Stoichiometry of Rtt109 complexes with Vps75 and histones H3-H4.

Histone acetylation is one of many posttranslational modifications that affect nucleosome accessibility. Vps75 is a histone chaperone that stimulates Rtt109 acetyltransferase activity toward histones H3-H4 in yeast. In this study, we use sedimentation velocity and light scattering to characterize various Vps75-Rtt109 complexes, both with and without H3-H4. These complexes were previously ill-defined because of protein multivalency and oligomerization. We determine both relative and absolute stoichiometry and define the most pertinent and homogeneous complexes. We show that the Vps75 dimer contains two unequal binding sites for Rtt109, with the weaker binding site being dispensable for H3-H4 acetylation. We further show that the Vps75-Rtt109-(H3-H4) complex is in equilibrium between a 2:1:1 species and a 4:2:2 species. Using a dimerization mutant of H3, we show that this equilibrium is mediated by the four-helix bundle between the two copies of H3. We optimize the purity, yield, and homogeneity of Vps75-Rtt109 complexes and determine optimal conditions for solubility when H3-H4 is added. Our comprehensive biochemical and biophysical approach ultimately defines the large-scale preparation of Vps75-Rtt109-(H3-H4) complexes with precise stoichiometry. This is an essential prerequisite for ongoing high-resolution structural and functional analysis of this important multi-subunit complex.

Comprehensive analysis of histone-binding proteins with multi-angle light scattering.

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.

Isomerization of Keggin Al13 Ions Followed by Diffusion Rates.

The solution chemistry of aluminum has long interested scientists due to its relevance to materials chemistry and geochemistry. The dynamic behavior of large aluminum-oxo-hydroxo clusters, specifically [Al13 O4 (OH)24 (H2 O)12 ]7+ (Al13 ), is the focus of this paper. 27 Al NMR, 1 H NMR, and 1 H DOSY techniques were used to follow the isomerization of the ϵ-Al13 in the presence of glycine and Ca2+ at 90 °C. Although the conversion of ϵ-Al13 to new clusters and/or Baker-Figgis-Keggin isomers has been studied previously, new 1 H NMR and 1 H DOSY analyses provided information about the role of glycine, the ligated intermediates, and the mechanism of isomerization. New 1 H NMR data suggest that glycine plays a critical role in the isomerization. Surprisingly, glycine does not bind to Al30 clusters, which were previously proposed as an intermediate in the isomerization. Additionally, a highly symmetric tetrahedral signal (δ=72 ppm) appeared during the isomerization process, which evidence suggests corresponds to the long-sought α-Al13 isomer in solution.