title: Ultrafast fluorescence decay profiles reveal differential unstacking of 2-aminopurine from neighboring bases in single-stranded DNA-binding protein subsites.
abstract: Gene 5 protein (g5p) is a dimeric single-stranded DNA-binding protein encoded by Ff strains of Escherichia coli bacteriophages. The 2-fold rotationally symmetric binding sites of a g5p dimer each bind to four nucleotides, and the dimers bind with high cooperativity to saturate antiparallel single-stranded DNA (ssDNA) strands. Ultrafast time-resolved fluorescence spectroscopies were used to investigate the conformational heterogeneity and dynamics of fluorescent 2-aminopurine (2AP) labels sequestered by bound g5p. The 2AP labels were positioned within the noncomplementary antiparallel tail sequences of d(AC)(8) or d(AC)(9) of hairpin constructs so that each fluorescent label could probe a different subsite location within the DNA-binding site of g5p. Circular dichroism and isothermal calorimetric titrations yielded binding stoichiometries of approximately six dimers per oligomer hairpin when tails were of these lengths. Mobility shift assays demonstrated the formation of a single type of g5p-saturated complex. Femtosecond time-resolved fluorescence spectroscopy showed that the 2AP in the free (non-protein-bound) DNAs had similar heterogeneous distributions of conformations. However, there were significant changes, dominated by a large increase in the population of unstacked bases from ~22 to 59-68%, depending on their subsite locations, when the oligomers were saturated with g5p. Anisotropy data indicated that 2AP in the bound state was less flexible than in the free oligomer. A control oligomer was labeled with 2AP in the loop of the hairpin and showed no significant change in its base stacking upon g5p binding. A proposed model summarizes the data.
authors.affiliation: Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75080-3021, United States.
authors.forename: Hieu-Ngoc
authors.initials: HN
authors.lastname: Nguyen
authors.forename: Liang
authors.initials: L
authors.lastname: Zhao
authors.forename: Carla W
authors.initials: CW
authors.lastname: Gray
authors.forename: Donald M
authors.initials: DM
authors.lastname: Gray
authors.forename: Tianbing
authors.initials: T
authors.lastname: Xia
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D003090:Coliphages / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000821:virology; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D055503:Protein Multimerization; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C057816:gene 5 protein, Enterobacteria phage M13; D015075:2-Aminopurine