Chromoproteins of the green fluorescent protein family: Properties and applications

The distribution in nature and the spectral and structural properties of chromoproteins of the green fluorescent protein (GFP) family and their differences from one another and other fluorescent proteins of this family are considered. Discussed in detail are practical applications of the chromoproteins and their mutant variants that have unique characteristics not found among natural proteins of the GFP family, such as far-red or photoconvertible fluorescence, a large Stokes shift, enhanced phototoxicity, etc.     

Spectral diversity among members of the green fluorescent protein family in hydroid jellyfish (Cnidaria,Hydrozoa)

The cDNAs encoding the genes of new proteins, homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria, were cloned. Two green fluorescent proteins from one unidentified anthomedusa, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent chromoprotein from another unidentified anthomedusa were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 49–53.Original Russian Text Copyright © 2005 by Yanushevich, Shagin, Fradkov, Shakhbazov, Barsova, Gurskaya, Labas, Matz, K. Lukyanov, S. Lukyanov.     

近红外发光卟啉镱-铂配合物的合成及其光物理性质研究

稀土发光材料相比于传统有机荧光染料在生物成像、分子检测和传感等领域具有独特的优势。目前,稀土发光生物探针主要以可见光发射为主,此类探针受限于组织穿透深度,应用范围较窄。具有较大组织穿透能力的近红外(NIR)稀土发光生物探针,由于其发光效率较低而少有报到。本工作合成了一种新型近红外发光的卟啉镱-铂配合物,TFPYb-Pt,表征并测试了该配合物的光物理性质。实验证实TFPYb-Pt具有较大的NIR发光效率(980/1 030 nm,Фem=0.37)和较长的NIR发光寿命(τ=49μs),表明该配合物可望被用于开发新型生物NIR发光探针。     

Kinetics and inhibition of glutamate carboxypeptidase II using a microplate assay

Proteomics, the study of protein function on a global scale, will play an important role in furthering our understanding of gene functions, complex biological pathways, and discovery of novel drug targets. A number of techniques have been developed for proteomic studies to identify and analyze proteins, compare protein expression levels, and study protein-protein interactions. Recent developments have applied a DNA array-type approach to immobilize proteins on a surface for high-throughput analysis. Here we report the development and construction of protein chips using derivatized glass and nitrocellulose-coated slides and the employment of recombinant proteins fused with green and red fluorescent proteins for detection. Fluorescent signals were found to be proportional to the amount of arrayed proteins and could be readily detected with a conventional fluorescence slide scanner. This technique allows the investigation of protein-protein interactions without the need for additional labeling steps of probe proteins.     

Identification of GFP-like Proteins in Nonbioluminescent,Azooxanthellate Anthozoa Opens New Perspectives for Bioprospecting

We screened nonbioluminescent, azooxanthellate cnidaria as potential sources for advanced marker proteins and succeeded in cloning a tetrameric green fluorescent protein (GFP) from the tentacles of Cerianthus membranaceus. The fluorescence of this protein (cmFP512) is characterized by excitation maximum at 503 nm, emission maximum at 512 nm, extinction coefficient of 58,800 M^–1 cm^–1, quantum yield of 0.66, and fluorescence lifetime of 2.4 ns. The chromophore is formed from the tripeptide Gln-Tyr-Gly. The amino acid sequence of this protein shares 17.8% identical residues with GFP from Aequorea victoria. Weak interactions between the subunits of the tetramer make cmFP512 a promising lead structure for the generation of monomeric variants of fluorescent proteins. Both red fluorescent proteins and nonfluorescent proteins of the GFP family were also purified from tissue homogenates of Adamsia palliata and Calliactis parasitica. The results presented here indicate that a photoprotective function of GFP-like proteins is unlikely in the examined anthozoa species.     

Divergent genetic control of protein solubility and conformational quality in Escherichia coli

In bacteria, protein overproduction results in the formation of inclusion bodies, sized protein aggregates showing amyloid-like properties such as seeding-driven formation, amyloid-tropic dye binding, intermolecular β-sheet architecture and cytotoxicity on mammalian cells. During protein deposition, exposed hydrophobic patches force intermolecular clustering and aggregation but these aggregation determinants coexist with properly folded stretches, exhibiting native-like secondary structure. Several reports indicate that inclusion bodies formed by different enzymes or fluorescent proteins show detectable biological activity. By using an engineered green fluorescent protein as reporter we have examined how the cell quality control distributes such active but misfolded protein species between the soluble and insoluble cell fractions and how aggregation determinants act in cells deficient in quality control functions. Most of the tested genetic deficiencies in different cytosolic chaperones and proteases (affecting DnaK, GroEL, GroES, ClpB, ClpP and Lon at different extents) resulted in much less soluble but unexpectedly more fluorescent polypeptides. The enrichment of aggregates with fluorescent species results from a dramatic inhibition of ClpP and Lon-mediated, DnaK-surveyed green fluorescent protein degradation, and it does not perturb the amyloid-like architecture of inclusion bodies. Therefore, the Escherichia coli quality control system promotes protein solubility instead of conformational quality through an overcommitted proteolysis of aggregation-prone polypeptides, irrespective of their global conformational status and biological properties.     

Linear polarized near‐infrared irradiation stimulates mechanical expansion of the rat sagittal suture

Mechanical sutural separation has been carried out in clinical orthodontics for controlling the growth of the craniofacial skeleton. This study was designed to evaluate the effects of linear polarized near‐infrared ray irradiation on the sutural expansion of rat sagittal suture. Twenty 8‐week‐old Wistar strain male rats were equally divided into experimental and control groups. Suture expansion was carried out for 5 days for all animals using an expansion appliance. The experimental animals were subjected to linear polarized near‐infrared irradiation. This study has demonstrated that linear polarized near‐infrared irradiation stimulates sutural expansion without any pathological changes. Copyright © 2002 John Wiley & Sons, Ltd.     

绿色荧光蛋白——照亮生命科学的一盏明灯

绿色荧光蛋白的发现及应用具有划时代的重要意义,它不仅为当代生物学研究提供了极为实用的基本研究手段,并且在此基础上改造发展和发现了一系列荧光蛋白,拓展了应用范围。这使得对微观生物学的研究也可以进入一个时空结合,研究鲜活动态过程的新时代。本文主要回顾总结了绿色荧光蛋白的发现、优化改造及其应用。     

Illuminating the ubiquitin/proteasome system

The ubiquitin/proteasome system (UPS) is responsible for the regulated processive degradation of proteins residing in the cytosol, nucleus, and endoplasmic reticulum. The two central players are ubiquitin, a small protein that is conjugated to substrates, and the proteasome, a large multi-subunit proteolytic complex that executes degradation of ubiquitylated proteins. Ubiquitylation and proteasomal degradation are highly dynamic processes. During the last decade, many researchers have started taking advantage of fluorescent proteins, which allow studying the dynamic nature of this system in the context of its natural environment: the living cell. In this review, we will summarize studies that have implemented this approach to examine the UPS and discuss novel insights in the dynamic organization of the UPS.     

Cell signaling: What is the signal and what information does it carry?

This paper reviews key findings from quantitative study of the yeast pheromone response system. Most come from single cell experiments that quantify molecular events the system uses to operate. After induction, signal propagation is relatively slow; peak activity takes minutes to reach the nucleus. At each measurement point along the transmission chain, signal rises, overshoots, peaks, and declines toward steady state. At at least one measurement point, this decline depends on negative feedback. The system senses and relays percent receptor occupancy, and one effect of the feedback is to maximize precision of this transmitted information. Over time, the system constantly adjusts quantitative behaviors to convey extracellular ligand concentration faithfully. These behaviors and mechanisms that control them are likely to be general for metazoan signaling systems.