The Hydrophobic Core of Twin-Arginine Signal Sequences Orchestrates Specific Binding to Tat-Pathway Related Chaperones

Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA) and TorD (specific for trimethylamine N-oxide reductase, TorA). Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function

Red Fluorescent Protein-Aequorin Fusions as Improved Bioluminescent Ca2+ Reporters in Single Cells and Mice

Bioluminescence recording of Ca2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca2+ in various cell compartments. In addition, they would also serve to monitor Ca2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R0) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca2+ oscillations in single HeLa cells expressing tdTA. Ca2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca2+ activity of HeLa cells injected subcutaneously into mice, and Ca2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca2+ sensor reported to date and is, therefore, a promising probe to study Ca2+ dynamics in whole organisms or tissues expressing the transgene

Generation of novel bioluminescent calcium sensors based on red fluorescent protein-aequorin fusions: organelle, single cell & in vivo imaging applications

The term `Luminescenz' was first established by the German physicist Eilhardt Wiedemann in 1888, which meant "all those phenomena of light which are not solely conditioned by the rise in temperature" [Harvey, 1957]. That was opposite to incandescence that is `hot light' radiated by any material heated to temperatures at which they become `red hot'. Later on, Wiedemann proposed a classification of various types of luminescence, according to the meth od of excitation, which remains valid till the present day [Brovko, 2010]. He documented thermoluminescence, electroluminescence, crystallo-luminescence, triboluminescence, photoluminescence and chemiluminescence. Nowadays, despite many new subtypes of luminescence have been discovered and investigated, they all belong to the original six categories of Wiedemann. The descriptions of distinct molecular excitation and emission processes are indicated by prefix; before -luminescence. In thermoluminescence, the emission of light is due to moderate heating, while electroluminescence is caused by the energy of electrical fields. Triboluminescence and crystalloluminescence occur when crystals are shattered or when solutions crystallize. The photoluminescence takes place when the excitation phase is achieved by absorbing the energy of light itself and is subdivided into phosphorescence and fluorescence

Fluorescent Protein-photoprotein Fusions and Their Applications in Calcium Imaging.

International audienceCalcium-activated photoproteins, such as aequorin, have been used as luminescent Ca(2+) indicators since 1967. After the cloning of aequorin in 1985, microinjection was substituted by its heterologous expression, which opened the way for a widespread use. Molecular fusion of GFP to aequorin recapitulated the nonradiative energy transfer process that occurs in the jellyfish Aequorea victoria, from which these two proteins were obtained, resulting in an increase of light emission and a shift to longer wavelength. The abundance and location of the chimera are seen by fluorescence, whereas its luminescence reports Ca(2+) levels. GFP-aequorin is broadly used in an increasing number of studies, from organelles and cells to intact organisms. By fusing other fluorescent proteins to aequorin, the available luminescence color palette has been expanded for multiplexing assays and for in vivo measurements. In this report, we will attempt to review the various photoproteins available, their reported fusions with fluorescent proteins, and their biological applications to image Ca(2+) dynamics in organelles, cells, tissue explants and in live organisms. This article is protected by copyright. All rights reserved

RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities

Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice.</jats:p

RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities

Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice

Alignment of DmsA and TorA signal sequences.

<p>The signal sequences of the DmsD substrates DmsA, YnfF and YnfE were aligned using ClustalW. Similarity of residues for these three sequence is summarized in the fourth row (* = identical, : = strong similarity, • = weak similarity). For comparison the signal sequence of TorA is shown in the bottom row. Regions of the TorA sequence that show remarkable similarity with the consensus sequence of DmsD substrates are indicated in grey boxes. N-regions are shown in black type, H-regions in red and C-regions blue, and the two arginines of the twin-arginine motif are typed in bold.</p

DmsD reduces membrane binding of ssDmsA-GFP.

<p>(<b>A</b>) SPR response curves of various proteins injected over a phospholipid bilayer that mimics the composition of the <i>E. coli</i> inner membrane. Injected solutions contain either 50 nM signal peptide-free Strep-GFP (orange line), 50 nM ssDmsA-GFP (blue line), 50 nM ssDmsA-GFP with 100 nM DmsD (red line), 50 nM ssDmsA-GFP with 200 nM DmsD (green line), 50 nM TorD (grey line), or 500 nM DmsD (black line). The red and green curves were corrected for the jump in refractive index by subtracting the response curves for injections of solutions containing 100 or 200 nM DmsD, respectively. (<b>B</b>) SPR response curve for an experiment in which DmsD was injected over a surface consisting of ssDmsA-GFP bound to a phospholipid bilayer. ssDmsA-GFP (100 nM) was injected over an immobilized phospholipid bilayer for a period of 100 s. The surface was washed with buffer for 500 s to remove weakly bound ssDmsA-GFP. Subsequently, buffer containing 25 nM DmsD was injected.</p