A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Vertebrate nucleoplasmin and NASP: egg histone storage proteins with multiple chaperone activities.

Recent reviews have focused on the structure and function of histone chaperones involved in different aspects of somatic cell chromatin metabolism. One of the most dramatic chromatin remodeling processes takes place immediately after fertilization and is mediated by egg histone storage chaperones. These include members of the nucleoplasmin (NPM2/NPM3), which are preferentially associated with histones H2A-H2B in the egg and the nuclear autoantigenic sperm protein (NASP) families. Interestingly, in addition to binding and providing storage to H3/H4 in the egg and in somatic cells, NASP has been shown to be a unique genuine chaperone for histone H1. This review revolves around the structural and functional roles of these two families of chaperones whose activity is modulated by their own post-translational modifications (PTMs), particularly phosphorylation. Beyond their important role in the remodeling of paternal chromatin in the early stages of embryogenesis, NPM and NASP members can interact with a plethora of proteins in addition to histones in somatic cells and play a critical role in processes of functional cell alteration, such as in cancer. Despite their common presence in the egg, these two histone chaperones appear to be evolutionarily unrelated. In contrast to members of the NPM family, which share a common monophyletic evolutionary origin, the different types of NASP appear to have evolved recurrently within different taxa.

ATP hydrolysis by RAD50 protein switches MRE11 enzyme from endonuclease to exonuclease.

MRE11-RAD50 is a key early response protein for processing DNA ends of broken chromosomes for repair, yet how RAD50 nucleotide dynamics regulate MRE11 nuclease activity is poorly understood. We report here that ATP binding and ATP hydrolysis cause a striking butterfly-like opening and closing of the RAD50 subunits, and each structural state has a dramatic functional effect on MRE11. RAD50-MRE11 has an extended conformation in solution when MRE11 is an active nuclease. However, ATP binding to RAD50 induces a closed conformation, and in this state MRE11 is an endonuclease. ATP hydrolysis opens the RAD50-MRE11 complex, and MRE11 maintains exonuclease activity. Thus, ATP hydrolysis is a molecular switch that converts MRE11 from an endonuclease to an exonuclease. We propose a testable model in which the open-closed transitions are used by RAD50-MRE11 to discriminate among DNA ends and drive the choice of recombination pathways.

The N-terminal amphipathic region of the Escherichia coli type III secretion system protein EspD is required for membrane insertion and function.

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. These pathogenic E. coli express a syringe-like protein machine, known as the type III secretion system (T3SS), used for the injection of virulence factors into the cytosol of the host epithelial cell. Breaching the epithelial plasma membrane requires formation of a translocation pore that contains the secreted protein EspD. Here we demonstrate that the N-terminal segment of EspD, encompassing residues 1-171, contains two amphipathic domains spanning residues 24-41 and 66-83, with the latter of these helices being critical for EspD function. Fluorescence and circular dichroism analysis revealed that, in solution, His6-EspD1₋171 adopts a native disordered structure; however, on binding anionic small unilamellar vesicles composed of phosphatidylserine, His6-EspD1₋171 undergoes a pH depended conformational change that increases the α-helix content of this protein approximately sevenfold. This change coincides with insertion of the region circumscribing Trp47 into the hydrophobic core of the lipid bilayer. On the HeLa cell plasma membrane, His6-EspD1₋171 forms a homodimer that is postulated to promote EspD-EspD oligomerization and pore formation. Complementation of ΔespD null mutant bacteria with an espDΔ66-83 gene showed that this protein was secreted but non-functional.

sNASP, a histone H1-specific eukaryotic chaperone dimer that facilitates chromatin assembly.

NASP has been described as a histone H1 chaperone in mammals. However, the molecular mechanisms involved have not yet been characterized. Here, we show that this protein is not only present in mammals but is widely distributed throughout eukaryotes both in its somatic and testicular forms. The secondary structure of the human somatic version consists mainly of clusters of alpha-helices and exists as a homodimer in solution. The protein binds nonspecifically to core histone H2A-H2B dimers and H3-H4 tetramers but only forms specific complexes with histone H1. The formation of the NASP-H1 complexes is mediated by the N- and C-terminal domains of histone H1 and does not involve the winged helix domain that is characteristic of linker histones. In vitro chromatin reconstitution experiments show that this protein facilitates the incorporation of linker histones onto nucleosome arrays and hence is a bona fide linker histone chaperone.

Nucleoplasmin-mediated unfolding of chromatin involves the displacement of linker-associated chromatin proteins.

We have previously characterized the interaction of nucleoplasmin with core histones and studied the possible involvement of this chaperone molecule in transcription. Here we study the interaction of nucleoplasmin with chromatin. We show that highly phosphorylated Xenopus laevis egg nucleoplasmin can unfold sperm and somatic chromatin in a way that involves the removal of chromosomal proteins from linker DNA regions without a stable interaction with the nucleosome. The complexes between egg nucleoplasmin and both somatic and sperm-specific linker proteins have been hydrodynamically characterized using sedimentation equilibrium in the analytical ultracentrifuge. The results are discussed within the context of the possible implication of nucleoplasmin in processes such as transcription and replication licensing which take place after egg fertilization at the onset of development.

The anthracycline antibiotics: antitumor drugs that alter chromatin structure.

Anthracycline antibiotics are an important group of antitumor drugs widely used in cancer chemotherapy. However, despite the increasing interest in these chemotherapeutic agents, their mechanism of action is not yet completely understood. Here, we review what is currently known about the molecular mechanisms involved with special emphasis on the interaction of these drugs with chromatin and its constitutive components: DNA and histones. The evidence suggests that one very important component of the activity of these drugs is the result of these manifold interactions that lead to a chromatin unfolding and aggregation. This chromatin structural disruption is likely to interfere with the metabolic processes of DNA (replication and transcription) and it may play an important role in the apoptosis undergone by the cells upon treatment with these drugs.

Histone H2A ubiquitination does not preclude histone H1 binding, but it facilitates its association with the nucleosome.

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.

Binding of antitumor antibiotic daunomycin to histones in chromatin and in solution.

Daunomycin is an anticancer drug that is well-known to interact with DNA in chromatin. Using a compositionally defined chicken erythrocyte chromatin fraction, we have obtained conclusive evidence that the drug is also able to interact with chromatin-bound linker histones without any noticeable binding to core histones. The drug can interact in an equal fashion with both histone H1 and H5 and to a greater extent with core histones H3/H4 and H2A/H2B as free proteins in solution. Thus, the binding of daunomycin to linker histones in the chromatin fiber is most likely due to the well-known higher accessibility of these histones to the surrounding environment of the fiber. Binding of daunomycin to linker histones appears to primarily involve the trypsin-resistant (winged-helix) domain of these proteins. The studies described here reveal the occurrence of a previously undisclosed mechanism for the antitumor activity of anthracycline drugs at the chromatin level.