Fluorescence imaging using a fluorescent protein with a large Stokes shift

Keima is a far-red fluorescent protein endowed with a large Stokes shift. It absorbs light maximally at around 440 nm and emits maximally at around 620 nm. While the original Keima is obligately tetrameric (tKeima), the dimeric and monomeric versions (mKeima and dKeima, respectively) have been generated. More recently, a tandem dimer of Keima (tdKeima) has been developed as the brightest version. Here we describe examples, which show the usefulness of Keima for dual-color fluorescence imaging technologies, such as fluorescence cross-correlation spectroscopy (FCCS) and two-photon laser scanning microscopy (TPLSM). Keima can be used in conjunction with existing fluorescent proteins in which the Stokes shift is much smaller, with the idea that while two fluorescent proteins are excited by a single laser each will fluoresce a different color.     

Spectral and structural analysis of a red fluorescent protein from Acropora digitifera

Fluorescent proteins (FPs) possess a wide variety of spectral properties that make them of widespread interest as optical markers. These proteins can be applied as pH indicators or metal biosensors. The discovery and characterization of new fluorescent proteins is expected to further extend their application. Here, we report the spectral and structural analysis of a red fluorescent protein from Acropora digitifera (designated AdRed). This protein shows a tetrameric state and is red emitting, with excitation and emission maxima at 567 and 612 nm, respectively. Its crystal structure shows the tetrameric interface stabilized by hydrogen bonding and salt bridges. The electron density map of the chromophore, consisting of Asp66–Tyr67–Gly68, shows the decarboxylated side chain of Asp66. Ser223, located near the chromophore, has the role of bridging His202 and Glu221, and is part of the hydrogen bond network. Mutated AdRed with Cys148Ser reveals a blue shift in fluorescence excitation and emission. Our results provide insights into understanding the molecular function of AdRed and other FPs.     

A Highlights from MBoC Selection: Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.     

A novel fluorescent probe with large Stokes shift for the detection of viscosity changes and its imaging in living cells

As an important cellular microenvironmental parameter, viscosity could reflect the status of living cells. Small molecular fluorescent probes are a vital tool to measure the change of viscosity in living cells. A novel fluorescence probe ZL-1 with a large Stokes shift (in methanol it reached to 153 nm and in glycerol it reached to 125 nm) and excellent sensitivity toward viscosity was developed. The sharp enhancement of the emission intensity for the probe ZL-1 from low viscous methanol to high viscous glycerol indicated that the probe ZL-1 could respond to the viscosity variations. Moreover, the probe ZL-1 has been successfully utilized to detect of the viscosity variations in living cells.     

A carbonothioate‐based highly selective fluorescent probe with a large Stokes shift for detection of Hg2+

Mercury (Hg) is one of the heavy metal pollutants in the environment. Even a very small amount of mercury can cause serious harm to human beings. Herein, we reported a new carbonothioate‐based fluorescent probe for the detection of Hg²⁺ without interference from other metal ions. This probe possessed a very large Stokes shift (192 nm), which could improve the detection sensitivity by minimizing the interferences resulted from self‐absorption or auto‐fluorescence. With the addition of Hg²⁺ to the probe solution, considerable fluorescence enhancement was observed. Additionally, the Hg²⁺ concentration of 0–16 μM and fluorescence intensity showed a good linear relationship (y = 22106× + 53108, R² = 0.9955). Finally, the proposed probe was used to detect Hg²⁺ in real water samples, and its result was satisfactory. Therefore, our proposed probe would provide a promising method for the determination of Hg²⁺ in the environment.     

Highlights of protein structural analysis

This article describes highlights of the state of the art in protein structural analysis, and comments on the current trends toward increased sensitivity and integrated isolation-structure methodologies.     

Spectral shift of fluorescent dye FM4-64 reveals distinct microenvironment of nuclear envelope in living cells

We report a distinct microenvironment within the nuclear envelope (NE) in living cells revealed by a spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br). The dye readily translocated to the NE at physiological temperature where it exhibited enhanced fluorescence when excited at 620-650 nm in contrast to 480-520 nm excitation in the endocytic pathway and in the endoplasmic reticulum (ER). In vitro data indicated that the dye reveals an enrichment of negatively charged lipids, presumably due to local phospholipid synthesis. Dual-excitation imaging of FM4-64 in relation to lamina-associated polypeptide-1-green fluorescent protein during mitosis suggested that the disassembly of NE preserves microscale lipid complexes in the ER. Convolutions of NE in cancer or primary cells were readily visualized, and killing of tumor cells by T cells was marked by sudden loss of the long-wavelength excited fluorescence in the NE coincident with apoptosis. This report of FM4-64 as the first vital dye sensitive to the NE environment opens new ways for real-time visualization and functional studies of the NE.     

Crystallization and preliminary X-ray analysis of fluorescent protein mBanana

mBanana is a novel monomeric red fluorescent protein mutant. It was cloned and expressed in Escherichia coli with 10 histidine residues at its N-terminal. After cleavage of the His tag by TEV protease, the mBanana was further purified and crystallized by the hanging-drop vapor-diffusion technique. The crystals can diffract to 2.0A resolution and one set of completed data was collected. It showed that the orthorhombic mBanana crystal was in space group P21 with unit cell parameters (48.629, 42.667, 61.714, 90, 111.676, 90) and contained one molecule in one asymmetric unit.     

An NIR emitting styryl dye with large Stokes shift to enable co-staining study on zebrafish neuromast hair cells

Hearing loss is a significant public health problem, and the “loss of sensory hair cells” is one of two leading causes in humans. Advanced imaging reagents are desirable for understanding the role of the surrounding support cells in the loss or regeneration of the hair cells. A styryl dye was found to exhibit NIR emission (λ_em ≈ 684 nm) with a very large Stokes shift (Δν ≈ 9190 cm⁻¹), due to the incorporation of excited state intramolecular proton transfer (ESIPT) mechanism. When used to stain live zebrafish embryos, the probe was found to exhibit good selectivity in targeting neuromasts, which are sensory organs on the surface of the fish’s body. The finding was verified by direct comparison with the known neuromast-labeling reagent, 4-Di-2-ASP. In contrast to the existing styryl dyes that label neuromast hair cells, the new probe labeled both neuromast hair cells and the surrounding support cells, while giving discernable signals. The study thus illustrated a useful tool to aid the developmental study of two closely related cell types on the mechanosensory sensory organ of zebrafish, which is a powerful animal model for hearing loss research.     

Direct structural analysis of modified RNA by fluorescent in-line probing

Chemical probing is a common method for the structural characterization of RNA. Typically, RNA is radioactively end-labelled, subjected to probing conditions, and the cleavage fragment pattern is analysed by gel electrophoresis. In recent years, many chemical modifications, like fluorophores, were introduced into RNA, but methods are lacking that detect the influence of the modification on the RNA structure with single-nucleotide resolution. Here, we first demonstrate that a 5'-terminal (32)P label can be replaced by a dye label for in-line probing of riboswitch RNAs. Next, we show that small, highly structured FRET-labelled Diels-Alderase ribozymes can be directly probed, using the internal or terminal FRET dyes as reporters. The probing patterns indeed reveal whether or not the attachment of the dyes influences the structure. The existence of two dye labels in typical FRET constructs is found to be beneficial, as 'duplexing' allows observation of the complete RNA on a single gel. Structural information can be derived from the probing gels by deconvolution of the superimposed band patterns. Finally, we use fluorescent in-line probing to experimentally validate the structural consequences of photocaging, unambiguously demonstrating the intentional destruction of selected elements of secondary or tertiary structure.     

蛋白质序列的聚类结构分析

基于模糊邻近关系的粒度空间,对蛋白质序列进行聚类结构分析。利用MEGA软件计算选取的木聚糖酶序列间的比对距离,引入内积将其转化为模糊邻近关系(或矩阵),再应用算法求解其粒度空间,进行序列的聚类结构分析和最佳聚类确定研究。这些研究为蛋白质序列提供了定量分析的工具。     

Molecular modeling of green fluorescent protein: structural effects of chromophore deprotonation

Molecular dynamics (MD) simulations were carried out to study the conformational rearrangement induced by deprotonation of the fluorescent chromophore in GFP, as well as the associated changes in the hydrogen-bonding network. For both the structures with either a neutral or an anionic chromophore, it was found that the beta-barrel was stable and rigid, and the conformation of the chromophore was consistent with the available x-ray structure. The conformational change in Thr203 due to deprotonation was also found to be consistent with the three-state isomerization model. Although GFP is highly fluorescent, denatured-GFP is nonfluorescent, indicating that the environment of the protein plays an important role in its fluorescence behavior. Our MD simulations, which explore the effect of the protein shell on the conformation of the chromophore, find the flexibility of the central chromophore to be significantly restricted due to the rigid nature of the protein shell. The hydrogen-bonding between the chromophore and neighboring residues was also shown to contribute to the chromophore rigidity. In addition to the MD studies, quantum mechanics/molecular mechanics (QM/MM) ONIOM calculations were carried out to investigate the effect of the beta-barrel on the internal rotation in the chromophore. Along with providing quantitative values for torsional rotation barriers about the bridging bond in the chromophore, the ONIOM calculations also validate our MD force field parameters.     

Highly sensitive quenched fluorescent substrate of Legionella major secretory protein (msp) based on its structural analysis

Legionella pneumophila has been shown to secrete a protease termed major secretory protein (Msp). This protease belongs to the M4 family of metalloproteases and shares 62.9% sequence similarity with pseudolysin (EC 3.4.24.26). With the aim of developing a specific enzymatic assay for the detection and quantification of Msp, the Fluofast substrate library was screened using both enzymes in parallel. Moreover, based on the crystal structure of pseudolysin, a model of the Msp structure was built. Screening of the peptide library identified a lead substrate specifically cleaved by Msp that was subsequently optimized by rational design. The proposed model for Msp is consistent with the enzymatic characteristics of the studied peptide substrates and provides new structural information useful for the characterization of the protease. This study leads to the identification of the first selective and high affinity substrate for Msp that is able to detect picomolar concentrations of the purified enzyme. The identified substrate could be useful for the development of a novel method for the rapid detection of Legionella.     

Tandem fluorescent protein timers for in vivo analysis of protein dynamics

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.     

Computational structural analysis of protein interactions and networks

Protein interactions have been at the focus of computational biology in recent years. In particular, interest has come from two different communities--structural and systems biology. Here, we will discuss key systems and structural biology methods that have been used for analysis and prediction of protein-protein interactions and the insight these approaches have provided on the nature and organization of protein-protein interactions inside cells.     

High-throughput modeling and analysis of protein structural dynamics

Protein function is a dynamic property closely related to the conformational mechanisms of protein structure in its physiological environment. To understand and control the function of target proteins, it becomes increasingly important to develop methods and tools for predicting collective motions at the molecular level. In this article, we review computational methods for predicting conformational dynamics and discuss software tools for data analysis. In particular, we discuss a high-throughput, web-based system called iGNM for protein structural dynamics. iGNM contains a database of protein motions for more than 20 000 PDB structures and supports online calculations for newly deposited PDB structures or user-modified structures. iGNM allows dynamics analysis of protein structures ranging from enzymes to large complexes and assemblies, and enables the exploration of protein sequence-structure-dynamics-function relations.     

Vibrational analysis of a solvated green fluorescent protein chromophore

Resonance Raman (RR) spectra of green fluorescent protein (GFP) model chromophores in solution have been simulated with the CASSCF/MM methodology. Although several reports on vibrational analysis of GFP model chromophores have been recently published, the RR spectra were simulated for the first time in explicit solution with the inclusion of the counterion, as these effects are crucial for unambiguously reproducing the vibrational band assignment in the anionic form of the GFP chromophore. This strategy allows for a one-to-one correspondence of the calculated vibrational modes to the observed RR bands, concerning both the location and intensity pattern. In addition, these simulations were complemented with total energy distribution calculations to aid in the unambiguous assignment of the measured spectra. The current study helps to clarify some of the previous RR bands assignments as well as producing some new assignment for the anionic form of GFP chromophore. The explicit solvent simulations and PCM-based calculations are compared to the measured spectra, and these results demonstrate that explicit solvent simulations provide better agreement with experiment, both in terms of vibrational frequencies and intensity distribution. Figure a Correlation of explicit hydration calculations (CASSCF/6-31G*/MM) for the HBI model chromophore and experimental RR data [21]; slope = 0.982, intercept = 27.210 and regression coefficient = 0.997. b Correlation of implicit PCM calculations (CASSCF/6-31G*) for the HBI model chromophore and experimental RR data [21], slope = 1.017, intercept = −48.838 and regression coefficient = 0.984     

文昌鱼GFP基因的鉴定及表达分析

近年在隶属头索动物亚门的文昌鱼体内发现有内源性绿色荧光蛋白存在,并发现文昌鱼荧光蛋白的发光现象在不同发育时期以及个体间有较大的差异。为了进一步揭示GFP基因在文昌鱼中的进化模式,探索其可能执行的功能,该文首先对白氏文昌鱼(Branchiostoma belcheri)GFP基因作了全面鉴定,并对其不同发育阶段胚胎及成体不同区域中的荧光信号进行了实时观察记录,进而对GFP基因在绿色荧光表达强烈的两个特定时期做了绝对定量检测。研究结果表明,文昌鱼基因组中至少有12个内源性GFP基因,在个体发育的不同时期,内源性荧光出现的位置有所变化,而且在变态后的个体之间出现荧光的情况差异较大,荧光蛋白基因的表达由多个GFP同源基因共同参与,这些基因在不同的发育时期表达量有较大的差异,提示不同的GFP基因在特定发育阶段可能行使各自的功能。     

Insights into protein kinase regulation and inhibition by large scale structural comparison

Protein structure determination of soluble globular protein domains has developed into an efficient routine technology which can now be applied to generate and analyze structures of entire human protein families. In the kinase area, several kinase families still lack comprehensive structural analysis. Nevertheless, Structural Genomics (SG) efforts contributed more than 40 kinase catalytic domain structures during the past 4 years providing a rich resource of information for large scale comparisons of kinase active sites. Moreover, many of the released structures are inhibitor complexes that offer chemical starting points for development of selective and potent inhibitors. Here we discuss the currently available structural data and strategies that can be utilized for the development of highly selective inhibitors.