chemistry, 1991,266(17):11063-11067.
doi: 10.1074/jbc.273.33.20903 pmid: 2040618
[8]   Sonar S, Marti T, Rath P , et al. A redirected proton pathway in the bacteriorhodopsin mutant Tyr-57-->Asp. Evidence for proton translocation without schiff base deprotonation. Journal of Biological Chemistry, 1994,269(46):28851-28858.
doi: 10.1016/0092-8674(94)90557-6 pmid: 7961844
[9]   Ihara K, Amemiya T, Miyashita Y , et al. Met-145 is a key residue in the dark adaptation of bacteriorhodopsin homologs. Biophysical Journal, 1994,67(3):1187-1191.
doi: 10.1016/S0006-3495(94)80587-9 pmid: 7811932
[10]   Kouyama T, Fujii R, Kanada S , et al. Structure of archaerhodopsin-2 at 1.8? resolution. Acta Crystallographica Section D Biolgical Crystallography, 2014,70(10):2692-2701.
doi: 10.1107/S1399004714017313
[11]   王娟, 郜玉娇, 孙超 , 等. D97N突变对光受体蛋白古紫质4质子泵和能量转换效率的影响. 中国生物工程杂志, 2017,37(9):23-30.
[11]   Wang J, Gao Y J, Sun C , et al. Effect of D97N mutation on proton transport and energy conversion in the photoreceptor archaerhodopsin 4. China Biotechnology, 2017,37(9):23-30.
[12]   Cline S W, Lam W L, Charlebois R L , et al. Transformation methods for halophilic archaebacteria. Canadian Journal of Microbiology, 1989,35(1):148-152.
doi: 10.1139/m89-022
[13]   Oesterhelt D, Stoeckenius W . Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nature New Biology, 1971,233(39):149-152.
[14]   Rothschild K J, Marrero H . Infrared evidence that the Schiff base of bacteriorhodopsin is protonated: bR570 and K intermediates. Proceedings of the National Academy of Sciences of the United States of America, 1982,79(13):4045-4049.
doi: 10.1073/pnas.79.13.4045 pmid: 6955790
[15]   Rothschild K J, Marrero H, Braiman M , et al. Primary photochemistry of bacteriorhodopsin: comparison of fourier transform infrared difference spectra with resonance raman spectra. Photochemistry and Photobiology, 1984,40(5):675-679.
doi: 10.1111/j.1751-1097.1984.tb05359.x pmid: 6514815
[16]   Smith S O, Myers A B, Mathies R A , et al. Vibrational analysis of the all-trans retinal protonated Schiff base. Biophysical Journal, 1985,47(5):653-664.
doi: 10.1016/S0006-3495(85)83961-8 pmid: 4016185
[17]   Braiman M S, Mogi T, Stern L J , et al. Vibrational spectroscopy of bacteriorhodopsin mutants: I. tyrosine-185 protonates and deprotonates during the photocycle. Proteins: Structure, Function, and Bioinformatics, 1988,3(4):219-229.
doi: 10.1002/prot.340030403 pmid: 2843849
[18]   Chan S K, Kitajima-Ihara T, Fujii R , et al. Crystal structure of cruxrhodopsin-3 from Haloarcula vallismortis. PLoS One, 2014,9(9):e108362.
doi: 10.1371/journal.pone.0108362 pmid: 4182453
[1] WANG Juan, GAO Yu-jiao, SUN Chao, ZHAO Xin. Effect of D97N Mutation on Proton Transport and Energy Conversion in the Photoreceptor Archaerhodopsin 4[J]. China Biotechnology, 2017, 37(9): 23-30.
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