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Papers by Roberto Pascual

Research paper thumbnail of A Peptide Pertaining to the Loop Segment of Human Immunodeficiency Virus gp41 Binds and Interacts with Model Biomembranes: Implications for the Fusion Mechanism

Journal of Virology, 2005

The human immunodeficiency virus gp41 envelope protein mediates the entry of the virus into the t... more The human immunodeficiency virus gp41 envelope protein mediates the entry of the virus into the target cell by promoting membrane fusion. In order to gain new insights into the viral fusion mechanism, we studied a 35-residue peptide pertaining to the loop domain of gp41, both in solution and membrane bound, by using infrared and fluorescence spectroscopy. We show here that the peptide, which has a membrane-interacting surface, binds and interacts with phospholipid model membranes and tends to aggregate in the presence of a membranous medium and induce the leakage of vesicle contents. The results reported in this work, i.e., the destabilization and fusion of negatively charged model membranes, suggest an essential role of the loop domain in the membrane fusion process induced by gp41.

Research paper thumbnail of Interaction of a Peptide Derived from the N-Heptad Repeat Region of gp41 Env Ectodomain with Model Membranes. Modulation of Phospholipid Phase Behavior †

Biochemistry, 2005

The HIV-1 gp41 envelope protein mediates the entry of the virus into the target cell by promoting... more The HIV-1 gp41 envelope protein mediates the entry of the virus into the target cell by promoting membrane fusion. With a view toward possible new insights into the protein membrane alteration leading to the viral fusion mechanism, we have studied by infrared and fluorescence spectroscopies a fragment of 21 amino acids corresponding to the N-heptad repeat region of the gp41 ectodomain. Information on the structure of the peptide both in solution and in the presence of model membranes, its incorporation and location in the phospholipid bilayer, and the modulation of the phase behavior of the membrane has been gathered. Here we demonstrate that the peptide binds to and interacts with phospholipid model membranes, changing its conformation and inducing leakage of vesicle contents. These characteristics suggest that different specific regions of gp41 are capable of modifying the biophysical properties of phospholipid membranes and, therefore, might be essential for the assistance and enhancement of the viral and cell fusion process.

Research paper thumbnail of Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotei... more The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pretransmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors. D

Research paper thumbnail of Interaction of Viscotoxins A3 and B with Membrane Model Systems: Implications to Their Mechanism of Action

Biophysical Journal, 2003

Viscotoxins are small proteins that are thought to interact with biomembranes, displaying differe... more Viscotoxins are small proteins that are thought to interact with biomembranes, displaying different toxic activities against a varied number of cell types, being viscotoxin A 3 (VtA 3 ) the most cytotoxic whereas viscotoxin B (VtB) is the less potent. By using infrared and fluorescence spectroscopies, we have studied the interaction of VtA 3 and VtB, both wild and reduced ones, with model membranes containing negatively charged phospholipids. Both VtA 3 and VtB present a high conformational stability, and a similar conformation both in solution and when bound to membranes. In solution, the infrared spectra of the reduced proteins show an increase in bandwidth compared to the nonreduced ones indicating a greater flexibility. VtA 3 and VtB bind with high affinity to membranes containing negatively charged phospholipids and are motional restricted, their binding being dependent on phospholipid composition. Whereas nonreduced proteins maintain their structure when bound to membranes, reduced ones aggregate. Furthermore, leakage experiments show that wild proteins were capable of disrupting membranes whereas reduced proteins were not. The effect of VtA 3 and VtB on membranes having different phospholipid composition is diverse, affecting the cooperativity and fluidity of the membranes. Viscotoxins interact with membranes in a complex way, most likely organizing themselves at the surface inducing the appearance of defects that lead to the destabilization and disruption of the membrane bilayer.

Research paper thumbnail of A Peptide Pertaining to the Loop Segment of Human Immunodeficiency Virus gp41 Binds and Interacts with Model Biomembranes: Implications for the Fusion Mechanism

Journal of Virology, 2005

The human immunodeficiency virus gp41 envelope protein mediates the entry of the virus into the t... more The human immunodeficiency virus gp41 envelope protein mediates the entry of the virus into the target cell by promoting membrane fusion. In order to gain new insights into the viral fusion mechanism, we studied a 35-residue peptide pertaining to the loop domain of gp41, both in solution and membrane bound, by using infrared and fluorescence spectroscopy. We show here that the peptide, which has a membrane-interacting surface, binds and interacts with phospholipid model membranes and tends to aggregate in the presence of a membranous medium and induce the leakage of vesicle contents. The results reported in this work, i.e., the destabilization and fusion of negatively charged model membranes, suggest an essential role of the loop domain in the membrane fusion process induced by gp41.

Research paper thumbnail of Interaction of a Peptide Derived from the N-Heptad Repeat Region of gp41 Env Ectodomain with Model Membranes. Modulation of Phospholipid Phase Behavior †

Biochemistry, 2005

The HIV-1 gp41 envelope protein mediates the entry of the virus into the target cell by promoting... more The HIV-1 gp41 envelope protein mediates the entry of the virus into the target cell by promoting membrane fusion. With a view toward possible new insights into the protein membrane alteration leading to the viral fusion mechanism, we have studied by infrared and fluorescence spectroscopies a fragment of 21 amino acids corresponding to the N-heptad repeat region of the gp41 ectodomain. Information on the structure of the peptide both in solution and in the presence of model membranes, its incorporation and location in the phospholipid bilayer, and the modulation of the phase behavior of the membrane has been gathered. Here we demonstrate that the peptide binds to and interacts with phospholipid model membranes, changing its conformation and inducing leakage of vesicle contents. These characteristics suggest that different specific regions of gp41 are capable of modifying the biophysical properties of phospholipid membranes and, therefore, might be essential for the assistance and enhancement of the viral and cell fusion process.

Research paper thumbnail of Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotei... more The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pretransmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors. D

Research paper thumbnail of Interaction of Viscotoxins A3 and B with Membrane Model Systems: Implications to Their Mechanism of Action

Biophysical Journal, 2003

Viscotoxins are small proteins that are thought to interact with biomembranes, displaying differe... more Viscotoxins are small proteins that are thought to interact with biomembranes, displaying different toxic activities against a varied number of cell types, being viscotoxin A 3 (VtA 3 ) the most cytotoxic whereas viscotoxin B (VtB) is the less potent. By using infrared and fluorescence spectroscopies, we have studied the interaction of VtA 3 and VtB, both wild and reduced ones, with model membranes containing negatively charged phospholipids. Both VtA 3 and VtB present a high conformational stability, and a similar conformation both in solution and when bound to membranes. In solution, the infrared spectra of the reduced proteins show an increase in bandwidth compared to the nonreduced ones indicating a greater flexibility. VtA 3 and VtB bind with high affinity to membranes containing negatively charged phospholipids and are motional restricted, their binding being dependent on phospholipid composition. Whereas nonreduced proteins maintain their structure when bound to membranes, reduced ones aggregate. Furthermore, leakage experiments show that wild proteins were capable of disrupting membranes whereas reduced proteins were not. The effect of VtA 3 and VtB on membranes having different phospholipid composition is diverse, affecting the cooperativity and fluidity of the membranes. Viscotoxins interact with membranes in a complex way, most likely organizing themselves at the surface inducing the appearance of defects that lead to the destabilization and disruption of the membrane bilayer.