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

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Time-Resolved Fluorescence Investigation of the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein: Influence of the Binding of Nucleic Acids

Depending on the HIV-1 isolate, MN or BH10, the nucleocapsid protein, NCp7, corresponds to a 55- or 71-amino acid length product, respectively. The MN NCp7 contains a single Trp residue at position 37 in the distal zinc finger motif, and the BH10 NCp7 contains an additional Trp, at position 61 in the C-terminal chain. The time-resolved intensity decay parameters of the zinc-saturated BH10 NCp7 were determined and compared to those of single-Trp-containing derivatives. The fluorescence decay of BH10 NCp7 could be clearly represented as a linear combination (with respect to both lifetimes and fractional intensities) of the individual emitting Trp residues. This suggested the absence of interactions between the two Trp residues, a feature that was confirmed by molecular modeling and fluorescence energy transfer studies. In the presence of tRNAPhe, taken as a RNA model, the same conclusions hold true despite the large fluorescence decrease induced by the binding of tRNAPhe. Indeed, the fluorescence of Trp37 appears almost fully quenched, in keeping with a stacking of this residue with the bases of tRNAPhe. Despite the multiple binding sites in tRNAPhe, the large prevalence of ultrashort lifetimes, associated with the stacking of Trp37, suggests that this stacking constitutes a major feature in the binding process of NCp7 to nucleic acids. In contrast, Trp61 only stacked to a small extent with tRNAPhe. The behavior of this residue in the tRNAPhe-NCp7 complexes appeared to be rather heterogeneous, suggesting that it does not constitute a major determinant in the binding process. Finally, our data suggested that the binding of NCp7 proteins from the two HIV-1 strains to nonspecific nucleic acid sequences was largely similar

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-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-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