RESUMEN
The fluorescent protein Dronpa undergoes reversible photoswitching reactions between the bright "on" and dark "off" states via photoisomerization and proton transfer reactions. We report the room temperature crystal structure of the fast switching Met159Thr mutant of Dronpa at 2.0-Å resolution in the bright on state. Structural differences with the wild type include shifted backbone positions of strand ß8 containing Thr159 as well as an altered A-C dimer interface involving strands ß7, ß8, ß10, and ß11. The Met159Thr mutation increases the cavity volume for the p-hydroxybenzylidene-imidazolinone chromophore as a result of both the side chain difference and the backbone positional differences.
Asunto(s)
Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Animales , Antozoos/genética , Cristalografía por Rayos X , Proteínas Luminiscentes/genética , Simulación de Dinámica Molecular , Mutación , Proteínas Recombinantes/genética , TemperaturaRESUMEN
Chromatin modifications regulate genome function by recruiting proteins to the genome. However, the protein composition at distinct chromatin modifications has yet to be fully characterized. In this study, we used natural protein domains as modular building blocks to develop engineered chromatin readers (eCRs) selective for DNA methylation and histone tri-methylation at H3K4, H3K9 and H3K27 residues. We first demonstrated their utility as selective chromatin binders in living cells by stably expressing eCRs in mouse embryonic stem cells and measuring their subnuclear localization, genomic distribution and histone-modification-binding preference. By fusing eCRs to the biotin ligase BASU, we established ChromID, a method for identifying the chromatin-dependent protein interactome on the basis of proximity biotinylation, and applied it to distinct chromatin modifications in mouse stem cells. Using a synthetic dual-modification reader, we also uncovered the protein composition at bivalently modified promoters marked by H3K4me3 and H3K27me3. These results highlight the ability of ChromID to obtain a detailed view of protein interaction networks on chromatin.
Asunto(s)
Cromatina , Histonas , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/genética , Proteómica/métodos , Animales , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Metilación de ADN/genética , Células Madre Embrionarias , Histonas/química , Histonas/genética , Histonas/metabolismo , RatonesRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.