Unfolding simulations reveal the mechanism of extreme unfolding cooperativity in the kinetically stable alpha-lytic protease.

Kinetically stable proteins, those whose stability is derived from their slow unfolding kinetics and not thermodynamics, are examples of evolution's best attempts at suppressing unfolding. Especially in highly proteolytic environments, both partially and fully unfolded proteins face potential inactivation through degradation and/or aggregation, hence, slowing unfolding can greatly extend a protein's functional lifetime. The prokaryotic serine protease alpha-lytic protease (alphaLP) has done just that, as its unfolding is both very slow (t(1/2) approximately 1 year) and so cooperative that partial unfolding is negligible, providing a functional advantage over its thermodynamically stable homologs, such as trypsin. Previous studies have identified regions of the domain interface as critical to alphaLP unfolding, though a complete description of the unfolding pathway is missing. In order to identify the alphaLP unfolding pathway and the mechanism for its extreme cooperativity, we performed high temperature molecular dynamics unfolding simulations of both alphaLP and trypsin. The simulated alphaLP unfolding pathway produces a robust transition state ensemble consistent with prior biochemical experiments and clearly shows that unfolding proceeds through a preferential disruption of the domain interface. Through a novel method of calculating unfolding cooperativity, we show that alphaLP unfolds extremely cooperatively while trypsin unfolds gradually. Finally, by examining the behavior of both domain interfaces, we propose a model for the differential unfolding cooperativity of alphaLP and trypsin involving three key regions that differ between the kinetically stable and thermodynamically stable classes of serine proteases

A bacteriophage tubulin harnesses dynamic instability to center DNA in infected cells.

Dynamic instability, polarity, and spatiotemporal organization are hallmarks of the microtubule cytoskeleton that allow formation of complex structures such as the eukaryotic spindle. No similar structure has been identified in prokaryotes. The bacteriophage-encoded tubulin PhuZ is required to position DNA at mid-cell, without which infectivity is compromised. Here, we show that PhuZ filaments, like microtubules, stochastically switch from growing in a distinctly polar manner to catastrophic depolymerization (dynamic instability) both in vitro and in vivo. One end of each PhuZ filament is stably anchored near the cell pole to form a spindle-like array that orients the growing ends toward the phage nucleoid so as to position it near mid-cell. Our results demonstrate how a bacteriophage can harness the properties of a tubulin-like cytoskeleton for efficient propagation. This represents the first identification of a prokaryotic tubulin with the dynamic instability of microtubules and the ability to form a simplified bipolar spindle

Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context

Multi-time delay, multi-point Linear Stochastic Estimation of a cavity shear layer velocity from wall-pressure measurements

Multi-time-delay Linear Stochastic Estimation (MTD-LSE) technique is thoroughly described, focusing on its fundamental properties and potentialities. In the multi-time-delay ap- proach, the estimate of the temporal evolution of the velocity at a given location in the flow field is obtained from multiple past samples of the unconditional sources. The technique is applied to estimate the velocity in a cavity shear layer flow, based on wall-pressure measurements from multiple sensor

Modular Equations and Distortion Functions

Modular equations occur in number theory, but it is less known that such equations also occur in the study of deformation properties of quasiconformal mappings. The authors study two important plane quasiconformal distortion functions, obtaining monotonicity and convexity properties, and finding sharp bounds for them. Applications are provided that relate to the quasiconformal Schwarz Lemma and to Schottky's Theorem. These results also yield new bounds for singular values of complete elliptic integrals.Comment: 23 page

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries

A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzymelabeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laserscanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells

Specific staining of human chromosomes in Chinese hamster x man hybrid cell lines demonstrates interphase chromosome territories

In spite of Carl Rabl's (1885) and Theodor Boveri's (1909) early hypothesis that chromosomes occupy discrete territories or domains within the interphase nucleus, evidence in favor pf this hypothesis has been limited and indirect so far in higher plants and animals. The alternative possibility that the chromatin fiber of single chromosomes might be extended throughout the major part of even the whole interphase nucleus has been considered for many years. In the latter case, chromosomes would only exist as discrete chromatin bodies during mitosis but not during interphase. Both possibilities are compatible with Boveri's well established paradigm of chromosome individuality. Here we show that an active human X chromosome contained as the only human chromosome in a Chinese hamster x man hybrid cell line can be visualized both in metaphse plates and in interphase nuclei after in situ hybridization with either 3H- or biotin-labeled human genomic DNA. We demonstrate that this chromosome is organized as a distinct chromatin body throughout interphase. In addition, evidence for the territorial organization of human chromosomes is also presented for another hybrid cell line containing several autosomes and the human X chromosome. These findings are discussed in the context of our present knowledge of the organization and topography of interphase chromosomes. General applications of a strategy aimed at specific staining of individual chromosomes in experimental and clinical cytogenetics are briefly considered