title: Fluorescence strategies for high-throughput quantification of protein interactions.
abstract: Advances in high-throughput characterization of protein networks in vivo have resulted in large databases of unexplored protein interactions that occur during normal cell function. Their further characterization requires quantitative experimental strategies that are easy to implement in laboratories without specialized equipment. We have overcome many of the previous limitations to thermodynamic quantification of protein interactions, by developing a series of in-solution fluorescence-based strategies. These methods have high sensitivity, a broad dynamic range, and can be performed in a high-throughput manner. In three case studies we demonstrate how fluorescence (de)quenching and fluorescence resonance energy transfer can be used to quantitatively probe various high-affinity protein-DNA and protein-protein interactions. We applied these methods to describe the preference of linker histone H1 for nucleosomes over DNA, the ionic dependence of the DNA repair enzyme PARP1 in DNA binding, and the interaction between the histone chaperone Nap1 and the histone H2A-H2B heterodimer.
authors: Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
authors: Aaron R
authors: AR
authors: Hieb
authors: Sheena
authors: S
authors: D'Arcy
authors: Michael A
authors: MA
authors: Kramer
authors: Alison E
authors: AE
authors: White
authors: Karolin
authors: K
authors: Luger
mesh_terms: D000818:Animals; D004247:DNA / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry / Q000379:methods*; D057166:High-Throughput Screening Assays; D006657:Histones / Q000032:analysis / Q000378:metabolism; D051379:Mice; D056490:Nucleosome Assembly Protein 1 / Q000032:analysis; D009707:Nucleosomes / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis
chemical_list: D006657:Histones; C533556:NAP1 protein, S cerevisiae; D056490:Nucleosome Assembly Protein 1; D009707:Nucleosomes; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases