We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photobleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1 in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 binding in vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.Histone H1 is the fifth histone subtype and is not one of the histones that form the histone octamer of the nucleosome. Rather, histone H1 binds to the surface of the nucleosome and interacts with nucleosomal DNA at the entry and exit points (1). In doing so, histone H1 is critical in determining the higher-order folding states of chromatin. Because of this property, histone H1 has traditionally been considered a general repressor of transcription (3). Consistent with this hypothesis, histone H1 was found to be modestly depleted in transcriptionally active genes (4 -6). More recently, genetic studies have revealed contributions of H1 histones to the establishment of epigenetic silencing (7-10). In addition to a structural role, histone H1 also functions in gene-specific regulation. A large number of studies have demonstrated that H1 histones or specific variants are directly involved in the regulation of specific genes (3,(11)(12)(13)(14), consistent with the observation of differential gene expression when the sole histone H1 gene was knocked out in Tetrahymena thermophila (15).The structure of H1 histones is typically considered to consist of three separate domains (16). A short stretch of amino acids on the N terminus and a much larger stretch that comprises the C terminus show significant variability between individual subtypes as well as between species. The amino and carboxyl termini have diverged considerably throughout the evolution of metazoans (17). If we restrict the analysis to mammals, the C termini diverge between individual histone H1 variants, but the sequences of the individual C termini are well conserved between species. When histone H1 sequences are examined in a broader range of ...

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Fluorescence recovery after photobleaching (FRAP) is an experimental technique used to measure the mobility of proteins within the cell nucleus. After proteins of interest are fluorescently tagged for their visualization and monitoring, a small region of the nucleus is photobleached. The experimental FRAP data are obtained by recording the recovery of the fluorescence in this region over time. In this paper, we characterize the fluorescence recovery curves for diffusing nuclear proteins undergoing binding events with an approximate spatially homogeneous structure. We analyze two mathematical models for interpreting the experimental FRAP data, namely a reaction-diffusion model and a compartmental model. Perturbation analysis leads to a clear explanation of two important limiting dynamical types of behavior exhibited by experimental recovery curves, namely, (1) a reduced diffusive recovery, and (2) a biphasic recovery characterized by a fast phase and a slow phase. We show how the two models, describing the same type of dynamics using different approaches, relate and share common ground. The results can be used to interpret experimental FRAP data in terms of protein dynamics and to simplify the task of parameter estimation. Application of the results is demonstrated for nuclear actin and type H1 histone.

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Previous studies from our laboratory have indicated that the lipopolysaccharide (LPS) from Mannheimia haemolytica serotype A1 contains both L-glycero-D-manno-heptose and D-glycero-D-manno-heptose residues. NMR methods making use of 1D 1H selective excitation and 2D (1H, 13C) and (1H, 31P) heteronuclear experiments were used for the structural determination of the major core oligosaccharide components of the deacylated low-molecular-mass LPS obtained following sequential treatment with anhydrous hydrazine and aq KOH. The core oligosaccharide region was found to be composed of a branched octasaccharide linked to the deacylated lipid A moiety via a 3-deoxy-4-phospho-D-manno-oct-2-ulosonate residue having the structure,[Formula: see text]Heterogeneity was found to be present at several linkages. NMR methods were devised to distinguish between the diastereomeric forms of the heptose residues. Synthesized monosaccharides of L-D- and D-D-heptose were used as model compounds for analysis of the 1H and 13C NMR chemical shifts and proton coupling constants. Molecular modeling using a Monte Carlo method for conformational analysis of saccharides was used to determine the conformation of the inner core of the oligosaccharide and to establish the stereochemical relationships between the heptoses.Key words: LPS, NMR, conformation, oligosaccharide, heptose.

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