Absorption tuning of the green fluorescent protein chromophore: synthesis and studies of model compounds

Abstract  

The green fluorescent protein (GFP) chromophore is a heterocyclic compound containing a p-hydroxybenzylidine attached to an imidazol-5(4H)-one ring. This review covers the synthesis of a variety of model systems for elucidating the intrinsic optical properties of the chromophore in the gas phase and its response in particular to hydrogen bond interactions. The overall goal is to understand how the protein binding pocket influences the absorption behavior, and the current status of our ongoing efforts is presented.     

Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation

Background  

Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1.     

Osmotic stress-dependent serine phosphorylation of the histidine kinase homologue DokA

Background  

Two-component systems consisting of histidine kinases and their corresponding receivers are widespread in bacterial signal transduction. In the past few years, genes coding for homologues of two-component systems were also discovered in eukaryotic organisms. DokA, a homologue of bacterial histidine kinases, is an element of the osmoregulatory pathway in the amoeba Dictyostelium. The work described here addresses the question whether DokA is phosphorylated in vivo in response to osmotic stress.     

The <Emphasis Type="Italic">FTO</Emphasis> (fat mass and obesity associated) gene codes for a novel member of the non-heme dioxygenase superfamily

Background  

Genetic variants in the FTO (fat mass and obesity associated) gene have been associated with an increased risk of obesity. However, the function of its protein product has not been experimentally studied and previously reported sequence similarity analyses suggested the absence of homologs in existing protein databases. Here, we present the first detailed computational analysis of the sequence and predicted structure of the protein encoded by FTO.     

Color transitions in coral's fluorescent proteins by site-directed mutagenesis

Background  

Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (~ 485 nm), green (~ 505 nm), yellow (~ 540 nm), and red (>580 nm) emitters.     

Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Background  

Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with ³H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity.     

Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening

Background  

The mitotic exit network (MEN) is a group of proteins that form a signaling cascade that is essential for cells to exit mitosis in Saccharomyces cerevisiae. The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2-Mob1 have been characterized, but physiological substrates for Dbf2-Mob1 have yet to be identified.     

Molecular characterization of <Emphasis Type="Italic">tlyA</Emphasis> gene product,Rv1694 of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>: A non-conventional hemolysin and a ribosomal RNA methyl transferase

Background  

Mycobacterium tuberculosis is a virulent bacillus causing tuberculosis, a disease responsible for million deaths each year worldwide. In order to understand its mechanism of pathogenesis in humans and to help control tuberculosis, functions of numerous Mycobacterium tuberculosis genes are being characterized. In this study we report the dual functionality of tlyA gene product of Mycobacterium tuberculosis annotated as Rv1694, a 268 amino acid long basic protein.     

alpha-Sarcin catalytic activity is not required for cytotoxicity

Background  

α-Sarcin is a protein toxin produced by Aspergillus giganteus. It belongs to a family of cytotoxic ribonucleases that inactivate the ribosome and inhibit protein synthesis. α-Sarcin cleaves a single phosphodiester bond within the RNA backbone of the large ribosomal subunit, which makes the ribosome unrecognizable to elongation factors and, in turn, blocks protein synthesis. Although it is widely held that the protein synthesis inhibition caused by the toxin leads to cell death, it has not been directly shown that catalytically inactive mutants of α-sarcin are non-toxic when expressed directly within the cytoplasm of cells. This is important since recent studies have cast doubt on whether protein synthesis inhibition is sufficient to initiate apoptosis.     

A ~35 kDa polypeptide from insect cells binds to yeast ACS like elements in the presence of ATP

Background  

The S. cerevisiae origin recognition complex binds to the ARS consensus sequence in an ATP dependent fashion. Recently, the yeast Cdc6 has been reported to have DNA binding activity. Conservation of replication proteins among different species strongly supports their functional similarity. Here we report the results of an investigation into the DNA binding activity of human Cdc6 protein. Cdc6 was expressed and purified from baculovirus infected Sf9 (Spodoptera frugiperda) insect cells as GST fusion protein (GST-Cdc6) and its DNA binding activity was tested.     

The protein kinase DYRK1A phosphorylates the splicing factor SF3b1/SAP155 at Thr434, a novel in vivo phosphorylation site

Background  

The U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1in vitro and in cultivated cells.     

Characterization of the aggregates formed during recombinant protein expression in bacteria

Background  

The first aim of the work was to analyze in detail the complexity of the aggregates formed upon overexpression of recombinant proteins in E. coli. A sucrose step gradient succeeded in separating aggregate subclasses of a GFP-GST fusion protein with specific biochemical and biophysical features, providing a novel approach for studying recombinant protein aggregates.     

Characterization of recombinant β-fructofuranosidase from <Emphasis Type="Italic">Bifidobacterium adolescentis</Emphasis>G1

Background  

We have previously reported on purification and characterization of β-fructofuranosidase (β-FFase) from Bifidobacterium adolescentis G1. This enzyme showed high activity of hydrolysis on fructo-oligosaccharides with a low degree of polymerization. Recently, genome sequences of B. longum NCC2705 and B. adolescentis ATCC 15703 were determined, and cscA gene in the both genome sequences encoding β-FFase was predicted. Here, cloning of cscA gene encoding putative β-FFase from B. adolescentis G1, its expression in E. coli and properties of the recombinant protein are described.     

Prion protein insertional mutations increase aggregation propensity but not fiber stability

Background  

Mutations in the PRNPgene account for ~15% of all prion disease cases. Little is understood about the mechanism of how some of these mutations in PRNPcause the protein to aggregate into amyloid fibers or cause disease. We have taken advantage of a chimeric protein system to study the oligopeptide repeat domain (ORD) expansions of the prion protein, PrP, and their effect on protein aggregation and amyloid fiber formation. We replaced the ORD of the yeast prion protein Sup35p with that from wild type and expanded ORDs of PrP and compared their biochemical properties in vitro. We previously determined that these chimeric proteins maintain the [PSI⁺] yeast prion phenotype in vivo. Interestingly, we noted that the repeat expanded chimeric prions seemed to be able to maintain a stronger strain of [PSI ⁺] and convert from [psi ^-] to [PSI ⁺] with a much higher frequency. In this study we have attempted to understand the biochemical properties of these chimeric proteins and to establish a system to study the properties of the ORD of PrP both in vivoand in vitro.     

Development of ERK Activity Sensor,an <Emphasis Type="Italic">in vitro</Emphasis>, FRET-based sensor of Extracellular Regulated Kinase activity

Background  

Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET) can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity.     

Integrated allosteric regulation in the <Emphasis Type="Italic">S. cerevisiae</Emphasis> carbamylphosphate synthetase – aspartate transcarbamylase multifunctional protein

Background  

The S. cerevisiae carbamylphosphate synthetase – aspartate transcarbamylase multifunctional protein catalyses the first two reactions of the pyrimidine pathway. In this organism, these two reactions are feedback inhibited by the end product UTP. In the present work, the mechanisms of these integrated inhibitions were studied.     

Biochemical characterization of Cdk2-Speedy/Ringo A2

Background  

Normal cell cycle progression requires the precise activation and inactivation of cyclin-dependent protein kinases (CDKs), which consist of a CDK and a cyclin subunit. A novel cell cycle regulator called Speedy/Ringo shows no sequence similarity to cyclins, yet can directly bind to and activate CDKs. Speedy/Ringo proteins, which bind to and activate Cdc2 and Cdk2 in vitro, are required for the G2 to M transition during Xenopus oocyte maturation and for normal S-phase entry in cultured human cells.     

Glyceraldehyde-3-phosphate dehydrogenase is largely unresponsive to low regulatory levels of hydrogen peroxide in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

The reversible oxidation of protein SH groups has been considered to be the basis of redox regulation by which changes in hydrogen peroxide (H₂O₂) concentrations may control protein function. Several proteins become S-glutathionylated following exposure to H₂O₂ in a variety of cellular systems. In yeast, when using a high initial H₂O₂ dose, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the major target of S-glutathionylation which leads to reversible inactivation of the enzyme. GAPDH inactivation by H₂O₂ functions to reroute carbohydrate flux to produce NADPH. Here we report the effect of low regulatory H₂O₂ doses on GAPDH activity and expression in Saccharomyces cerevisiae.     

GC-MS studies of the chemical composition of two inedible mushrooms of the genus Agaricus

Background  

Mushrooms in the genus Agaricus have worldwide distribution and include the economically important species A. bisporus. Some Agaricus species are inedible, including A. placomyces and A. pseudopratensis, which are similar in appearance to certain edible species, yet are known to possess unpleasant odours and induce gastrointestinal problems if consumed. We have studied the chemical composition of these mushrooms using GC-MS.     

PocketPicker: analysis of ligand binding-sites with shape descriptors

Background  

Identification and evaluation of surface binding-pockets and occluded cavities are initial steps in protein structure-based drug design. Characterizing the active site's shape as well as the distribution of surrounding residues plays an important role for a variety of applications such as automated ligand docking or in situ modeling. Comparing the shape similarity of binding site geometries of related proteins provides further insights into the mechanisms of ligand binding.