Engineering the structural stability and functional properties of the GI domain into the intrinsically unfolded GII domain of the yeast linker histone Hho1p

Yeast Hho1p contains two domains, GI and GII, that are homologous to the single globular domain of the linker histone H1 (GH1). We showed previously that the isolated GI and GII domains have different structural stabilities and functional properties. GI, like GH1 and the related GH5, is stably folded at low ionic strength (10 mM sodium phosphate) and gives strong protection of chromatosome-length DNA ( approximately 166 bp) during micrococcal nuclease digestion of chromatin. GII is intrinsically unfolded in 10 mM sodium phosphate and gives weak chromatosome protection, but in 250 mM sodium phosphate has a structure very similar to that of GI as determined by NMR spectroscopy. We now show that the loop between helices II and III in GII is the cause of both its instability and its inability to confer strong chromatosome protection. A mutant GII, containing the loop of GI, termed GII-L, is stable in 10 mM sodium phosphate and is as effective as GI in chromatosome protection. Two GII mutants with selected mutations within the original loop were also slightly more stable than GII. In GII, two of the four basic residues conserved at the second DNA binding site ("site II") on the globular domain of canonical linker histones, and in GI, are absent. Introduction of the two "missing" site II basic residues into GII or GII-L destabilised the protein and led to decreased chromatosome protection relative to the protein without the basic residues. In general, the ability to confer chromatosome protection in vitro is closely related to structural stability (the relative population of structured and unstructured states). We have determined the structure of GII-L by NMR spectroscopy. GII-L is very similar to GII folded in 250 mM sodium phosphate, with the exception of the substituted loop region, which, as in GI, contains a single helical turn.     

Solution structure of the isolated Pelle death domain

The interaction between the death domains (DDs) of Tube and the protein kinase Pelle is an important component of the Toll pathway. Published crystallographic data suggests that the Pelle-Tube DD interface is plastic and implies that in addition to the two predominant Pelle-Tube interfaces, a third interaction is possible. We present the NMR solution structure of the isolated death domain of Pelle and a study of the interaction between the DDs of Pelle and Tube. Our data suggests the solution structure of the isolated Pelle DD is similar to that of Pelle DD in complex with Tube. Additionally, they suggest that the plasticity observed in the crystal structure may not be relevant in the functioning death domain complex.     

Exploring sequence/folding space: folding studies on multiple hydrophobic core mutants of ubiquitin

The stability, dynamic, and structural properties of ubiquitin and two multiple hydrophobic core mutants were studied. One of the mutants (U4) has seven substitutions in the hydrophobic core (M1L, I3L, V5I, I13F, L15V, V17M, and V26L). On average, its side chains are larger than the wild-type, and it can thus be thought of as having an overpacked core. The other mutant (U7) has two substitutions (I3V and I13V). On average, it has smaller side chains than the wild-type, and it can therefore be considered to be underpacked. The three proteins are well-folded and show similar backbone dynamics (T(1), T(2), and HNOE values), indicating that the regular secondary structure extends over the same residue ranges. The crystallographic structure of U4 was determined. The final R(factor) and R(free) are 0.198 and 0.248, respectively, at 2.18 A resolution. The structure of U4 is very similar to wild-type ubiquitin. Remarkably, there are almost no changes in the positions of the C(alpha) atoms along the entire backbone, and the hydrogen-bonding network is maintained. The mutations of the hydrophobic core are accommodated by small movements of side chains in the core of mutated and nonmutated residues. Unfolding and refolding kinetic studies revealed that U4 unfolds with the highest rates; however, its refolding rate constants are very similar to those of the wild-type protein. Conversely, U7 seems to be the most destabilized protein; its refolding rate constant is smaller than the other two proteins. This was confirmed by stopped-flow techniques and by H/D exchange methodologies. This work illustrates the possibility of repacking the hydrophobic core of small proteins and has important implications in the de novo design of stable proteins.     

Differential Expression of Immune Response Genes in Steller Sea Lions (Eumetopias jubatus): An Indicator of Ecosystem Health?

Characterization of the polygenic and polymorphic features of the Steller sea lion major histocompatibility complex (MHC) provides an ideal window for evaluating immunologic vigor of the population and identifying emergence of new genotypes that reflect ecosystem pressures. MHC genotyping can be used to measure the potential immunologic vigor of a population. However, since ecosystem-induced changes to MHC genotype can be slow to emerge, measurement of differential expression of these genes can potentially provide real-time evidence of immunologic perturbations. MHC DRB genes were cloned and sequenced using peripheral blood mononuclear leukocytes derived from 10 Steller sea lions from Southeast Alaska, Prince William Sound, and the Aleutian Islands. Nine unique DRB gene sequences were represented in each of 10 animals. MHC DRB gene expression was measured in a subset of six sea lions. Although DRB in genomic DNA was identical in all individuals, relative levels of expressed DRB mRNA was highly variable. Selective suppression of MHC DRB genes could be indicative of geographically disparate environmental pressures, thereby serving as an immediate and sensitive indicator of population and ecosystem health.     

Infection with a novel gammaherpesvirus in northern elephant seals (Mirounga angustirostris)

Twenty juvenile northern elephant seals (Mirounga angustirostris) that died between 1998 and 2004 had ulcers on the tongue, palatine mucosa, and/or tonsils. Histologic examination of the lesions revealed cytoplasmic swelling, nuclear pyknosis, and eosinophilic to amphophilic intranuclear inclusions bodies suggestive of herpesviral infection. Electron microscopic examination and polymerase chain reaction analysis confirmed the presence of a herpesvirus. Subsequent DNA sequencing identified this to be a new gammaherpesvirus that was similar to Porcine lymphotropic virus 2, Alcephaline herpesvirus 1 (malignant catarrhal fever virus from wildebeest), and Chlorocebus rhadinovirus 1 from African green monkeys. Identical herpesviral DNA was also detected in blood and mucosal swabs collected from five healthy elephant seal pups.     

Gene conversion in human rearranged immunoglobulin genes

Over the past 20 years, many DNA sequences have been published suggesting that all or part of the V(H) segment of a rearranged immunoglobulin gene may be replaced in vivo. Two different mechanisms appear to be operating. One of these is very similar to primary V(D)J recombination, involving the RAG proteins acting upon recombination signal sequences, and this has recently been proven to occur. Other sequences, many of which show partial V(H) replacements with no addition of untemplated nucleotides at the V(H)-V(H) joint, have been proposed to occur by an unusual RAG-mediated recombination with the formation of hybrid (coding-to-signal) joints. These appear to occur in cells already undergoing somatic hypermutation in which, some authors are convinced, RAG genes are silenced. We recently proposed that the latter type of V(H) replacement might occur by homologous recombination initiated by the activity of AID (activation-induced cytidine deaminase), which is essential for somatic hypermutation and gene conversion. The latter has been observed in other species, but not in human Ig genes, so far. In this paper, we present a new analysis of sequences published as examples of the second type of rearrangement. This not only shows that AID recognition motifs occur in recombination regions but also that some sequences show replacement of central sections by a sequence from another gene, similar to gene conversion in the immunoglobulin genes of other species. These observations support the proposal that this type of rearrangement is likely to be AID-mediated rather than RAG-mediated and is consistent with gene conversion.     

HMGB1-Facilitated p53 DNA Binding Occurs via HMG-Box/p53 Transactivation Domain Interaction,Regulated by the Acidic Tail

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Highlights? Binding of p53 to its cognate DNA is facilitated by HMGB1 ? The N-terminal region of p53 (residues 38–61; TAD2) interacts with the HMG boxes ? The acidic tail of HMGB1 masks the p53 binding site in the free proteins ? The structure of the A-box/p53(1–93) complex shows that TAD2 acts as an ss-DNA mimic     

Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes

The purpose of this study was to generate the equivalent of a cholesterol/temperature phase map for a biological membrane using fluorescence spectroscopy. The pseudo-phase map was created using human erythrocytes treated with various concentrations of methyl-beta-cyclodextrin to remove defined amounts of cholesterol and a trio of fluorescent probes that assess different membrane properties (laurdan, diphenylhexatriene, and merocyanine 540). Parallel experiments with two-photon microscopy suggested that changes in cellular cholesterol content affected the entire membrane rather than being localized to specific macroscopic domains. The various regions of the composite erythrocyte pseudo-phase map were interpreted using analogous data acquired from multilamellar vesicles that served as simplified models of cholesterol-dependent phases. The vesicles consisted of various concentrations of cholesterol (0 to 50 mol%) with either palmitoyl sphingomyelin, 1:1 dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine, or phospholipid mixtures intended to simulate either the inner or outer leaflet of erythrocyte membranes. Four distinguishable regions were observed in sphingomyelin phase maps corresponding to the traditional solid-ordered and liquid-disordered phases and two types of liquid-ordered behavior. Physical properties were less diverse in the mixed phospholipid vesicles, as expected, based on previous studies. Erythrocytes displayed five regions of different combinations of membrane properties along the phase map. Some of the observations identified similarities between the cells and liquid-ordered behavior observed in the various types of liposomes as well as some interesting differences.