RESUMEN
Interactions between histones and their binding partners are an important aspect of chromatin biology. Determining the stoichiometry of histone-containing complexes is an important pre-requisite for performing in vitro biochemical, biophysical and structural analyses. In this article, we detail how Size Exclusion Chromatography (SEC) coupled to Multi-Angle Light Scattering (MALS) can be used to study histone chaperones and their complexes. Our protocol details system setup, sample preparation, data collection, and data interpretation. We provide tips on designing an informative SEC-MALS experiment, using histone chaperones Nap1 and Vps75 as demonstrative examples. We outline recommendations to overcome specific challenges such as protein oligomerization, heterogeneity, and non-specific binding. We find SEC-MALS to be a robust and user-friendly approach for characterizing histone-binding proteins and their complexes.
Asunto(s)
Cromatografía en Gel/métodos , Luz , Chaperonas Moleculares/análisis , Proteínas de Saccharomyces cerevisiae/análisis , Dispersión de Radiación , ARNt Metiltransferasas/análisis , Histonas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Agregado de Proteínas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismoRESUMEN
In intravacuolar pathogens, iron is essential for growth and virulence. In Legionella pneumophila, a putative transmembrane protein inserted on the surface of the host pathogen-containing vacuole, IroT/MavN, facilitates intravacuolar iron acquisition from the host by an unknown mechanism, bypassing the problem of Fe(III) insolubility and mobilization. We developed a platform for purification and reconstitution of IroT in artificial lipid bilayer vesicles (proteoliposomes). By encapsulating the fluorescent reporter probe Fluozin-3, we reveal, by real-time metal transport assays, that IroT is a high-affinity iron transporter selective for Fe(II) over other essential transition metals. Mutational analysis reveals important residues in the transmembrane helices, soluble domains, and loops important for substrate recognition and translocation. The work establishes the substrate transport properties in a novel transporter family important for iron acquisition at the host-pathogen intravacuolar interface and provides chemical tools for a comparative investigation of the translocation properties in other iron transporter families.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Hierro/metabolismo , Legionella pneumophila/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Colorantes Fluorescentes , Glucolípidos/química , Transporte Iónico , Cinética , Mutación , Compuestos Policíclicos , Unión Proteica , Liposomas Unilamelares/químicaRESUMEN
We report the crystal structure of nuclear import receptor Importin-9 bound to its cargo, the histones H2A-H2B. Importin-9 wraps around the core, globular region of H2A-H2B to form an extensive interface. The nature of this interface coupled with quantitative analysis of deletion mutants of H2A-H2B suggests that the NLS-like sequences in the H2A-H2B tails play a minor role in import. Importin-9â¢H2A-H2B is reminiscent of interactions between histones and histone chaperones in that it precludes H2A-H2B interactions with DNA and H3-H4 as seen in the nucleosome. Like many histone chaperones, which prevent inappropriate non-nucleosomal interactions, Importin-9 also sequesters H2A-H2B from DNA. Importin-9 appears to act as a storage chaperone for H2A-H2B while escorting it to the nucleus. Surprisingly, RanGTP does not dissociate Importin-9â¢H2A-H2B but assembles into a RanGTPâ¢Importin-9â¢H2A-H2B complex. The presence of Ran in the complex, however, modulates Imp9-H2A-H2B interactions to facilitate its dissociation by DNA and assembly into a nucleosome.
Asunto(s)
Histonas/química , Histonas/metabolismo , Carioferinas/química , Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Cristalografía por Rayos X , Análisis Mutacional de ADN , Humanos , Carioferinas/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Conformación Proteica , XenopusRESUMEN
Nucleosome assembly proteins (Naps) influence chromatin dynamics by directly binding to histones. Here we provide a comprehensive structural and biochemical analysis of a Nap protein from Caenorhabditis elegans (CeNap1). CeNap1 naturally lacks the acidic N-terminal tail and has a short C-terminal tail compared to many other Nap proteins. Comparison of CeNap1 with full length and tail-less constructs of Saccharomyces cerevisiae Nap1 uncovers the role of these tails in self-association, histone binding, and Nap competition with DNA for H2A-H2B. We find that the presence of tails influences the stoichiometry of H2A-H2B binding and is required to complete the interactions between H2A-H2B and DNA. The absolute stoichiometry of the Nap protein and H2A-H2B complex is 2:1 or 2:2, with only a very small population of higher-order oligomers occurring at 150 mM NaCl. We also show that H3-H4 binds differently than H2A-H2B and that an (H3-H4)2 tetramer can simultaneously bind two Nap2 protein homodimers.