荧光蛋白研究进展

荧光蛋白在生物学众多研究领域中有着广泛的应用,基于荧光蛋白的分子探针和标记方法已成为活细胞或活体内动态成像研究生物大分子或细胞功能的重要工具。本文对现有荧光蛋白的种类和理化特性,及其在生物学研究中的应用进行了综述介绍。重点介绍了近年来荧光蛋白在亮度、Stokes位移、光谱改变等方面的研究进展,介绍了光转换与光活化荧光蛋白及其在超分辨荧光成像技术中的应用。最后对荧光蛋白未来的发展方向进行了展望。     

红色荧光蛋白的光谱多样性及体外分子进化

从珊瑚中来源的各种红色荧光蛋白（red fluorescent protein,RFP）经过一系列体外进化,其波谱范围覆盖了570－655mn,极大地丰富了细胞内或体内光学成像的荧光探针．简要阐述了来源于Discosoma sp．,Entacmaea quadricolor,Anemonia sulcata,Heteractis crispo,Actinia equina5种珊瑚的红色荧光蛋白的光学特征、结构、体外分子进化及其应用．     

动态检测活细胞内泛素-蛋白酶体系统活性方法的建立

目的建立一种动态检测活细胞内泛素-蛋白酶体系统活性的方法。方法将表达绿色荧光蛋白（GFP）或红色荧光蛋白（DsRed2）的质粒分别改建为表达带有内泛素-蛋白酶体系统降解信号CL1的GFP或DsRed2的pGFP^u或pDsRed2质粒,然后转染HEK293细胞,通过G418筛选得到稳定表达GFP^u或DsRed2^u的细胞系。在蛋白酶体抑制N—Acetyl—Leu-Leu—Norleu—al（ALLN）处理GFP^u或DsRed2^u细胞后,应用免疫印记技术检测细胞内GFP或DsRed,含量的变化,应用荧光显微镜和激光扫描共聚焦显微镜技术观察GFP或DsRed,荧光强度的变化。结果ALLN处理能使GFP“和DsRed2^u细胞内GFP和DsRed。含量明显增加,荧光强度显著增强,并呈现明显的剂量／时间-效应关系。结论本文成功地建立了检测内泛素-蛋白酶体系统活性的方法,该方法能有效地对活细胞的内泛素-蛋白酶体系统活性进行实时动态检测。     

量子点荧光探针在生物成像中的应用进展

量子点荧光探针是近几年发展起来的一种新型荧光标记物,拥有荧光染料及荧光蛋白所不能比拟的独特优势,已经在细胞功能研究及细胞表面和内部功能分子的探测、组织的成像和病灶的定位等方面得到了较为广泛的应用。本文对量子点的光学特性、生物化修饰及其在生物成像等方面的应用进展进行了较为详细的介绍,并展望了其应用发展。     

基于荧光蛋白的荧光共振能量转移探针的构建及应用

荧光共振能量转移(fluorescence resonance energy transfer,FRET)是基于荧光基团供体和荧光基团受体间偶极子–偶极子耦合作用的非辐射方式的能量传递现象。基于荧光蛋白的FRET技术已被广泛用于研究细胞信号通路中蛋白质–蛋白质活体相互作用检测、蛋白质构象变化监测以及生物探针的研制中。基于荧光蛋白的荧光共振能量转移探针使得人们可以在时间和空间层面上研究细胞信号的转导过程。该文简要介绍了四大类基于荧光蛋白的FRET生物探针的设计、研制以及其在生物信号分子检测、活细胞成像以及药物筛选中的应用和进展情况。     

人结直肠癌细胞HCT116红色和绿色荧光标记肿瘤模型的建立

目的筛选表达绿色荧光蛋白和红色荧光蛋白基因的人的单克隆结直肠癌细胞系,为体内监测肿瘤的早期生长建立一种新的肿瘤动物模型。方法以脂质体2000介导chickenβ-actin-GFP-neo和chickenβ-actin-DsRed-neo转染人结直肠癌细胞HCT-116,经梯度浓度G418筛选获得稳定表达红色和绿色荧光蛋白的细胞克隆并扩大培养。BALB/CA-nu裸鼠皮下接种1×10^6个发光细胞使其成瘤,活体荧光成像系统观察肿瘤的生长情况。结果获得了稳定表达GFP、DsRed的人结肠癌细胞株,将其接种到裸鼠体内可成瘤,利用活体成像系统观察了肿瘤的生长过程,肿瘤的发光随着观察时间的延长而增加。结论红色和绿色荧光蛋白能够在人结直肠癌细胞HCT-116中长期稳定表达,用红色和绿色荧光蛋白标记的人结直肠癌细胞HCT-116建立的裸鼠肿瘤模型为进一步研究结肠肿瘤和相应的药物筛选提供了一种简便、可行的新方法。     

综述——基于纳米材料的生物检测与医学诊断技术：贵金属团簇探针用于细胞成像及体外检测

贵金属团簇(noble metal clusters)是近年来新兴的一类荧光标记材料．由于具有物理尺寸小、荧光可调及生物相容性等优异的性能使得其在生物成像及检测领域都有着广泛的应用前景．本文讨论了贵金属团簇的制备和荧光特性,重点论述了其作为标记材料在细胞成像方面及体外检测应用中的研究进展．     

基于荧光检测的新型细胞传感器

主要介绍了一类基于荧光检测的新型细胞传感器,这类传感器利用免疫细胞表面分子特异性识别、结合抗原的特性和生物(或化学)发光技术,通过检测荧光信号在数分钟内达到检测病原体或其他抗原的目的.这类传感器的发光原理主要是利用钙离子敏感型化学荧光探针发光,如Fluo-4等,或钙离子敏感型发光蛋白发光,如水母发光蛋白、绿色荧光蛋白等.现在已经应用的主要是B细胞传感器和肥大细胞传感器.这类传感器具有灵敏度高、检测准确、反应速度快的优点.同时又存在交叉反应、细胞不易保存等不足之处.这类传感器在疾病诊断、环境监测、生物战剂检测等领域具有较大的应用前景.     

荧光标记技术在肿瘤模型研究中的应用

目的利用绿色荧光小鼠和红色荧光蛋白标记肿瘤细胞,建立荧光标记的小鼠肿瘤模型,并建立活体荧光成像和荧光显微镜成像在整体和细胞水平直接观察肿瘤的技术。方法将小鼠B16黑色素瘤细胞接种到绿色荧光蛋白转基因小鼠皮下,建立GFP小鼠肿瘤模型。以红色荧光蛋白作为标记基因导入小鼠黑色素瘤细胞B16细胞,建立稳定表达红色荧光蛋白的细胞株。将表达红色荧光蛋白B16细胞接种到绿色荧光转基因小鼠皮下,建立双荧光小鼠肿瘤模型。用荧光显微镜和活体荧光成像系统检测小鼠肿瘤的发生发展。结果分别建立了GFP小鼠肿瘤模型和双色荧光小鼠肿瘤模型。利用活体荧光影像仪可以观察双色荧光小鼠模型中受体绿色荧光组织和红色荧光移植肿瘤相互融合。利用荧光显微镜,可以观察到肿瘤内绿色荧光标记的来源于受体小鼠的血管和免疫细胞。经香菇多糖刺激的GFP小鼠肿瘤模型的移植瘤组织中,来源于受体小鼠绿色荧光标记的免疫细胞明显多于经生理盐水刺激的对照小鼠。结论利用绿色荧光小鼠和红色荧光RFP标记肿瘤细胞建立荧光标记的小鼠肿瘤模型,采用活体荧光成像仪和荧光显微镜可在整体和细胞水平直接观察肿瘤的生长以及肿瘤与宿主的相互作用。     

橙色荧光蛋白的研究进展及其应用

荧光蛋白(Fluorescent protein,FPs)可作为探针用以探究细胞内分子间相互作用,追踪特定代谢物的代谢途径,对活细胞内的各种代谢过程和细胞通路进行详细、准确的描述。目前已有的FPs几乎已经覆盖了从紫外光到远红外光的所有光谱波段,这些FPs借助高分辨率显微技术应用于生命科学的诸多领域,为生物学的发展作出巨大贡献。橙色FPs通常指光谱区间在540–570nm的FPs,近几年来关于橙色FPs的研究进展较快,并且其作为标记蛋白以及荧光共振能量转移技术(Fluorescence resonance energy transfer,FRET)中的荧光受体在生物学及医学领域得到较多的应用。文中综述了近15年橙色FPs领域的相关研究,重点聚焦橙色FPs的发展和应用,为今后橙色FPs的研究提供依据。     

A purple-blue chromoprotein from Goniopora tenuidens belongs to the DsRed subfamily of GFP-like proteins

A number of recently cloned chromoproteins homologous to the green fluorescent protein show a substantial bathochromic shift in absorption spectra. Compared with red fluorescent protein from Discosoma sp. (DsRed), mutants of these so-called far-red proteins exhibit a clear red shift in emission spectra as well. Here we report that a far-red chromoprotein from Goniopora tenuidens (gtCP) contains a chromophore of the same chemical structure as DsRed. Denaturation kinetics of both DsRed and gtCP under acidic conditions indicates that the red form of the chromophore (absorption maximum at 436 nm) converts to the GFP-like form (384 nm) by a one-stage reaction. Upon neutralization, the 436-nm form of gtCP, but not the 384-nm form, renaturates instantly, implying that the former includes a chromophore in its intact state. gtCP represents a single-chain protein and, upon harsh denaturing conditions, shows three major bands in SDS/PAGE, two of which apparently result from hydrolysis of an acylimine C=N bond. Instead of having absorption maxima at 384 nm and 450 nm, which are characteristic for a GFP-like chromophore, fragmented gtCP shows a different spectrum, which presumably corresponds to a 2-keto derivative of imidazolidinone. Mass spectra of the chromophore-containing peptide from gtCP reveal an additional loss of 2 Da relative to the GFP-like chromophore. Tandem mass spectrometry of the chromopeptide shows that an additional bond is dehydrogenated in gtCP at the same position as in DsRed. Altogether, these data suggest that gtCP belongs to the same subfamily as DsRed (in the classification of GFP-like proteins based on the chromophore structure type).     

Single amino acid replacement transforms mCherry to a far-red fluorescent protein

Far-red fluorescent proteins are beneficial for imaging in mammals. Here, starting from mCherry, the most commonly used among the different types of red fluorescent proteins (RFP), not having a H-bond network in its original form, we sought to recover the hydrogen bond network in mCherry. By comparing the structure of wtGFP and mCherry, we focused on a few key residues involved in a proton wire, and discovered an I197T mutant that showed a more red-shifted fluorescence. The detailed optical and photo-switching properties of related engineered RFPs are described. This study will guide further development of monomeric far-red FPs.     

Recovery of Red Fluorescent Protein Chromophore Maturation Deficiency through Rational Design

Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore’s covalent structure. We also created an optimized mPlum mutant (mPlum-E16P) that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore.     

Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms.     

Modern fluorescent proteins: from chromophore formation to novel intracellular applications

The diverse biochemical and photophysical properties of fluorescent proteins (FPs) have enabled the generation of a growing palette of colors, providing unique opportunities for their use in a variety of modern biology applications. Modulation of these FP characteristics is achieved through diversity in both the structure of the chromophore as well as the contacts between the chromophore and the surrounding protein barrel. Here we review our current knowledge of blue, green, and red chromophore formation in permanently emitting FPs, photoactivatable FPs, and fluorescent timers. Progress in understanding the interplay between FP structure and function has allowed the engineering of FPs with many desirable features, and enabled recent advances in microscopy techniques such as super-resolution imaging of single molecules, imaging of protein dynamics, photochromic FRET, deep-tissue imaging, and multicolor two-photon microscopy in live animals.     

The role of quarternary structure in fluorescent protein stability

The stability of fluorescent proteins (FPs) is of great importance for their use as reporters in studies of gene expression, protein dynamics and localization in cell. A comparative analysis of conformational stability of fluorescent proteins, having different association state was done. The list of studied proteins includes EGFP (monomer of green fluorescent protein, GFP), zFP506 (tetramer GFP), mRFP1 and "dimer2" (monomer and dimmer of red fluorescent protein), DsRed1 (red tetramer). The character of fluorescence intensity changes induced by guanidine hydrochloride (GdnHCl) of these proteins differs significantly. Green tetramer zFP506 has been shown to be more stable than green monomer EGFP, red dimmer "dimer2" has been shown to be less stable than red tetramer DsRed1, while red monomer mRFP1 has been shown to be practically as stable as tetramer DsRedl. It is concluded that the quaternary structure, being an important stabilizing factor, does not represent the only circumstance dictating the dramatic variations between fluorescent proteins in their conformational stability.     

Crystallographic structures of Discosoma red fluorescent protein with immature and mature chromophores: linking peptide bond trans-cis isomerization and acylimine formation in chromophore maturation

The mature self-synthesizing p-hydroxybenzylideneimidazolinone-like fluorophores of Discosoma red fluorescent protein (DsRed) and Aequorea victoria green fluorescent protein (GFP) are extensively studied as powerful biological markers. Yet, the spontaneous formation of these fluorophores by cyclization, oxidation, and dehydration reactions of tripeptides within their protein environment remains incompletely understood. The mature DsRed fluorophore (Gln 66, Tyr 67, and Gly 68) differs from the GFP fluorophore by an acylimine that results in Gln 66 Calpha planar geometry and by a Phe 65-Gln 66 cis peptide bond. DsRed green-to-red maturation includes a green-fluorescing immature chromophore and requires a chromophore peptide bond trans-cis isomerization that is slow and incomplete. To clarify the unique structural chemistry for the individual immature "green" and mature "red" chromophores of DsRed, we report here the determination and analysis of crystal structures for the wild-type protein (1.4 A resolution), the entirely green DsRed K70M mutant protein (1.9 A resolution), and the DsRed designed mutant Q66M (1.9 A resolution), which shows increased red chromophore relative to the wild-type DsRed. Whereas the mature, red-fluorescing chromophore has the expected cis peptide bond and a sp(2)-hybridized Gln 66 Calpha with planar geometry, the crystal structure of the immature green-fluorescing chromophore of DsRed, presented here for the first time, reveals a trans peptide bond and a sp(3)-hybridized Gln 66 Calpha with tetrahedral geometry. These results characterize a GFP-like immature green DsRed chromophore structure, reveal distinct mature and immature chromophore environments, and furthermore provide evidence for the coupling of acylimine formation with trans-cis isomerization.     

zFP538, a yellow-fluorescent protein from Zoanthus, contains a novel three-ring chromophore

Crystal structures of the tetrameric yellow-fluorescent protein zFP538 from the button polyp Zoanthus sp. and a green-emitting mutant (K66M) are presented. The atomic models have been refined at 2.7 and 2.5 A resolution, with final crystallographic R factors of 0.206 (R(free) = 0.255) and 0.190 (R(free) = 0.295), respectively, and have excellent stereochemistry. The fold of the protomer is very similar to that of green (GFP) and red (DsRed) fluorescent proteins; however, evidence from crystallography and mass spectrometry suggests that zFP538 contains a three-ring chromophore derived from that of GFP. The yellow-emitting species (lambda(em)(max) = 538 nm) is proposed to result from a transimination reaction in which a transiently appearing DsRed-like acylimine is attacked by the terminal amino group of lysine 66 to form a new six-membered ring, cleaving the polypeptide backbone at the 65-66 position. This extends the chromophore conjugation by an additional double bond compared to GFP, lowering the absorption and emission frequencies. Substitution of lysine 66 with aspartate or glutamate partially converts zFP538 into a red-fluorescent protein, providing additional support for an acylimine intermediate. The diverse and unexpected roles of the side chain at position 66 give new insight into the chemistry of chromophore maturation in the extended family of GFP-like proteins.     

GFP-like chromoproteins as a source of far-red fluorescent proteins.

We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing chromoproteins. An identical single amino acid substitution converted novel chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far-red FPs (emission lambda(max)=615-640 nm). Moreover, coupled site-directed and random mutagenesis of the chromoprotein from H. crispa resulted in a unique far-red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi-color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi-color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.     

Improved "optical highlighter" probes derived from discosoma red fluorescent protein

The tetrameric red fluorescent protein, DsRed, undergoes a rapid red to green color change evoked by short wavelength (lambda < 760 nm) femtosecond irradiation--a phenomenon that underpins the application of DsRed as an "optical highlighter" probe for tracking live cells, organelles, and fusion proteins. This color change results from selective bleaching of the "mature" red-emitting species of DsRed and an enhancement of emission from the "immature" green species, likely caused by dequenching of fluorescence resonance energy transfer occurring within the protein tetramer. Here, we have examined the role of residues known to influence the rate and completeness of chromophore maturation on the cellular and biophysical properties of DsRed mutants. Surprisingly, a single amino acid mutation (N42Q) with increased basal green emission yet rapid chromophore maturation displayed a multiphoton-evoked color change that was brighter, more consistent, more vivid, and easier to evoke than DsRed, despite the larger proportion of green chromophores. Rapidly maturing mutants with more complete chromophore maturation, exhibited little color change and increased resistance to multiphoton bleaching. We describe improved optical and cell biological properties for two DsRed-derived variants which we showcase in photolabeling studies, and discuss these data in terms of implications for fluorescence resonance energy transfer-based probes.