Effects of Mutations of Lys41 and Asp102 of Bacteriorhodopsin

Transcription

1 Biosci. Biotechnol. Biochem., 75 (7), , 2011 Effects of Muttions of Lys41 nd Asp102 of Bcteriorhodopsin Yingchun ZHAO, 1;2 Yzhuo WANG, 1 Dewng MA, 1 Ji WU, 1 Weid HUANG, 2 nd Jindong DING 1;y 1 Key Lortory of Moleculr Engineering of Polymers of the Ministry of Eduction, Deprtment of Mcromoleculr Science, Lortory of Advnced Mterils, Fudn University, Shnghi , Chin 2 Deprtment of Biochemistry, School of Life Sciences, Fudn University, Shnghi , Chin Received Mrch 8, 2011; Accepted April 12, 2011; Online Puliction, July 7, 2011 [doi: / ] Bcteriorhodopsin (BR) is retinl protein tht functions s light-driven proton pump. In this study, six novel mutnts including K41E nd D102K, were otined to verify or rule out the possiility tht residues Lys41 nd Asp102 re determinnts of the time order of proton relese nd uptke, ecuse we found tht the order ws reversed in nother retinl protein rcherhodopsin 4 (AR4), which hd different 41th nd 102th residues. Our results rule out tht possiility nd confirm tht the pk of the proton relese complex (PRC) determines the time order. Nevertheless, muttions, especilly D102K, were found to ffect the kinetics of proton uptke sustntilly nd the pk of Asp96. Compred to the wild-type BR (BR-WT), the decy of the M intermedite nd proton uptke in the photocycle ws slowed out 3-fold in D102K. Hence those residues might e involved in proton uptke nd delivery to the internl proton donor. Key words: cteriorhodopsin; point mutnt; proton pump; M decy BR is retinl-contining protein cting s lightdriven proton pump. It ws found in the purple memrne of the extremely hlophilic rchecterium Hlocterium slinrum. 1) BR hs een investigted extensively over the pst four decdes. 2 18) Upon photon sorption, the retinl is isomerized from the ll-trns to the 13-cis configurtion. This initites photocycle in which series of photointermedites re formed. They re distinguished y their sorption mxim: BR 570! hv K 610 $ L 550 $ M412 I! MII 412 $ N 560 $ O 640! BR 570 The photocycle is coupled to the directionl trnsfer of proton cross the memrne. Illumintion induces proton trnsfer from the nitrogen of the Schiff se linking retinl nd Lys-216 to Asp-85 during the L-to-M trnsition, which leds, on sumillisecond timescle, to prompt proton relese from the proton relese complex (PRC or XH) to the extrcellulr surfce t neutrl ph ) After proton relese, nother proton is tken up from the cytoplsmic side in the N 560 -to-o 640 trnsition 22) or trnsition etween the two N-susttes, 23) nd lifetime constnt of few milliseconds is usul. Therefore, proton pumping y BR under physiologicl conditions involves proton relese on the extrcellulr side, followed y proton uptke on the cytoplsmic side. Site-specific mutgenesis is used extensively to illustrte the reltionship of function nd structure for vrious proteins. 7,24 26) It hs een found tht croxylic residues in the proton-conducting cytoplsmic nd extrcellulr hlf-chnnels, D96, D85, E194, nd E204 re involved in the photocycle nd proton trnsfer. Negtively chrged D85 is prt of the counterion to the protonted Schiff se nd proton cceptor. Buried in the cytoplsmic domin, neutrl D96 serves s n internl proton donor for the Schiff se in the M intermedite. Two glutmtes t the extrcellulr surfce, E194 nd E204, nd some intercting wter molecules re thought to comprise PRC. 6,27 29) While much progress hs een mde in finding key residues in BR, only out 58 residues hve een exmined to dte while the functionl significnce of mjority of the residues in BR remins undetermined. Especilly the key residues in the cytoplsmic side re less investigted thn the extrcellulr side, where proton relese tkes plce nd the PRC is locted. The mutnts locted on the cytoplsmic side including D36N, D38N, D102N, nd D104N, were expressed in E. coli. They presented slightly decresed time of M decy, 30) ut proton pumping rtes similr or higher thn the BR-WT. 31) Among the other mutnts expressed in Hlocterium slinrum L33, with D36, D38, D102, nd D104 replced y cysteine or rginine, D36 ws found to ct s proton ttrctor t the orifice of the proton-conducting chnnel, 32) D38 ws found to influence the lte steps in the functionl photocycle, such s the occurrence of intermedites N nd O, 33) nd D104C lowered the trnsient proton inding cpcity of the cluster tht consists of D104, E161, nd E ) Hints from comprison of the BR sequence to those of other rchel nd cteril retinl proteins is helpful in serching for residues involved in proton pumping. 7) In the present study we exmined effects of mutting Lys- 41 (K41) nd Asp-102 (D102), two chrged residues on the cytoplsmic side of the light-driven proton pump. These two mino cid residues were selected for comprison of BR nd AR4. AR4 ws found in strin of the hlocterium Hlorurum species xz515 isolted from slt lke in y To whom correspondence should e ddressed. Fx: ; E-mil: jdding1@fudn.edu.cn Arevitions: BR, cteriorhodopsin; AR4, rcherhodopsin 4; PRC, proton relese complex; BR-WT, wild-type BR expressed in L33

2 Effects of Muttions of Bcteriorhodopsin 1365 difficulty hs lso een reported for other rcherhodopsins. 42,43) Fig. 1. Schemtic Presenttion of All the Amino Acids in BR-WT nd Its Structure of Seven Trnsmemrne Helixes. In the photocycle, proton is relesed to the extrcellulr side nd nother proton is tken up from the cytoplsmic side. Squres nd dimonds denote cidic nd lkline residues respectively. Two chrged residues on the cytoplsmic side, K41 nd D102, in BR re highlighted y lrge ellipses. In AR4 these residues re replced y E nd K respectively. Tiet in ) Its mino cid sequence hs een determined. 36,37) It exhiits 59% sequence similrity to BR. 37) Like BR, AR4 functions s light-driven proton pump. In contrst to BR, it exhiits reversed order of light-driven proton relese nd uptke t neutrl ph, 35) even though oth BR nd AR4 hve key residues E194 nd E204, cting s PRC. It lso exhiits severl other fetures (fster decy of the M intermedite nd higher pk of the counterion). We hve found tht the inversion of the order of proton relese nd uptke is cused y the elevted pk of the PRC in the M intermedite (from 5.6 in BR to 8.3 in AR4) ) The higher pk of the PRC in the M intermedite of AR4 might e cused y replcement of nery or distnt residues. Severl polr or chrged residues locted in the extrcellulr side re conserved in BR nd AR4, including R7, E9, nd K129. 7) A few polr or chrged residues t the cytoplsmic side re different in these pigments. Especilly, K41 in helix B nd D102 in loop-linking helixes C nd D (the CD loop) re interesting. Their loctions re schemticlly presented in Fig. 1. In AR4, these two residues re replced y E nd K respectively. In this study, we exmined the consequences of muttions of the two residues (K41 nd D102) on the cytoplsmic surfce of BR. A series of point mutnts of BR, including K41E, K41D, D102K, D102E, K41E/ D102K, K41D/D102E re designed nd reported for the first time. Our investigtion indictes tht muttions of these two mino cid residues in the cytoplsmic side sustntilly slow the kinetics of M decy nd proton uptke, ut do not determine the temporl order of proton relese nd uptke. The structurl nd mechnicl implictions of these results re discussed. Mterils nd Methods BR-R 1 M 1 nd AR4-xz515. Nturl Hlocterium slinrum strin R 1 M 1 nd xz515 nd gene engineering strin L33 stndrd procedures 40,41) following previous report. 35) Retinl proteins BR-R 1 M 1 nd AR4-xz515 nd the gene engineering proteins expressed in L33 BR-WT nd other mutnts were isolted. BR-R 1 M 1 ws the only protein in the purple memrne isolted from strin R 1 M 1, nd AR4- xz515 ws the only protein in the clret memrne isolted from strin xz515. The clret memrne contins some crotenoid cterioruerin, which ws hrd to seprte from AR4 y our purifiction method. This Point muttion vi gene engineering. The regents needed for gene engineering, including the restricted nuclese, T4 ligse nd Tq DNA polymerse were purchsed from BioRd (Hercules, CA). The oligonucleotide primers for the mutnts were synthesized nd purified with oligonucleotide purifiction crtridge (OPC) from Applied Biosystems (USA). Gene engineering BR-WT nd mutnt proteins were expressed in H. slinrium strin L33. This stin is negtive for the synthesis of cterio-opsin (op), ut positive for the synthesis of retinl. Expression of the BR mutnt proteins in strin L33 ws done y the method of Ni et l. 44) The expressing plsmid for the K41E mutnt, Lys41 replced y Glu, ws derivtive from plsmid pxlnov-r-op, gift from Dr. Richrd Needlemn. Primer 1: 5 0 -GGATCCGCGTGAA- GATGGGGC-3 0 (BmHI site, underlined); primer 2: 5 0 -AAGCTT- CTAGATCAGTCGCTGG-3 0 (HindIII site, underlined); Primer 3: 5 0 -GCAAAGGAGTTCTACGCCATCACGACGC-3 0 ; nd Primer 4: 5 0 -GTAGAACTCCTTTGCATCTGGGTCCGAG-3 0 were used in order to produce the op gene sequence with K41E. A new 1.2-k DNA sequence ws otined nd, ws nmed op-(k41e), with BmHI nd HindIII sites. After the sequence of the op-(k41e) frgment ws checked, frctions produced y PCR mplifiction were ligted to the psk plsmid nd nlyzed for identifiction. Other point mutnts of BR, including K41D, D102K, D102E, K41E/D102K, nd K41D/D102E, were constructed y the sme method. All of the primers used to construct the mutnts re shown s the primers 3 to 10 in Supplementl Tle S1 (see Biosci. Biotechnol. Biochem. We site) in supporting informtion. The pxlnov-r plsmid does not contin the gene frgment of cterio-opsin in contrst with the pxlnov-r-op plsmid. This plsmid nd the PCR mplified products of the op-(k41e) gene were digested with BmHI nd HindIII restriction enzymes nd ligted together. The trnsformnts grew on pltes contining 1 mg/ml of novoiocin for 2 weeks, nd then were inoculted into liquid culture medium for lrge-scle culture in the presence of 1 mg/ml of novoiocin. The resulting purple memrnes were isolted y the sme procedure s previously. 40,41) Mss spectr. Mss spectr of BR-WT nd the series of mutnts were mesured in mtrix-ssisted lser desorption/ioniztion timeof-flight mss spectrometer (Applied Biosystems Voyger System 4310). The ice-dried protein smples were dissolved with pure cetic cid t concentrtion of 10 mg/ml. Then the mtrix, 2,5-dihydroxyenzoic cid (DHB), ws dissolved with methnol nd chloroform (v/v 2:1) t concentrtion of 10 mg/ml. Finlly, 2 ml of smple solution nd 5 ml of mtrix solution were mixed thoroughly nd dropped on the smple plte for MALDI-TOF mesurement. Circulr dichroism spectr. Circulr dichroism (CD) spectr in the ultrviolent (UV) region were mesured with spectropolrimeter (J- 715; Jsco, Tokyo) t 20 C. The smples were dissolved in Milli-Q wter t concentrtion of 20 mg/ml. The pth length of the mesured cells ws 1 cm. The molr ellipticity (degcm 2 dmol 1 ) ws determined on men-residue sis. Asorption spectr. The sorption spectr of the smples were mesured on Shimdzu UV-3150 spectrophotometer (Shimdzu, Ohtsu, Jpn). For light dpttion, 200 W tungsten lmp with 500 nm cut-off filter ws employed. Drk dpttion of smples ws chieved when necessry t 25 C for 24 h. The rte constnt of drk dpttion ws determined y mesuring the sorption chnges in the light-dpted smples t 570 nm t intervls of 1 min in the drk environment. The curve ws fitted with one-component exponentil decy. Smples were prepred in 0.2 mol/l phosphte uffer sline (PBS) solution t ph 7.0. Flsh-induced photocycle intermedites nd proton pump ehviors. Trces of flsh-induced proton relese nd uptke were detected in spectrophotometer, which we mnufctured in our l. A photoflsh for light-dpted smples ws used for excittion. The M intermedite ws monitored t 412 nm. In order to monitor flshinduced proton relese nd uptke y BR, sorption chnges in the

3 1366 Y. ZHAO et l. ph-sensitive dye pyrnine were detected t 456 nm, t which pyrnine hs mximum sorption while no BR intermedites hve significnt sorption. The signl without pyrnine ws sutrcted from tht with dye. All the mesurements were done t 25 C. Results Identifiction of mutnts The results for gene sequences of the mutnt frgments (Figs. S1 S6 in supporting informtion) nd the mss spectr of the mutnt (Figs. S7 S8) confirmed tht the pre-designed mutnts were successfully otined. The CD spectr of the mutnts in the UV region (Fig. S9) were similr to tht of BR-WT, suggesting tht the secondry structure did not chnge significntly in those mutnts nd confirming tht the mutnts might lso hve seven trnsmemrne -helixes. As the visile sorption spectr in Figs. S10 S11 indicte, the chromophore ws not pertured significntly. The rtes of drk dpttion of the BR mutnts indicte tht the replcements of mino cids t the cytoplsmic side did not significntly ffect the isomeriztion of the retinl chromophore (Fig. S12). Flsh-induced proton pumping nd M formtion nd decy The proton pumping kinetics of the AR4 nd BR nlogs t ph 7.0 re presented in Fig. 2. The smples were suspended in slt solution with 0.1 mol/l of NCl nd 0.02 mol/l of KCl. Proton-pumping ws trced with the ph-sensitive dye pyrnine. An increse in dye sornce t 456 nm reflected proton uptke y the protein, nd decrese in sornce corresponded to proton relese into the medium. As Fig. 2 shows, the BR mutnts exhiited proton-pumping ctivity with proton relese preceding proton uptke, similrly to BR-WT. These results confirm not only tht the mutnt mitined the sic iologicl function s light-driven proton pump, ut lso tht the replcement muttions did not lter the temporl order of proton relese nd uptke. The decrese in the rte of proton uptke presented most prominent in the D102K mutnt, which suggest D102 is involved in this process, perhps cting s proton ntenn in the cytoplsmic side. Flsh-induced chnges in sorption t 412 nm shown in Fig. 2 were due mostly to the M intermedite. The lifetime of M decy of the pigment ws otined y exponentil fitting. The fitting results re summrized in Fig. 3, long with the dely times of proton uptke s clculted y fitting the dt in Fig. 2 sed on single exponentil eqution. The chrcteristic times of M decy nd proton uptke were consistent with ech other. Compred to the wide-type BR, expressed in strin L33, every BR mutnt exhiited slower M decy. Two mutnts, D102K nd K41E/D102K showed 3-fold increses in M lifetime. ph dependence of M decy The kinetics of the M decy of BR-WT nd ll the mutnts were monitored t ph rnge of 4 to 10. As shown in Fig. 4, the M decy of BR-WT ws monophsic t neutrl ph nd ecme iphsic t higher ph, s fitted with two components, fst nd slow. Biphsic Fig. 2. Photoresponse of BR nd Its Mutnts nd of AR4. () Flsh-induced chnges in sorption re indicted y pyrnine t 456 nm. The sorption chnge is due to the trnsient chnge in the medium ph, indicting the time order of proton relese nd uptke of the light-driven proton pumps. () Flsh-induced chnges in sorption t 412 nm representing the M intermedite. In oth () nd (), the mesurements were done t neutrl ph. The titrtion trces were shifted for etter viewing. decy ws lso oserved t ph levels elow 7. The K41E/D102K mutnt exhiited iphsic decy t ll ph levels. The frction of fst M decy in the mutnt ws smller thn in the WT, suggesting tht the muttion on the surfce of the protein hd n effect on the internl proton trnsfer from Asp96 to the Schiff se. Aout 3-fold elongtion of the slow component of M decy in the mutnt ws oserved from ph 4 to 8, ut t higher ph levels the difference with the BR-WT ws smller, indicting the possiility tht it ws cused y the

4 Effects of Muttions of Bcteriorhodopsin 1367 Fig. 5. pk Vlues of D96 in Proteins Expressed in Gene-Engineering Strin L33 s Well s Wild-Type Proteins from Strins R1M1 nd xz515. Vlues were otined y fitting the ph dependence of the mximum light-induced sorption chnge t 660 nm (O intermedite) divided y mximum sorption chnge t 410 nm (M stte) t the corresponding ph y the eqution y ¼ A 1 þða 2 A 1 Þ=½1 þ 10 nðx pkþ Š in the lkline regime, s shown in Fig. S13 in supporting informtion. chrge on Lys-102, which ws deprotonted t higher ph levels. Similr results were found for D102K (Fig. S13). Fig. 3. Histogrm of the Chrcteristic Times of () Proton Uptke nd () M Decy of the BR, BR Mutnts nd AR4 t Neutrl ph. Fig. 4. ph Dependence of M Decy of BR-WT nd Mutnts. () The kinetics of M decy of BR-WT nd its mutnt K41E/ D102K t ph 7.0 nd 10.0; () The lifetime of the M intermedite of BR-WT nd K41E/D102K fitted with i-exponentil eqution. The decy of BR-WT is monophsic, with similr fst nd slow t neutrl ph, while it is iphsic t the other indicted ph levels. K41E/D102K presented iphsic t ll ph levels exmined. ph dependence of the formtion nd decy of the O intermedite (pk of Asp-96) The kinetics of the O formtion nd decy of the BR- WT nd mutnt proteins t neutrl ph mesured t 660 nm re shown in Fig. S14. The ph dependence of the O intermedite ws used to estimte the pk of D96, the internl proton donor to the Schiff se in the photocycle. Figure S15 descries the chnge in mximum sorption t 660 nm reltive to the chnge mximum sorption t 412 nm during the N-to-O process in the photocycle. The trnsition in the cidic ph region corresponded to the lrger ccumultion of the O intermedite long with the decrese in the rte of its decy, nd reflects pk of the PRC in the O intermedite, nd the trnsition in the lkline region ws cused y slowing of proton uptke nd reflected the pk of D96 in the N intermedite. 45) All of the pk vlues in the lkline region of D96 of the AR4, BR- R 1 M 1, BR-WT nd BR mutnts re summrized in Fig. 5. These dt illustrte tht the mount of the O intermedite nd its pk strongly decresed in the K41E/D102K mutnt, consistently with slowing of M decy nd proton uptke in tht mutnt. The pk of PRC in the M intermedite Our group recently developed n efficient photoelectrochemicl pproch 14) to study the ph dependence of proton relese nd uptke of retinl proteins nd to determine the pk of PRC in the photocycles. In this study, we employed this pproch to investigte the BR mutnts. Figure 6 showed photo-response profiles from K41E/D102K film deposited on n ITO electrode in n electrolyte solution of 100 mmol/l NCl nd 20 mmol/l KCl t vrious ph. The mplitude nd polrity of the oserved signls chnged with the medium ph. The signls were negtive t ph levels ove 6, ut ecme positive t ph levels elow 5. A trnsition thus took plce etween 5 nd 6. The ph

5 1368 Y. ZHAO et l. Fig. 7. pk Vlues of PRC in the Indicted Proteins Expressed in Gene-Engineering Strin L33 s Well s Wild-Type Proteins from Strins R1M1 nd xz515. Vlues were otined y fitting the titrtion of the frction of fst proton relese versus medium ph in the photoelectrochemicl pproch with typicl trce, shown in Fig. 6, nd other trces, shown in Fig. S14. All pk vlues in the BR pigments were elow ph 7 (dotted line), while AR4 ws ove tht. Discussion c Fig. 6. Kinetics of Light-Induced Trnsient Potentil Chnges from K41E/D102K Film Deposited on n ITO Electrode nd Mesured in 100 mmol NCl nd 20 mmol KCl nd Estimtion of the pk of the PRC. () Flsh-induced photovoltge under the indicted ph vlues. Just one dt point for every 10 dt points collected is shown, for clrity, in () for ech ph; only five sets of dt re plotted, lthough nine ph conditions were exmined. () The time constnts of proton relese nd uptke t different ph levels otined from the fit. When ph ws elow 6, only proton uptke nd slow relese were oserved. (c) The frction of fst proton relese over ll protons relesed, f, s function of ph to determine the pk of PRC during the photocycle (in the M stte). The dt were fitted with the Henderson- Hssellch eqution, y ¼ A 1 þða 2 A 1 Þ=½1 þ 10 nðx pkþ Š, resulting in pk of 5:2 0:1 (n ¼ 1:3). dependence of the time constnts of proton relese nd uptke re presented in Fig. 6. Bsed on the frction of fst relese t series of ph levels, we determined the pk of PRC of K41E/D102K (Fig. 6c). The photo-response profiles nd the pk of PRC of the other mutnts re presented in Fig. S16 in supporting informtion. Figure 7 summrizes ll the pk vlues of the mutnts of BR nd AR4. The resulting pk vlues of PRC of ll the BR mutnts fter illumintion were lower thn 7, like tht of BR-WT (pk 6.3) nd tht of BR- R 1 M 1 (pk 5.7). Thus the temporl order of proton relese nd uptke of mutnts t neutrl ph were the sme s tht of BR-WT. Our mutgenesis of K41 nd D102 in BR ws initilly done to interpret the reversed time order of proton relese nd uptke of AR4. Govindjee et l. found tht muttion of surfce residue, Lys-129 of BR, on the extrcellulr side reversed the temporl sequence of proton relese nd uptke, 46) nd thus the replcement of one chrged residue might lter the time order. Hence, we devoted ttention to chrged residues on the cytoplsmic side where proton is tken up in the photocycle. Two chrged residues, K41 nd D102, which re different etween BR nd AR4, ecme cndidtes. The present study rules out the possiility tht mino cids Lys41 nd Asp102 re determinnts of the time order of proton relese nd uptke. The residues determining the different time orders of proton relese nd uptke ( much higher pk for PRC in rcherhodopsin 14) ) remin to e identified, nd further exmintion is clled for. Although mutgenesis did not reverse the order of proton relese nd uptke, the replcements resulted in significnt chnges in certin photo-response properties of BR, including the decy time of the M intermedite, the time constnt of proton uptke, nd the proton ffinity of D96 in the photocycle. As shown in Fig. 3, M-intermedite decy ws out 3-fold slowed down in D102K nd the corresponding doule mutnt K41E/ D102K, s compred to BR-WT. Thus, the replcement of Asp t site 102 with Lys ffected the efficiency of proton collection from the cytoplsmic surfce. This is especilly interesting considering tht D102 is locted in the CD loop. Our results gree with those of Riesle et l., 33) who proposed tht polr or chrged residues on the cytoplsmic side of BR might prticipte in or ffect re-protontion of the Schiff se nd proton uptke. In their studies, M decy ws prolonged strongly y the mutnt of D 2 102,104R 2, in which two sprttes were replced y rginines. 33,47) However, nother group found tht replcement of D102 with neutrl Asn did not cuse sustntil chnges, nd hence questioned the role of this Asp residue in proton collection. 48) Due to

6 our novel muttions, the pk of D96 of BR-WT decresed from 7.9 to D102K (6.5) nd K41E/D102K (6.9) (Fig. 5). The lowered pk of D96 pprently wekens the ssocition of proton with sprtic cid, especilly t ph levels higher thn tht of pk. Proton trnsfer from the cytoplsmic surfce to D96 ws 3-fold slowed t ph levels elow 9, which is consistent with the slowing of the decy of M intermedite, since it is in equilirium with the N intermedite. The decy of the ltter is strightforwrdly relted to proton uptke. 6) Thus, the present study suggests tht the cidic 102 site of BR is involved in the process of proton uptke from the cytoplsmic side nd dontion of it to Asp96, since replcement of Asp102 with Lys interfered with de-protontion nd re-protontion of Asp96, nd even with re-protontion of the Schiff se. Using the photoelectrochemicl pproch recently developed y us, 14) the pk vlues of the PRC of BR mutnts fter illumintion were mesured. While the muttion t the cytoplsmic side might hve distured the chrge distriution of the cytoplsmic surfce of BR nd hve influenced the pk of PRC, the vlues were still fr lower thn tht of AR4. Hence those muttions of BR do not led to the reversed time order of proton relese nd uptke. In summry, we performed gene engineering on BR nd exmined the effects of the K41 nd D102 muttions on the functions of the light-driven proton pump. These replcements did not lter the sic sptil structure of the protein, nd the chrged mino cids ffected proton uptke on the cytoplsmic side. Hence the two residues re not key to the temporl order of proton relese nd uptke of BR nd AR4. Nevertheless, these replcements influenced the photocycle y ffecting the proton ffinity of D96 nd the rte of proton delivery to it through the proton chnnel. The mutnts contining D102K prolonged the lifetime of the M intermedite out 3-fold. 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