title: Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy.
abstract: Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies.
authors.affiliation: Department of Integrative Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR 7104, Université de Strasbourg, 67404 Illkirch, France voltz@igbmc.fr.
authors.forename: Karine
authors.initials: K
authors.lastname: Voltz
authors.affiliation: Institut de Physique et Chimie des Matériaux de Strasbourg & Labex NIE, CNRS UMR 7504, Université de Strasbourg, Strasbourg, France Jeremie.Leonard@ipcms.unistra.fr.
authors.forename: Jérémie
authors.initials: J
authors.lastname: Léonard
authors.affiliation: Institut de Physique et Chimie des Matériaux de Strasbourg & Labex NIE, CNRS UMR 7504, Université de Strasbourg, Strasbourg, France.
authors.forename: Patricia Tourón
authors.initials: PT
authors.lastname: Touceda
authors.affiliation: Institut de Physique et Chimie des Matériaux de Strasbourg & Labex NIE, CNRS UMR 7504, Université de Strasbourg, Strasbourg, France.
authors.forename: Jamie
authors.initials: J
authors.lastname: Conyard
authors.affiliation: Institut de Physique et Chimie des Matériaux de Strasbourg & Labex NIE, CNRS UMR 7504, Université de Strasbourg, Strasbourg, France.
authors.forename: Ziyad
authors.initials: Z
authors.lastname: Chaker
authors.affiliation: Department of Integrative Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR 7104, Université de Strasbourg, 67404 Illkirch, France.
authors.forename: Annick
authors.initials: A
authors.lastname: Dejaegere
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch Cedex, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Godet
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, Illkirch Cedex, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
authors.affiliation: Institut de Physique et Chimie des Matériaux de Strasbourg & Labex NIE, CNRS UMR 7504, Université de Strasbourg, Strasbourg, France.
authors.forename: Stefan
authors.initials: S
authors.lastname: Haacke
authors.affiliation: Department of Integrative Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964, Centre National de Recherche Scientifique (CNRS) UMR 7104, Université de Strasbourg, 67404 Illkirch, France rstote@igbmc.fr.
authors.forename: Roland H
authors.initials: RH
authors.lastname: Stote
mesh_terms: D015075:2-Aminopurine; D004247:DNA / Q000737:chemistry*; D004279:DNA, Viral / Q000737:chemistry; D015497:HIV-1 / Q000235:genetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D004279:DNA, Viral; D015075:2-Aminopurine; D004247:DNA