绿色荧光蛋白及其应用

绿色荧光蛋白是在水母中发现的新型报告分子,能在多种生物体内表达并发出荧光。对GFP中一些特定氨基酸进行突变可以产生多种类型的突变体,有利于研究蛋白之间或细胞器之间的相互作用。目前,GFP已经用于基因表达的报告、细胞动态的研究、活细胞内蛋白的定位及westernbloting检测中。GFP美好的应用前景也促进了有关GFP的研究,特别是寻找新的突变体并将之运用到细胞生物学和分子生物学的各个领域。     

绿色荧光蛋白

来源于水母Aequorea victoria的绿色荧光蛋白(green fluorescent protein, GFP)现已成为在生物化学和细胞生物学中研究和开发应用得最广泛的蛋白质之一. 其内源荧光基团在受到紫外光或蓝光激发时（λ_max=395 nm, 小峰在479 nm）可高效发射清晰可见的绿光. GFP的高分辨率晶体结构为了解和研究蛋白质结构和光谱学功能关系提供了一个极好的机会. GFP已成为一个监测在完整细胞和组织内基因表达和蛋白质定位的理想标记. 通过突变和蛋白质工程构建的GFP嵌合蛋白在生理指示剂、生物传感器、光化学领域以及生产发光纤维等方面展示了广阔前景.     

含Dsred类似生色团的红色荧光蛋白的发展及应用

自从绿色荧光蛋白(GFP)被发现以来,荧光蛋白在生物医学领域已经成为一种重要的荧光成像工具．随着红色荧光蛋白DsRed的出现,各种优化的DsRed突变体和远红荧光蛋白也不断涌现．其中荧光蛋白生色团的形成机制对改建更优的荧光蛋白变种影响很大,对于红色荧光蛋白而言,大多数的红色荧光蛋白的生色团类型为DsRed类似生色团,在此基础上又出现了Far-red DsRed类似生色团．目前,含DsRed类似生色团的荧光蛋白主要有单体红色荧光蛋白、光转换荧光蛋白、斯托克斯红移蛋白、荧光计时器等．这些优化的荧光蛋白作为分子探针可以实现对活细胞、细胞器或胞内分子的时空标记和追踪,已经在生物工程学、细胞生物学、基础医学领域得到广泛应用．本文综述了含DsRed类似生色团的荧光蛋白的研究进展及其应用,以及由此发展起来的远红荧光蛋白在活体显微成像技术中的应用,并展望了荧光探针技术研究的新方向．     

非酶糖基化对α-synuclein分子构象的影响

将纯化后的α－synuclein分别与果糖和葡萄糖孵育,通过内源荧光、非酶糖基化衍生物特征荧光、圆二色光谱以及电子显微镜等技术进行检测发现：α—synuclein与还原糖共同孵育后,308nm内源荧光强度明显降低,同时在447nm产生一个非酶糖基化衍生物特征荧光．与果糖孵育的蛋白质样品其非酶糖基化特征荧光的出现速度快于葡萄糖孵育样品．内源荧光与非酶糖基化特征荧光之间存在能量传递现象,提示Tyr残基与非酶糖基化特征荧光发色团在空间距离上彼此接近．圆二色光谱测定结果显示,α－synuclein与果糖孵育后,其α－螺旋含量增加．非酶糖基化的α－synuclein在电子显微镜下表现为短纤维状．非酶糖基化可以诱导α－synuclein蛋白分子聚集,且果糖较葡萄糖更容易使α－synuclein发生非酶糖基化．以上结果提示,非酶糖基化似乎可以导致α－synuclein在细胞内的错误折叠和分子聚集．     

烟曲霉蛋白质分泌载体的构建及酸性磷酸酯酶的细胞定位

[目的]在酵母细胞中蛋白质的糖基磷酸肌醇化(GPI)修饰是将GPI定位于细胞膜或细胞壁的信号.目前已对酵母GPI蛋白的细胞定位信号有一定了解,但对丝状真菌GPI蛋白的定位则了解甚少.AfPhoA是丝状真菌烟曲霉(Aspergillus fumigatus)的酸性磷酸酯酶,是GPI修饰的蛋白.该蛋白首先分离自细胞膜,随后又发现该蛋白与细胞壁结合.分析其C-端序列也未发现已知的定位信号,因此目前还不能确定其细胞定位.[方法]我们以绿色荧光蛋白(GFP)作为报告分子,将AfPhoA的C-端序列与GFP的C-端融合后检测融合GFP的细胞定位.[结果]我们用烟曲霉几丁质酶AfChiB1的启动子和N-端信号肽构建了可在烟曲霉中分泌表达GFP的表达载体pchiGFP.在此基础上将AfPhoA的C-端与GFP融合,融合质粒与pCDA14共转化烟曲霉后筛选到一株转化子.该转化子可表达融合GFP,在诱导和非诱导条件下,融合GFP均主要分布在细胞膜上,随培养时间的延长,融合GFP在细胞壁上也有少量分布;在培养上清液中只能检出约30KD的GFP融合蛋白,而没有完整的GFP融合蛋白,推测为从GPI锚上水解释放的.[结论]我们的研究结果表明,AfPhoA蛋白GPI修饰的作用是使该蛋白定位于细胞膜.本研究不仅初步确定了AfPhoA蛋白GPI修饰的细胞膜定位功能,而且为烟曲霉基因与蛋白质功能的研究建立了一个有效表达系统.     

光激活荧光蛋白及其在植物分子细胞生物学研究中的应用

光激活荧光蛋白是指用特定光照射时,其荧光特性发生显著改变的一类荧光蛋白。借助光激活荧光蛋白的这种特性,可以实现对活细胞、细胞器或胞内分子的时空标记和追踪。该文介绍了目前光激活荧光蛋白的性质,并从多个方面对其应用进行了概括,包括分子标记与动态分析、蛋白质相互作用、细胞器及细胞组分动态研究、细胞追踪以及在光激活定位显微镜中的应用等,且对目前光激活荧光蛋白在植物分子细胞生物学中的应用进行了详细介绍。     

光激活荧光蛋白及其在植物分子细胞生物学研究中的应用

光激活荧光蛋白是指用特定光照射时, 其荧光特性发生显著改变的一类荧光蛋白。借助光激活荧光蛋白的这种特性,可以实现对活细胞、细胞器或胞内分子的时空标记和追踪。该文介绍了目前光激活荧光蛋白的性质, 并从多个方面对其应用进行了概括, 包括分子标记与动态分析、蛋白质相互作用、细胞器及细胞组分动态研究、细胞追踪以及在光激活定位显微镜中的应用等, 且对目前光激活荧光蛋白在植物分子细胞生物学中的应用进行了详细介绍。     

过表达TRAF2降解外源性绿色荧光蛋白

TRAF2是机体免疫与炎症反应中具有重要作用的蛋白,有E3连接酶活性;GFP是一种受到激发光照射后可产生绿色荧光的蛋白,常用于蛋白质的细胞共定位研究,并显示与GFP融合表达靶蛋白在细胞中的位置．我们实验发现共转染表达质粒pCMV-myc-TRAF2与pEGFP-C3,可引起转染细胞绿色荧光减弱．Western印迹实验证明TRAF2可以降解GFP,并且这种降解是通过蛋白酶体途径进行的．为进一步确定这种降解的特异性,在HEK293细胞中共转pCMV-myc-TRAF2与pEGFP-C3-LNX后,发现随着TRAF2表达量增加,融合蛋白GFP-LNX减少;而共转质粒pCMV-myc-TRAF2与pCMV-myc-LNX后,未发现LNX蛋白表达减少,表明TRAF2对GFP的降解具有相对特异性．GFP是人类细胞中并不存在的蛋白,TRAF2能够将其降解可能意味着TRAF2参与了细胞抗病原体感染的过程．     

植物蛋白磷酸化的检测方法

蛋白磷酸化是一种重要的蛋白质翻译后修饰方式,几乎参与植物所有生命过程的调节。蛋白磷酸化过程主要指在蛋白激酶的催化作用下,将三磷酸腺苷(ATP)上的γ位磷酸基团转移到底物蛋白特定氨基酸残基上的过程。底物蛋白上被磷酸化的常见氨基酸有丝氨酸、苏氨酸及酪氨酸,磷酸基团与氨基酸中的羟基通过酯键连接。该文详细描述了几种常用的蛋白质体外及体内磷酸化的检测方法及注意事项。     

细胞器荧光蛋白标记稳定细胞株系的建立与应用

绿色荧光蛋白（green fluorescent protein,GFP）以及由其演变而来的突变体或来自于其他物种的各色荧光蛋白,因为良好的荧光激发特性、稳定的真原核细胞表达及较小的细胞毒性等特点,被广泛应用为蛋白质定位的重要标记分子和报告基因,在生命科学的诸多研究领域得以应用。     

Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins

The variant of Aequorea green fluorescent protein (GFP) known as blue fluorescent protein (BFP) was originally engineered by substituting histidine for tyrosine in the chromophore precursor sequence. Herein we report improved versions of BFP along with a variety of engineered fluorescent protein variants with novel and distinct chromophore structures that all share the property of a blue fluorescent hue. The two most intriguing of the new variants are a version of GFP in which the chromophore does not undergo excited-state proton transfer and a version of mCherry with a phenylalanine-derived chromophore. All of the new blue fluorescing proteins have been critically assessed for their utility in live cell fluorescent imaging. These new variants should greatly facilitate multicolor fluorescent imaging by legitimizing blue fluorescing proteins as practical and robust members of the fluorescent protein "toolkit".     

Tyrosine 192 in apolipoprotein A-I is the major site of nitration and chlorination by myeloperoxidase, but only chlorination markedly impairs ABCA1-dependent cholesterol transport

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-nitrotyrosine and 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in humans, indicating that MPO oxidizes HDL in vivo. In the current studies, we used tandem mass spectrometry to identify the major sites of tyrosine oxidation when lipid-free apolipoprotein A-I (apoA-I), the major protein of HDL, was exposed to MPO or peroxynitrite (ONOO(-)). Tyrosine 192 was the predominant site of both nitration and chlorination by MPO and was also the major site of nitration by ONOO(-). Electron paramagnetic spin resonance studies of spin-labeled apoA-I revealed that residue 192 was located in an unusually hydrophilic environment. Moreover, the environment of residue 192 became much more hydrophobic when apoA-I was incorporated into discoidal HDL, and Tyr(192) of HDL-associated apoA-I was a poor substrate for nitration by both myeloperoxidase and ONOO(-), suggesting that solvent accessibility accounted in part for the reactivity of Tyr(192). The ability of lipid-free apoA-I to facilitate ATP-binding cassette transporter A1 cholesterol transport was greatly reduced after chlorination by MPO. Loss of activity occurred in concert with chlorination of Tyr(192). Both ONOO(-) and MPO nitrated Tyr(192) in high yield, but unlike chlorination, nitration minimally affected the ability of apoA-I to promote cholesterol efflux from cells. Our results indicate that Tyr(192) is the predominant site of nitration and chlorination when MPO or ONOO(-) oxidizes lipid-free apoA-I but that only chlorination markedly reduces the cholesterol efflux activity of apoA-I. This impaired biological activity of chlorinated apoA-I suggests that MPO-mediated oxidation of HDL might contribute to the link between inflammation and cardiovascular disease.     

Lysine residues direct the chlorination of tyrosines in YXXK motifs of apolipoprotein A-I when hypochlorous acid oxidizes high density lipoprotein

Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid (HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus, HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein oxidation in humans. One important target of the myeloperoxidase pathway may be high density lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The major site of chlorination was a single tyrosine residue located in one of the protein's YXXK motifs (where X represents a nonreactive amino acid). To investigate the mechanism of chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid sequences YKXXY, YXXKY, or YXXXY. MS/MS analysis demonstrated that chlorination of tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the substrate was KXXY or YXXK. NMR and MS analyses revealed that the N(epsilon) amino group of lysine was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine chlorination. Molecular modeling of the YXXK motif in apolipoprotein A-I demonstrated that these tyrosine and lysine residues are adjacent on the same face of an amphipathic alpha-helix. Our observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently damage proteins when they destroy microbial proteins during infection or damage host tissue during inflammation.     

Evaluation and biosynthetic incorporation of chlorotyrosine into recombinant proteins

Recently, non-canonical amino acids (NCAA) incorporation was developed to enhance the functional properties of proteins. Incorporation of NCAA containing chlorine atom is conceptually an attractive approach to prepare pharmacologically active substances, which is a difficult task since chlorine is bulky atom. In this study, we evaluated the efficiency and extent of in vivo incorporation of tyrosine analogue 3-chlorotyrosine [(3-Cl)Tyr] into the recombinant proteins GFP and GFPHS (highly stable GFP). The incorporation of (3-Cl)Tyr into GFP leads to dramatic reduction in the expression level of protein. On the other hand, the incorporation of (3-Cl)Tyr into GFPHS was expressed well as a soluble form. In addition we used bioinformatics tools for the analysis to explore the possible constraints in micro-environment of each natural amino acid residue to be replaced with chlorine atom accommodation into GFPHS. In conclusion, our approaches are reliable and straightforward way to enhance the translation of chlorinated amino acids into proteins.     

Structural characterization of a blue chromoprotein and its yellow mutant from the sea anemone Cnidopus japonicus

Green fluorescent protein (GFP) and its relatives (GFP protein family) have been isolated from marine organisms such as jellyfish and corals that belong to the phylum Cnidaria (stinging aquatic invertebrates). They are intrinsically fluorescent proteins. In search of new members of the family of green fluorescent protein family, we identified a non-fluorescent chromoprotein from the Cnidopus japonicus species of sea anemone that possesses 45% sequence identity to dsRed (a red fluorescent protein). This newly identified blue color protein has an absorbance maximum of 610 nm and is hereafter referred to as cjBlue. Determination of the cjBlue 1.8 A crystal structure revealed a chromophore comprised of Gln(63)-Tyr(64)-Gly(65). The ring stacking between Tyr(64) and His(197) stabilized the cjBlue trans chromophore conformation along the Calpha2-Cbeta2 bond of 5-[(4-hydroxyphenyl)methylene]-imidazolinone, which closely resembled that of the "Kindling Fluorescent Protein" and Rtms5. Replacement of Tyr(64) with Leu in wild-type cjBlue produced a visible color change from blue to yellow with a new absorbance maximum of 417 nm. Interestingly, the crystal structure of the yellow mutant Y64L revealed two His(197) imidazole ring orientations, suggesting a flip-flop interconversion between the two conformations in solution. We conclude that the dynamics and structure of the chromophore are both essential for the optical appearance of these color proteins.     

Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidation and site-specific tyrosine chlorination of apolipoprotein A-I

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in vivo, indicating that MPO oxidizes HDL in humans. We previously reported that Tyr-192 is the major site that is chlorinated in apolipoprotein A-I (apoA-I), the chief protein in HDL, and that chlorinated apoA-I loses its ability to promote cholesterol efflux from cells by the ATP-binding cassette transporter A1 (ABCA1) pathway. However, the pathways that promote the chlorination of specific Tyr residues in apoA-I are controversial, and the mechanism for MPO-mediated loss of ABCA1-dependent cholesterol efflux of apoA-I is unclear. Using site-directed mutagenesis, we now demonstrate that lysine residues direct tyrosine chlorination in apoA-I. Importantly, methionine residues inhibit chlorination, indicating that they can act as local, protein-bound antioxidants. Moreover, we observed near normal cholesterol efflux activity when Tyr-192 of apoA-I was mutated to Phe and the oxidized protein was incubated with methionine sulfoxide reductase. Thus, a combination of Tyr-192 chlorination and methionine oxidation is necessary for depriving apoA-I of its ABCA1-dependent cholesterol transport activity. Our observations suggest that biologically significant oxidative damage of apoA-I involves modification of a limited number of specific amino acids, raising the feasibility of producing oxidation-resistant forms of apoA-I that have enhanced anti-atherogenic activity in vivo.     

Biosynthetic substitution of tyrosine in green fluorescent protein with its surrogate fluorotyrosine in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Introduction of a fluorine moiety into green fluorescent protein offers an interesting novel spectral variant. The calculated binding energy of fluorotyrosine (F-Tyr) (−8.42 kcal/mol) for tyrosyl tRNA synthetase was moderately higher than that of tyrosine (Tyr) (−8.36 kcal/mol). This result directly correlated with the expression level of F-Tyr containing GFP (38 mg/l), which was comparably higher than that of the parent GFP expression level (34 mg/l). Finally, we generated a model structure for GFP to assess possible interaction in the chromophore of the protein structure, which plays an important role in determining the spectral and folding behaviors of the F-Tyr incorporated GFP variant.     

Modeling spectral tuning in monomeric teal fluorescent protein mTFP1

We present results of theoretical studies of the variants of the monomeric teal fluorescent protein from Clavularia coral (mTFP1) which present promising members from the GFP family. Predictions of quantum chemical approaches including density functional theory and semiempirical approximations are presented for the model systems which mimic the chromophores in different environments. We describe the excitation energy spectrum of the cyan mTFP1 fluorescent protein with the original chromophore and with chromophore mutants Tyr67His and Tyr67Trp.     

Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine

Expression of green fluorescent protein (GFP) in Escherichia coli (E. coli) resulted in only small amount of soluble and fluorescent GFP protein and hence most of the protein in insoluble particles. The expressed GFP in insoluble particles, however, was fluorescent, indicating that it is at least in part folded with an intact chromophore. The GFP in insoluble particles could not be solubilized by an aqueous (denaturant-free) buffer. Solubilization of active GFP from insoluble particles was then attempted with guanidine hydrochloride (GdnHCl), a strong protein-denaturant, or L-arginine, an aggregation suppressor. Solubilization from insoluble particles by 6M GdnHCl led to complete denaturation of the GFP existing in insoluble particles, while GdnHCl solution at lower concentration could solubilize fluorescent GFP. Solubilization of fluorescently active GFP from insoluble particles was also achieved by L-arginine. It is noteworthy that L-arginine was stronger in solubilizing insoluble GFP than GdnHCl below 2M. These results demonstrate that some proteins expressed in E. coli may form insoluble particles containing native conformation and L-arginine may be used to recover the proteins in the native form from such insoluble particles.     

Loss of 3-chlorotyrosine by inflammatory oxidants: implications for the use of 3-chlorotyrosine as a bio-marker in vivo

Activated neutrophils generate the potent oxidant hypochlorous acid (HOCl) from the enzyme myeloperoxidase (MPO). A proposed bio-marker for MPO-derived HOCl in vivo is 3-chlorotyrosine, elevated levels of which have been measured in several human inflammatory pathologies. However, it is unlikely that HOCl is produced as the sole oxidant at sites of chronic inflammation as other reactive species are also produced during the inflammatory response. The work presented shows that free and protein bound 3-chlorotyrosine is lost upon addition of the pro-inflammatory oxidants, HOCl, peroxynitrite, and acidified nitrite. Furthermore, incubation of 3-chlorotyrosine with activated RAW264.7 macrophages or neutrophil-like HL-60 cells resulted in significant loss of 3-chlorotyrosine. Therefore, at sites of chronic inflammation where there is concomitant ONOO⁻ and HOCl formation, it is possible measurement of 3-chlorotyrosine may represent an underestimate of the true extent of tyrosine chlorination. This finding could account for some of the discrepancies reported between 3-chlorotyrosine levels in tissues in the literature.