Fluorene Analogues of Prodan with Superior Fluorescence Brightness and Solvatochromism

In a search for environmentally sensitive (solvatochromic) dyes with superior properties, we extended the electronic conjugation of one of the best solvatochromic dyes, Prodan, by substituting its naphthalene core with fluorene. The newly synthesized fluorene derivatives bearing strong electron-donor (dialkylamino) and -acceptor (carbonyl) groups at the 2 and 7 positions showed red-shifted absorption (close to 400 nm), twice as large of a absorption coefficient (43 000 M⁻¹ cm⁻¹), and a manifold larger two-photon absorption cross section (∼400 GM) compared to Prodan. Studies in solvents revealed much stronger fluorescence solvatochromism of the new dyes, which is connected with their twice as large transition dipole moment (14.0 D). Similarly to Prodan, they exhibit high fluorescence quantum yields, while their photostability is largely improved. Thus, substitution of the naphthalene core in Prodan with fluorene resulted in new fluorophores with superior spectroscopic and solvatochromic properties. We expect them to find a variety of applications as environmentally sensitive probes and labels in biology

Oxyluciferin Derivatives: A Toolbox of Environment-Sensitive Fluorescence Probes for Molecular and Cellular Applications

In this work, we used firefly oxyluciferin (<b>OxyLH</b>_<b>2</b>) and its polarity-dependent fluorescence mechanism as a sensitive tool to monitor biomolecular interactions. The chromophores, <b>OxyLH</b>_<b>2</b>, and its two analogues, <b>4-MeOxyLH</b> and <b>4,6′-DMeOxyL</b>, were modified trough carboxylic functionalization and then coupled to the N-terminus part of Tat and NCp7 peptides of human immunodeficiency virus type-1 (HIV-1). The photophysical properties of the labeled peptides were studied in live cells as well as in complex with different oligonucleotides in solution. By monitoring the emission properties of these derivatives we were able, for the first time, to study <i>in vitro</i> biomolecular interactions using oxyluciferin as a sensor. As an additional application, cyclopropyl-oxyluciferin (<b>5,5-Cpr-OxyLH</b>) was site-specifically conjugated to the thiol group (Cys-232) of the human protein α-1 antytripsin to investigate its interaction with porcine pancreatic elastase. Our data demonstrate that <b>OxyLH</b>_<b>2</b> and its derivatives can be used as fluorescence reporters for monitoring biomolecular interactions

Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat-4

Rformed in the absence (black solid line), or in the presence of 4 μM holo-Tat (blue dashed line), 8 μM holo-Tat (red dashed-dotted line), 8 μM zinc sulfate (purple dotted line), or 16 μM zinc sulfate (green dashed-dotted-dotted line), in PMG buffer at 37°C. At the time indicated by the first arrow, samples were cooled to 10°C. The second arrow represents one hour of incubation at 4°C. The trace with 8 μM apo-Tat was indistinguishable from the control and was thus not represented. B. Electron micrographs of 6 μM tubulin in the presence of 8 μM holo-Tat, or 16 μM zinc sulfate, in PMG buffer at 37°C and after cold depolymerisation at 10°C or 4°C.<p><b>Copyright information:</b></p><p>Taken from "Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat"</p><p>http://www.retrovirology.com/content/5/1/62</p><p>Retrovirology 2008;5():62-62.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2483996.</p><p></p

Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat-5

20 mM buffer, pH6.5 (●), or in Hepes buffer 50 mM, pH7.5, in the absence (△), or in the presence of 2 (■) or 5 (□) zinc equivalents.<p><b>Copyright information:</b></p><p>Taken from "Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat"</p><p>http://www.retrovirology.com/content/5/1/62</p><p>Retrovirology 2008;5():62-62.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2483996.</p><p></p

Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat-2

S sedimentation coefficient distribution C(S) of tubulin (5 μM) in the absence (black solid line), or in the presence of 10 μM holo-Tat (blue dashed line) or 10 μM apo-Tat (red dotted line). Inset: Full range C(S) of tubulin (5 μM) in the presence of 10 μM apo-Tat (red dotted line). Tat contributed to less than 10% of the signal. B. Electron micrograph of 5 μM tubulin in the presence of 10 μM apo-Tat, in PG buffer.<p><b>Copyright information:</b></p><p>Taken from "Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat"</p><p>http://www.retrovirology.com/content/5/1/62</p><p>Retrovirology 2008;5():62-62.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2483996.</p><p></p

Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat-1

Hepes buffer 50 mM, 0.05% IGEPAL CA-230, pH7.5, at 20°C. The continuous lines are fits to the experimental points with Equation 1.<p><b>Copyright information:</b></p><p>Taken from "Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat"</p><p>http://www.retrovirology.com/content/5/1/62</p><p>Retrovirology 2008;5():62-62.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2483996.</p><p></p

Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat-0

20 mM buffer, pH6.5 (●), or in Hepes buffer 50 mM, pH7.5, in the absence (△), or in the presence of 2 (■) or 5 (□) zinc equivalents.<p><b>Copyright information:</b></p><p>Taken from "Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat"</p><p>http://www.retrovirology.com/content/5/1/62</p><p>Retrovirology 2008;5():62-62.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2483996.</p><p></p

Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat-3

Were performed in the absence (black solid line), or in the presence of 2 μM (blue dashed line), 3 μM (red dotted line), or 4 μM (green dashed-dotted line) of A) apo-Tat or B) holo-Tat, in PMG buffer at 37°C. At the time indicated by the arrow, samples were cooled to 10°C. C. Electron micrographs of 15 μM tubulin in the absence or the presence of 4 μM apo-Tat, or 4 μM holo-Tat at 37°C and after cold depolymerisation at 10°C, in PMG buffer.<p><b>Copyright information:</b></p><p>Taken from "Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat"</p><p>http://www.retrovirology.com/content/5/1/62</p><p>Retrovirology 2008;5():62-62.</p><p>Published online 9 Jul 2008</p><p>PMCID:PMC2483996.</p><p></p

Simulation and Analysis of the Spectroscopic Properties of Oxyluciferin and Its Analogues in Water

Firefly bioluminescence is a quite efficient process largely used for numerous applications. However, some fundamental photochemical properties of the light emitter are still to be analyzed. Indeed, the light emitter, oxyluciferin, can be in six different forms due to interexchange reactions. In this work, we present the simulation of the absorption and emission spectra of the possible natural oxyluciferin forms in water and some of their analogues considering both the solvent/oxyluciferin interactions and the dynamical effects by using MD simulations and QM/MM methods. On the one hand, the absorption band shapes have been rationalized by analyzing the electronic nature of the transitions involved. On the other hand, the simulated and experimental emission spectra have been compared. In this case, an ultrafast excited state proton transfer (ESPT) occurs in oxyluciferin and its analogues, which impairs the detection of the emission from the protonated state by steady-state fluorescence spectroscopy. Transient absorption spectroscopy was used to evidence this ultrafast ESPT and rationalize the comparison between simulated and experimental steady-state emission spectra. Finally, this work shows the suitability of the studied oxyluciferin analogues to mimic the corresponding natural forms in water solution, as an elegant way to block the desired interexchange reactions allowing the study of each oxyluciferin form separately

IN/LEDGF/INI1-IBD/DNA structure.

<p><b>A</b>. INI1-IBD (pink) sits on the top of the complex and interacts with the C and N termini of the two IN monomers B. An extension of INI1-IBD interacts with the C-terminus of the two monomers A. <b>B</b>. A 90° view sliced as shown by the dotted square in figure A. <b>C and D</b>: two 90° views showing clearly the IN interacting domains with INI1.</p