Structure-Guided Generation of a Redox-Independent Blue Fluorescent Protein from mBFP

Fluorescent proteins, such as the green fluorescent protein, are used for detection of cellular components and events. However, green fluorescent protein and its derivatives have limited usage under anaerobic conditions and require a long maturation time. On the other hand, the NADPH-dependent blue fluorescent protein (BFP) without oxidative modification of residues is instantly functional in both aerobic and anaerobic systems. BFP proteins belong to a short-chain dehydrogenase/reductase (SDR) protein family, and their fluorescent property changes with reaction time in the presence of a substrate. With the aim of developing a better fluorescent reporter independent of redox state, we elucidated the crystal structure of a tetrameric mBFP from soil metagenomes with and without NADPH. Apart from the previously known regions, structure-guided mutational studies have identified several residues that contribute to the fluorescence of mBFP, including two aromatic residues (F97 and Y157) near the nicotinamide moiety of the bound NADPH. A single histidine mutation at Y157 (Y157H) has conferred more stabilized, time-independent fluorescence even in the presence of substrates. Furthermore, we discovered another SDR protein that can also emit blue fluorescence. These results open a new possibility for the development of BFP as a stable cellular reporter for widespread use, independent of subcellular environments.     

Disulfide Bond Formation and Cysteine Exclusion in Gram-positive Bacteria

Most secretion pathways in bacteria and eukaryotic cells are challenged by the requirement for their substrate proteins to mature after they traverse a membrane barrier and enter a reactive oxidizing environment. For Gram-positive bacteria, the mechanisms that protect their exported proteins from misoxidation during their post-translocation maturation are poorly understood. To address this, we separated numerous bacterial species according to their tolerance for oxygen and divided their proteomes based on the predicted subcellular localization of their proteins. We then applied a previously established computational approach that utilizes cysteine incorporation patterns in proteins as an indicator of enzymatic systems that may exist in each species. The Sec-dependent exported proteins from aerobic Gram-positive Actinobacteria were found to encode cysteines in an even-biased pattern indicative of a functional disulfide bond formation system. In contrast, aerobic Gram-positive Firmicutes favor the exclusion of cysteines from both their cytoplasmic proteins and their substantially longer exported proteins. Supporting these findings, we show that Firmicutes, but not Actinobacteria, tolerate growth in reductant. We further demonstrate that the actinobacterium Corynebacterium glutamicum possesses disulfide-bonded proteins and two dimeric Dsb-like enzymes that can efficiently catalyze the formation of disulfide bonds. Our results suggest that cysteine exclusion is an important adaptive strategy against the challenges presented by oxidative environments.     

Optimized conjugation of a fluorescent label to proteins via intein-mediated activation and ligation

Intein-mediated ligation provides a site-specific method for the attachment of molecular probes to proteins. The method is inherently flexible with regard to either the protein sequence or the attached probe, but practical difficulties have limited the widespread use of this valuable labeling system for the attachment of small- to medium-sized molecules. We report herein studies to improve the efficiency and practical application of these reactions, including the assembly of plasmids for the expression of target-intein fusion proteins and the analysis of their reaction with a fluorescent cysteine derivative under a range of conditions. Optimal ligation of the fluorophore to the target protein is critically dependent on the degree of oxidation of the fluorescent cysteine derivative. Efficient ligation has been achieved with freshly prepared fluorescent cysteine derivative under rigorously anaerobic conditions. Similar ligation yields have also been achieved using more practically convenient conditions including anaerobic reaction with addition of thiophenol, or aerobic reaction with the further addition of tricarboxyethylphosphine.     

Mechanism of a genetically encoded dark-to-bright reporter for caspase activity

Fluorescent proteins have revolutionized modern biology with their ability to report the presence of tagged proteins in living systems. Although several fluorescent proteins have been described in which the excitation and emission properties can be modulated by external triggers, no fluorescent proteins have been described that can be activated from a silent dark state to a bright fluorescent state directly by the activity of an enzyme. We have developed a version of GFP in which fluorescence is completely quenched by appendage of a hydrophobic quenching peptide that tetramerizes GFP and prevents maturation of the chromophore. The fluorescence can be fully restored by catalytic removal of the quenching peptide, making it a robust reporter of proteolysis. We have demonstrated the utility of this uniquely dark state of GFP as a genetically encoded apoptosis reporter that monitors the function of caspases, which catalyze the fate-determining step in programmed cell death. Caspase Activatable-GFP (CA-GFP) can be activated both in vitro and in vivo, resulting in up to a 45-fold increase in fluorescent signal in bacteria and a 3-fold increase in mammalian cells. We used CA-GFP successfully to monitor real-time apoptosis in mammalian cells. This dark state of GFP may ultimately serve as a useful platform for probes of other enzymatic processes.     

Fluorescent protein biosensors applied to microphysiological systems

This mini-review discusses the evolution of fluorescence as a tool to study living cells and tissues in vitro and the present role of fluorescent protein biosensors (FPBs) in microphysiological systems (MPSs). FPBs allow the measurement of temporal and spatial dynamics of targeted cellular events involved in normal and perturbed cellular assay systems and MPSs in real time. FPBs evolved from fluorescent analog cytochemistry (FAC) that permitted the measurement of the dynamics of purified proteins covalently labeled with environmentally insensitive fluorescent dyes and then incorporated into living cells, as well as a large list of diffusible fluorescent probes engineered to measure environmental changes in living cells. In parallel, a wide range of fluorescence microscopy methods were developed to measure the chemical and molecular activities of the labeled cells, including ratio imaging, fluorescence lifetime, total internal reflection, 3D imaging, including super-resolution, as well as high-content screening. FPBs evolved from FAC by combining environmentally sensitive fluorescent dyes with proteins in order to monitor specific physiological events such as post-translational modifications, production of metabolites, changes in various ion concentrations, and the dynamic interaction of proteins with defined macromolecules in time and space within cells. Original FPBs involved the engineering of fluorescent dyes to sense specific activities when covalently attached to particular domains of the targeted protein. The subsequent development of fluorescent proteins (FPs), such as the green fluorescent protein, dramatically accelerated the adoption of studying living cells, since the genetic “labeling” of proteins became a relatively simple method that permitted the analysis of temporal–spatial dynamics of a wide range of proteins. Investigators subsequently engineered the fluorescence properties of the FPs for environmental sensitivity that, when combined with targeted proteins/peptides, created a new generation of FPBs. Examples of FPBs that are useful in MPS are presented, including the design, testing, and application in a liver MPS.     

New tools for in vivo fluorescence tagging

Engineering of fluorescent proteins continues to produce new tools for in vivo studies. The current selection contains brighter, monomeric, spectral variants that will facilitate multiplex imaging and FRET, and a collection of optical highlighter proteins that might replace photoactivatable-GFP. These new highlighter proteins, which include proteins that have photoswitchable fluorescence characteristics and a protein whose fluorescence can be repeatedly turned on and off, should simplify refined analyses of protein dynamics and kinetics. Fluorescent protein-based systems have also been developed to allow facile detection of protein-protein interactions in planta. In addition, new tags in the form of peptides that bind fluorescent ligands and quantum dots offer the prospect of overcoming some of the limitations of fluorescent proteins such as excessive size and insufficient brightness.     

Struggle for photostability: Bleaching mechanisms of fluorescent proteins

In modern biotechnological science, there is a need for visualization of objects under study at the levels of cells, organelles, and individual molecules. Prominent among imaging methods are the methods based on the detection of fluorescence from the fluorophores with which objects under study are labeled. Fluorescent proteins (FPs) are very popular as genetically encoded fluorescent labels for lifetime imaging of target structures and processes in living systems. One of the key characteristics of FPs is their photostability, i.e., their resistance to photochemical reactions that quench the fluorescence signal. This review describes the currently known molecular mechanisms underlying photobleaching and the methods used to improve the photostability of fluorescent proteins.     

The yiaKLX1X2PQRS and ulaABCDEFG gene systems are required for the aerobic utilization of L-ascorbate in Klebsiella pneumoniae strain 13882 with L-ascorbate-6-phosphate as the inducer

The capacity to both ferment and oxidize L-ascorbate has been widely documented for a number of enteric bacteria. Here we present evidence that all the strains of Klebsiella pneumoniae tested in this study ferment L-ascorbate using the ula regulon-encoded proteins. Under aerobic conditions, several phenotypes were observed for the strains. Our results showed that the yiaK-S system is required for this aerobic metabolic process. Gel shift experiments performed with UlaR and YiaJ and probes corresponding to the specific promoters indicated that L-ascorbate-6-phosphate is the effector molecule recognized by both regulators, since binding of the repressors to their recognition sites was impaired by the presence of this compound. We demonstrated that in K. pneumoniae cells L-ascorbate-6-phosphate is formed only by the action of the UlaABC phosphotransferase system. This finding explains why strains that lack the ula genetic system and therefore are unable to form the inducer intracellularly cannot efficiently use this vitamin as a carbon source under either anaerobic or aerobic conditions. Thus, efficient aerobic metabolism of L-ascorbate in K. pneumoniae is dependent on the presence of both the yiaK-S and ula systems. The expression of the yiaK-S operon, but not the expression of the ula regulon, is controlled by oxygen availability. Both systems are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and by IHF.     

Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines

Fluorescent pseudomonads are gamma-proteobacteria known for their capacity to colonize various ecological niches. This adaptability is reflected by their sophisticated and diverse iron uptake systems. The majority of fluorescent pseudomonads produce complex peptidic siderophores called pyoverdines or pseudobactins, which are very efficient iron scavengers. A tremendous variety of pyoverdines has been observed, each species producing a different pyoverdine. This variety can be used as an interesting tool to study the diversity and taxonomy of fluorescent pseudomonads. Other siderophores, including newly described ones, are also produced by pseudomonads, sometimes endowed with interesting properties in addition to iron scavenging, such as formation of complexes with other metals or antimicrobial activity. Factors other than iron limitation, and different regulatory proteins also seem to influence the production of siderophores in pseudomonads and are reviewed here as well. Another peculiarity of pseudomonads is their ability to use a large number of heterologous siderophores via different TonB-dependent receptors. A first genomic analysis of receptors in four different fluorescent pseudomonads suggests that their siderophore ligand repertoire is likely to overlap, and that not all receptors recognize siderophores as ligands.     

Simple and tunable Förster resonance energy transfer-based bioprobes for high-throughput monitoring of caspase-3 activation in living cells by using flow cytometry

Sensing systems based on F?rster resonance energy transfer (FRET) can be used to monitor enzymatic reactions, protein-protein interactions, changes in conformation, and Ca2+ oscillations in studies on cellular dynamics. We developed a series of FRET-based chimeric bioprobes, each consisting of fluorescent protein attached to a fluorescent dye. Green and red fluorescent proteins were used as donors and a series of Alexa Fluor dyes was used as acceptors. The basic fluorescent proteins were substituted with appropriate amino acids for recognition of the target (caspase-3) and subjected to site-directed modification with a fluorescent dye. Variants that retained similar emission profiles to the parent proteins were readily derived for use as FRET-based bioprobes with various fluorescent patterns by incorporating various fluorescent proteins and dyes, the nature of which could be adjusted to experimental requirements. All the constructs prepared functioned as bioprobes for quantitative measurement of caspase-3 activity in vitro. Introduction of the bioprobes into cells was so simple and efficient that activation of caspase-3 upon apoptosis could be monitored by means of cytometric analysis. FRET-based bioprobes are valuable tool for high-throughput flow-cytometric analysis of many cellular events when used in conjunction with other fluorescent labels or markers. Statistical dynamic studies on living cells could provide indications of paracrine signaling.     

Directed molecular evolution to design advanced red fluorescent proteins

Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.     

Fungal hydrogenosomes contain mitochondrial heat-shock proteins

At least three groups of anaerobic eukaryotes lack mitochondria and instead contain hydrogenosomes, peculiar organelles that make energy and excrete hydrogen. Published data indicate that ciliate and trichomonad hydrogenosomes share common ancestry with mitochondria, but the evolutionary origins of fungal hydrogenosomes have been controversial. We have now isolated full-length genes for heat shock proteins 60 and 70 from the anaerobic fungus Neocallimastix patriciarum, which phylogenetic analyses reveal share common ancestry with mitochondrial orthologues. In aerobic organisms these proteins function in mitochondrial import and protein folding. Homologous antibodies demonstrated the localization of both proteins to fungal hydrogenosomes. Moreover, both sequences contain amino-terminal extensions that in heterologous targeting experiments were shown to be necessary and sufficient to locate both proteins and green fluorescent protein to the mitochondria of mammalian cells. This finding, that fungal hydrogenosomes use mitochondrial targeting signals to import two proteins of mitochondrial ancestry that play key roles in aerobic mitochondria, provides further strong evidence that the fungal organelle is also of mitochondrial ancestry. The extraordinary capacity of eukaryotes to repeatedly evolve hydrogen-producing organelles apparently reflects a general ability to modify the biochemistry of the mitochondrial compartment.     

Use of fluorescent protein tags to study nuclear organization of the spliceosomal machinery in transiently transformed living plant cells

Although early studies suggested that little compartmentalization exists within the nucleus, more recent studies on metazoan systems have identified a still increasing number of specific subnuclear compartments. Some of these compartments are dynamic structures; indeed, protein and RNA-protein components can cycle between different domains. This is particularly evident for RNA processing components. In plants, lack of tools has hampered studies on nuclear compartmentalization and dynamics of RNA processing components. Here, we show that transient expression of fluorescent protein fusions of U1 and U2 small nuclear ribonucleoprotein particle (snRNP)-specific proteins U1-70K, U2B", and U2A ', nucleolar proteins Nop10 and PRH75, and serine-arginine-rich proteins in plant protoplasts results in their correct localization. Furthermore, snRNP-specific proteins also were correctly assembled into mature snRNPs. This system allowed a systematic analysis of the cellular localization of Arabidopsis serine-arginine-rich proteins, which, like their animal counterparts, localize to speckles but not to nucleoli and Cajal bodies. Finally, markers for three different nuclear compartments, namely, nucleoli, Cajal bodies, and speckles, have been established and were shown to be applicable for colocalization studies in living plant protoplasts. Thus, transient expression of proteins tagged with four different fluorescent proteins is a suitable system for studying the nuclear organization of spliceosomal proteins in living plant cells and should therefore allow studies of their dynamics as well.     

Use of fluorescein hydrazide and fluorescein thiosemicarbazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized protein on polyacrylamide gels

Highly fluorescent thiosemicarbazide and hydrazide prepared by reaction of fluorescein isothiocyanate with hydrazine or adipic acid dihydrazide have been used to monitor the presence of carbonyl groups in oxidatively modified proteins. After oxidation, proteins react with these reagents under anaerobic conditions in the dark to yield fluorescent protein conjugates (presumably thiosemicarbazones or hydrazones) which can be visualized as fluorescent bands following electrophoresis (0-4 degrees C) on lithium dodecyl sulfate-polyacrylamide gels. These reagents do not react with unoxidized proteins. The conjugates formed dissociate readily at room temperature but are fairly stable at pH 6-9, 0 degrees C. Current data suggest that these reagents will be useful in the detection and quantitation of oxidatively modified proteins in biological systems.     

Evidence of microbial rhodopsins in Antarctic Dry Valley edaphic systems

Microorganisms able to synthesize rhodopsins have the capacity to translocate ions through their membranes, using solar energy to generate a proton motive force. Rhodopsins are the most abundant phototrophic proteins in oceanic surface waters and are key constituents in marine bacterial ecology. However, it remains unclear how rhodopsins are used in most microorganisms. Despite their abundance in marine and fresh‐water systems, the presence of functional rhodopsin systems in edaphic habitats has never been reported. Here, we show the presence of several new putative H⁺, Na⁺ and Cl⁺ pumping rhodopsins identified by metagenomic analysis of Antarctic desert hypolithic communities. Reconstruction of two Proteobacteria genomes harboring xanthorhodopsin‐like proteins and one Bacteroidetes genome with a Na‐pumping‐like rhodopsin indicated that these bacteria were aerobic heterotrophs possessing the apparent capacity for the functional expression of rhodopsins. The existence of these protein systems in hypolithic bacteria expands the known role of rhodopsins to include terrestrial environments and suggests a possible predominant function as heterotrophic energy supply proteins, a feasible microbial adaptation to the harsh conditions prevalent in Antarctic edaphic systems.     

Characterization of Ternary Protein Systems In?Vivo with Tricolor Heterospecies Partition Analysis

Tools and assays that characterize protein-protein interactions are of fundamental importance to biology, because protein assemblies play a critical role in the control and regulation of nearly every cellular process. The availability of fluorescent proteins has facilitated the direct and real-time observation of protein-protein interactions inside living cells, but existing methods are mostly limited to binary interactions between two proteins. Because of the scarcity of techniques capable of identifying ternary interactions, we developed tricolor heterospecies partition analysis. The technique is based on brightness analysis of fluorescence fluctuations from three fluorescent proteins that serve as protein labels. We identified three fluorescent proteins suitable for tricolor brightness experiments. In addition, we developed the theory of identifying interactions in a ternary protein system using tricolor heterospecies partition analysis. The theory was verified by experiments on well-characterized protein systems. A graphical representation of the heterospecies partition data was introduced to visualize interactions in ternary protein systems. Lastly, we performed fluorescence fluctuation experiments on cells expressing a coactivator and two nuclear receptors and applied heterospecies partition analysis to explore the interactions of this ternary protein system.     

Budding yeast Saccharomyces cerevisiae as a model to study oxidative modification of proteins in eukaryotes

The budding yeast Saccharomyces cerevisiae is a well studied unicellular eukaryotic organism the genome of which has been sequenced. The use of yeast in many commercial systems makes its investigation important not only from basic, but also from practical point of view. Yeast may be grown under both aerobic and anaerobic conditions. The investigation of the response of eukaryotes to different kinds of stresses was pioneered owing to yeast and here we focus mainly on the so-called oxidative stress. It is a result of an imbalance between the formation and decomposition of reactive oxygen species increasing their steady-state concentration. Reactive oxygen species may attack any cellular component. In the present review oxidation of proteins in S. cerevisiae is analyzed. There are two connected approaches to study oxidative protein modification - characterization of the overall process and identification of individual oxidized proteins. Because all aerobic organisms possess special systems which defend them against reactive oxygen species, the involvement of so-called antioxidant enzymes, particularly superoxide dismutase and catalase, in the protection of proteins is also analyzed.     

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.     

Synthesis of membrane proteins in eukaryotic cell‐free systems

Cell‐free protein synthesis (CFPS) is a valuable method for the fast expression of difficult‐to‐express proteins as well as posttranslationally modified proteins. Since cell‐free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell‐free translation systems are capable to provide high molecular weight membrane proteins, fluorescent‐labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis.     

Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns

To realize a practical high-throughput protein-detection system, novel peptide arrays have been constructed using designed peptide libraries with loop, alpha-helix, or beta-strand structures. Here, we describe the overview of the reported designed peptide arrays with loop and alpha-helix structures and the new results of those with beta-strand structures. Initially, several model peptides known to interact with model structured proteins were selected to establish the present strategy for high-throughput detection of proteins. The fluorescent probes and suitable scaffolds of peptides were examined for the effective detection of proteins. The detection methods were established in solution and in an immobilized manner using the model systems. In the case of alpha-helix peptide, the response of a peptide with fluorescent resonance energy transfer between two probes at both termini was several times higher than that of a peptide with a single probe. In the cases of peptides with other structures, however, proteins were effectively detectable even by the fluorescent change of one probe. Furthermore, structurally focused libraries consisting of a total of ca. 250 different peptides based on the model peptides with secondary and/or tertiary structures were constructed with systematic replacement of residues. Using these libraries, various proteins were characterized effectively to give their own fluorescent "protein fingerprint" patterns. The resulting protein fingerprints correlated with the recognition properties of the proteins. These studies demonstrate that arrays with peptide libraries based on designed structures can be promising tools for detecting the target proteins. Designed synthetic peptides play roles as the capturing agents to be developed for practical protein chips.