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Concentrative uptake of osmoprotectants via transporter ProP contributes to the rehydration of Escherichia coli cells that encounter high osmolality media. A member of the major facilitator superfamily, ProP is activated by osmotic upshifts in whole bacteria, in cytoplasmic membrane vesicles and in proteoliposomes prepared with the purified protein. Soluble protein ProQ is also required for full osmotic activation of ProP in vivo. ProP is differentiated from structural and functional homologues by its osmotic activation and its C-terminal extension, which is predicted to form an alpha-helical coiled-coil. A synthetic polypeptide corresponding to the C-terminus of ProP (ProP-p) formed a dimeric alpha-helical coiled-coil. A derivative of transporter ProP lacking 26 C-terminal amino acids was expressed but inactive. A derivative harbouring amino acid changes K460I, Y467I and H495I (each at the core, coiled-coil 'a' position) required a larger osmotic upshift for activation than did the wild type transporter. The same changes extended, stabilized and altered the oligomeric state of the coiled-coil formed by ProP-p. Amino acid change R488I (also at the 'a' position) further increased the magnitude of the osmotic upshift required to activate ProP, reduced the activity attained and rendered ProP activation transient. Unexpectedly, replacement R488I destabilized the coiled-coil formed by ProP-p. The activity and osmotic activation of ProP were even more strongly attenuated by helix-destabilizing change I474P. These data demonstrate that the carboxyl terminal domain of ProP can form a homodimeric alpha-helical coiled-coil with unusual properties. They implicate the C-terminal domain in the osmotic activation of ProP.

Histone H3 Lys4 trimethylation (H3-K4me3) is a conserved mark of actively transcribed chromatin. Using a conditional mutant of the yeast H3-K4 methyltransferase, Set1p, we demonstrate rapid turnover of H3-K4me3 and H3-K4me2 in vivo and show this process requires Yjr119Cp, of the JARID1 family of JmjC proteins. Ectopic overexpression of mouse Jarid1B, a Yjr119Cp homolog, greatly diminished H3-K4me3 and H3-K4me2 in HeLa cells, suggesting these proteins function as K4 demethylases in vivo.

The first step in the formation of the nucleosome is commonly assumed to be the deposition of a histone H3-H4 heterotetramer onto DNA. Antisilencing function 1 (ASF1) is a major histone H3-H4 chaperone that deposits histones H3 and H4 onto DNA. With a goal of understanding the mechanism of deposition of histones H3 and H4 onto DNA, we have determined the stoichiometry of the Asf1-H3-H4 complex. We have established that a single molecule of Asf1 binds to an H3-H4 heterodimer using gel filtration, amino acid, reversed-phase chromatography, and analytical ultracentrifugation analyses. We demonstrate that Asf1 blocks formation of the H3-H4 heterotetramer by a mechanism that likely involves occlusion of the H3-H3 dimerization interface.

We describe here a systematic investigation into the role of position a in the hydrophobic core of a model coiled-coil protein in determining coiled-coil stability and oligomerization state. We employed a model coiled coil that allowed the formation of an extended three-stranded trimeric oligomerization state for some of the analogs; however, due to the presence of a Cys-Gly-Gly linker, unfolding occurred from the same two-stranded monomeric oligomerization state for all of the analogs. Denaturation from a two-stranded state allowed us to measure the relative contribution of 20 different amino acid side chains to coiled-coil stability from chemical denaturation profiles. In addition, the relative hydrophobicity of the substituted amino acid side chains was assessed by reversed-phase high-performance liquid chromatography and found to correlate very highly (R = 0.95) with coiled-coil stability. We also determined the effect of position a in specifying the oligomerization state using ultracentrifugation as well as high-performance size-exclusion chromatography. We found that nine of the analogs populated one oligomerization state exclusively at peptide concentrations of 50 microM under benign buffer conditions. The Leu-, Tyr-, Gln-, and His-substituted analogs were found to be exclusively three-stranded trimers, while the Asn-, Lys-, Orn-, Arg-, and Trp-substituted analogs formed exclusively two-stranded monomers. Modeling results for the Leu-substituted analog showed that a three-stranded oligomerization state is preferred due to increased side-chain burial, while a two-stranded oligomerization state was observed for the Trp analog due to unfavorable cavity formation in the three-stranded state.

Two dimensional