海洋微生物中变形菌视紫红质（Proteorhodopsin）结构和功能的研究
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摘要
视紫红质(Rhodopsins)是一类吸光色素膜蛋白,分子质量约为26kDa,是由7个跨膜α-螺旋(A-G)组成的视蛋白(opsins)与色素视黄醛(retinal)通过质子化的希夫碱共价结合而形成。2000年,研究者利用基因测序技术首次从海洋有光区微生物群落的DNA片断中发现了编码与嗜盐古细菌视紫红质(Bacteiorrhodosin, bR)有同源性蛋白的基因,其编码的蛋白被命名为变形菌视紫红质(Proteorhodopsin, PR)。变型菌视紫红质(PR)具有光驱动的质子泵功能,每个分子都含有一个被称为视黄醛的发色团,它的重要功能与视黄醛密不可分。PR在可见光照射下,PR中的视黄醛的构型发生改变,视蛋白利用视黄醛捕获的光能作为动力,将质子从细胞内侧泵向胞质间隙,从而形成跨膜的质子梯度,该质子梯度势能进而被ATP合酶利用产生ATP。初步研究显示,视黄醛在吸收光能后发生结构异构化作用,导致活性位点的希夫碱和Asp97、Glu108、Arg94、His75等氨基酸残基的构象发生变化,促使Asp97和Glu108等定向去质子化和质子化作用,从而将质子从细胞膜内转运到细胞膜外。PR蛋白的重要性不仅由于它存在的广泛性,而且体现在研究PR蛋白具有帮助人们开启利用光能又一新途径的潜力。但与bR相比,PR质子泵功能的神秘面纱还未为世人所揭开,需要从三维结构上进行解析和阐释。
我们主要对太平洋HOT站75米深海水的一种Proteobacteria型细菌中的PR(蓝光型)蛋白(BPR-Hot75)采用了基因克隆和E.coli表达系统对其进行了表达,然后用于PR蛋白结构与功能的研究。
PR蛋白的质子泵功能相关研究主要采用生物信息学的方法寻找出可能与质子泵作用相关的位点。前期的研究表明：PR的同源蛋白bR的质子释放基团主要由Arg85,E194和E204组成,PR与bR的序列比较后显示：在PR中只有与bR中的Arg85相对应的Arg94是保守的,而另外两个酸性氨基酸则不存在于PR中。因此,我们首先把PR中保守的Arg94氨基酸分别突变为赖氨酸(Lys)、亮氨酸(Leu)、谷氨酸(Glu)和丙氨酸(Ala),然后对其突变体的功能和特性进行了研究。结果显示：Arg94的突变都导致了PR质子转运功能的完全丧失,野生型PR能够在光照的情况下将质子从细胞内泵到细胞外,而PR的Arg突变体则不能将质子泵到细胞外。另外Arg94的突变也导致PR的光周期发生改变,导致绿光吸收型PR的光循环明显减慢,其中基态(ground state)在光照后的恢复期要比野生型慢十倍。另外蛋白构象变化中的M中间体发生了积累,O中间体则减少到很小的量,这是由于O中间体快速的衰减所导致。这种现象在蓝光吸收型PR中的也是这样。另外,我们还对从海水中分离到一个天然的Arg突变体的表达产物N5R8_5进行了研究,也显示了相类似的结果,而对其突变体(G94R)的表达产物N5R8_5_GR的研究结果显示,甘氨酸突变成精氨酸能使得其质子泵功能得以恢复,同时也改变了其光循环周期。而突变了PR的其它位于胞质间隙的其它酸性氨基酸残基,包括BPR的D85、D88、 E142、E213,结果显示这些氨基酸残基突变对PR的质子泵功能都没有明显的影响。
为了对PR的结构进行解析,采用PCR方法获得了Hot D97N nO20(nO20表示该蛋白N末端缺少了20个氨基酸残基的信号肽)突变体,采用Ni-NTA亲和层析纯化目的蛋白,并获得高纯度的Hot D97N nO20膜蛋白。对PR突变体Hot D97N nO20使用坐滴气相扩散法进行结晶,在295K的条件下生长获得了五个结晶条件。优化得到Index51结晶条件,并在瑞士同步辐射光源和上海光源中心采集到晶体的X-射线衍射数据,运用XDS晶体衍射数据处理软件包对衍射数据进行处理,并运用CCP4和Phenix等结构解析软件包分析数据。主要是采用分子置换(Molecular Replacement)的方法,以氨基酸序列与PR有约30%同源性的bR (PDB:1C3W,1CWQ)、XR (PDB:3DDL)、 SR (PDB:2F93,1GU8)等为模板,对其进行了衍射数据分析。其结果分别阐述如下：
Hot D97N no20蛋白最优的结晶条件为：0.2mol/L醋酸铵,0.1mol/L Bis-Tris pH6.5,45%v/v2-甲基-2,4-戊二醇；蛋白浓度均为20mg/ml,去垢剂为anapoe-80, Additive Screen为EDTA。晶体衍射度达到3.3A,空间群为P21212,晶胞参数为a=162, b=169,c=68；α=90°,β=90°,γ=90°,每个不对称空间中均只有五或六个单体。
由于MR无法解析Hot D97N nO20蛋白的三维结构,存在相位问题,所以尝试采用同晶置换的方法来解决相位问题。首先,采用重金属进行浸泡蛋白晶体,可以检测到重金属Pt、Hg的信号,但不能获得高衍射度的数据；使用卤素Ⅰ进行浸泡,发现其反常散射信号不是很强,无法使用其数据解决相位问题；使用重金属Ta对蛋白晶体进行浸泡,发现在衍射过程中蛋白晶体辐射损伤严重,导致其衍射度下降严重。其次,我们采用与重金属共结晶的方法来获得重金属衍射物晶体,信号扫描无重金属信号。另外,通过硒代甲硫氨酸方法表达获得的蛋白晶体进行X-射线衍射,所得衍射数据中Se的信号太弱。
为了制备Hot D97N no20硒代甲硫氨酸蛋白衍生物晶体,利用反常散射信息解决相位问题。因Hot D97N no20蛋白含有的11个甲硫氨酸,在X-衍射的过程中甲硫氨酸替换的位点太多,影响重原子的定位,故选择其中六个同源性低的甲硫氨酸进行了突变。通过重叠PCR、获得重组质粒和转化到表达宿主菌C43(DE3)中进行诱导表达,同样获得了Hot D97N six M突变体蛋白,并纯化得到高纯度的蛋白,可用于硒代甲硫氨酸蛋白衍生物晶体生长。
为了更进一步对PR质子释放氨基酸基团进行深入的研究,对Hot75中与质子释放相关的氨基酸94位Arg(R)进行了突变,将其突变Ala(A),进而获得了Hot R94A突变体。另外,PR蛋白吸收峰的差异的主要由于其105位的氨基酸不同造成的,其中BPR的第105位氨基酸残基为Gln(Q),而GPR的则为Leu (L)。为进一步深入研究105位氨基酸残基作用机理,又将BPR-Hot75蛋白的Gln突变为Leu。通过亲和层析纯化获得高纯度的Hot R94A、 H ot Q105L no20膜蛋白。Hot R94A蛋白质结晶条件：0.2mol/L氯化镁六水合物,0.1mol/L Hepes pH7.0,25%v/v聚乙二醇400；蛋白浓度为20mg/mL;使用坐滴气相扩散法,在295K下培养下约三天长出。使用Additive Screen和Detergent Screen对蛋白晶体进行优化,发现在Additive Screen作用下蛋白的晶体有着明显的改变,在上海光源中心17u光束线进行衍射,发现Hot R94A蛋白晶体衍射度只能达到20A。Ho Q105L no20蛋白我们只发现有一个结晶条件：0.2mol/L氯化镁六水合物,0.1mol/L HEPES sodium pH7.5,30%v/v聚乙二醇400,蛋白晶体衍射度也只能达到20A。
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.
我们主要对太平洋HOT站75米深海水的一种Proteobacteria型细菌中的PR(蓝光型)蛋白(BPR-Hot75)采用了基因克隆和E.coli表达系统对其进行了表达,然后用于PR蛋白结构与功能的研究。
PR蛋白的质子泵功能相关研究主要采用生物信息学的方法寻找出可能与质子泵作用相关的位点。前期的研究表明：PR的同源蛋白bR的质子释放基团主要由Arg85,E194和E204组成,PR与bR的序列比较后显示：在PR中只有与bR中的Arg85相对应的Arg94是保守的,而另外两个酸性氨基酸则不存在于PR中。因此,我们首先把PR中保守的Arg94氨基酸分别突变为赖氨酸(Lys)、亮氨酸(Leu)、谷氨酸(Glu)和丙氨酸(Ala),然后对其突变体的功能和特性进行了研究。结果显示：Arg94的突变都导致了PR质子转运功能的完全丧失,野生型PR能够在光照的情况下将质子从细胞内泵到细胞外,而PR的Arg突变体则不能将质子泵到细胞外。另外Arg94的突变也导致PR的光周期发生改变,导致绿光吸收型PR的光循环明显减慢,其中基态(ground state)在光照后的恢复期要比野生型慢十倍。另外蛋白构象变化中的M中间体发生了积累,O中间体则减少到很小的量,这是由于O中间体快速的衰减所导致。这种现象在蓝光吸收型PR中的也是这样。另外,我们还对从海水中分离到一个天然的Arg突变体的表达产物N5R8_5进行了研究,也显示了相类似的结果,而对其突变体(G94R)的表达产物N5R8_5_GR的研究结果显示,甘氨酸突变成精氨酸能使得其质子泵功能得以恢复,同时也改变了其光循环周期。而突变了PR的其它位于胞质间隙的其它酸性氨基酸残基,包括BPR的D85、D88、 E142、E213,结果显示这些氨基酸残基突变对PR的质子泵功能都没有明显的影响。
为了对PR的结构进行解析,采用PCR方法获得了Hot D97N nO20(nO20表示该蛋白N末端缺少了20个氨基酸残基的信号肽)突变体,采用Ni-NTA亲和层析纯化目的蛋白,并获得高纯度的Hot D97N nO20膜蛋白。对PR突变体Hot D97N nO20使用坐滴气相扩散法进行结晶,在295K的条件下生长获得了五个结晶条件。优化得到Index51结晶条件,并在瑞士同步辐射光源和上海光源中心采集到晶体的X-射线衍射数据,运用XDS晶体衍射数据处理软件包对衍射数据进行处理,并运用CCP4和Phenix等结构解析软件包分析数据。主要是采用分子置换(Molecular Replacement)的方法,以氨基酸序列与PR有约30%同源性的bR (PDB:1C3W,1CWQ)、XR (PDB:3DDL)、 SR (PDB:2F93,1GU8)等为模板,对其进行了衍射数据分析。其结果分别阐述如下：
Hot D97N no20蛋白最优的结晶条件为：0.2mol/L醋酸铵,0.1mol/L Bis-Tris pH6.5,45%v/v2-甲基-2,4-戊二醇；蛋白浓度均为20mg/ml,去垢剂为anapoe-80, Additive Screen为EDTA。晶体衍射度达到3.3A,空间群为P21212,晶胞参数为a=162, b=169,c=68；α=90°,β=90°,γ=90°,每个不对称空间中均只有五或六个单体。
由于MR无法解析Hot D97N nO20蛋白的三维结构,存在相位问题,所以尝试采用同晶置换的方法来解决相位问题。首先,采用重金属进行浸泡蛋白晶体,可以检测到重金属Pt、Hg的信号,但不能获得高衍射度的数据；使用卤素Ⅰ进行浸泡,发现其反常散射信号不是很强,无法使用其数据解决相位问题；使用重金属Ta对蛋白晶体进行浸泡,发现在衍射过程中蛋白晶体辐射损伤严重,导致其衍射度下降严重。其次,我们采用与重金属共结晶的方法来获得重金属衍射物晶体,信号扫描无重金属信号。另外,通过硒代甲硫氨酸方法表达获得的蛋白晶体进行X-射线衍射,所得衍射数据中Se的信号太弱。
为了制备Hot D97N no20硒代甲硫氨酸蛋白衍生物晶体,利用反常散射信息解决相位问题。因Hot D97N no20蛋白含有的11个甲硫氨酸,在X-衍射的过程中甲硫氨酸替换的位点太多,影响重原子的定位,故选择其中六个同源性低的甲硫氨酸进行了突变。通过重叠PCR、获得重组质粒和转化到表达宿主菌C43(DE3)中进行诱导表达,同样获得了Hot D97N six M突变体蛋白,并纯化得到高纯度的蛋白,可用于硒代甲硫氨酸蛋白衍生物晶体生长。
为了更进一步对PR质子释放氨基酸基团进行深入的研究,对Hot75中与质子释放相关的氨基酸94位Arg(R)进行了突变,将其突变Ala(A),进而获得了Hot R94A突变体。另外,PR蛋白吸收峰的差异的主要由于其105位的氨基酸不同造成的,其中BPR的第105位氨基酸残基为Gln(Q),而GPR的则为Leu (L)。为进一步深入研究105位氨基酸残基作用机理,又将BPR-Hot75蛋白的Gln突变为Leu。通过亲和层析纯化获得高纯度的Hot R94A、 H ot Q105L no20膜蛋白。Hot R94A蛋白质结晶条件：0.2mol/L氯化镁六水合物,0.1mol/L Hepes pH7.0,25%v/v聚乙二醇400；蛋白浓度为20mg/mL;使用坐滴气相扩散法,在295K下培养下约三天长出。使用Additive Screen和Detergent Screen对蛋白晶体进行优化,发现在Additive Screen作用下蛋白的晶体有着明显的改变,在上海光源中心17u光束线进行衍射,发现Hot R94A蛋白晶体衍射度只能达到20A。Ho Q105L no20蛋白我们只发现有一个结晶条件：0.2mol/L氯化镁六水合物,0.1mol/L HEPES sodium pH7.5,30%v/v聚乙二醇400,蛋白晶体衍射度也只能达到20A。
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.
引文
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