Simulated pathogenic conformational switch regions matched well with the biochemical findings

Pathogenic conformational conversion is a general causation of many disease, such as transmissible spon- giform encephalopathy (TSE) caused by misfolding of prion, sickle cell anemia, and etc. In such structural changes, misfolding occurs in regions important for the stability of native structure firstly. This destabi- lizes the normal conformation and leads to subsequent errors in folding pathway. Sites involved in the first stage can be deemed switch regions of the protein, and are vital for conformational conversion. Namely it could be a switch of disease at residue level. Here we report an algorithm that can identify such sites computationally with an accuracy of 93%, by calculating the probability of the native structure of a short segment jumping to a mistake one. Knowledge of such switch sites could be used to target clinical therapy, study physiological and pathologic mechanism of protein, and etc.

Switch Region for Pathogenic Structural Change in Conformational Disease and Its Prediction

Many diseases are believed to be related to abnormal protein folding. In the first step of such pathogenic structural changes, misfolding occurs in regions important for the stability of the native structure. This destabilizes the normal protein conformation, while exposing the previously hidden aggregation-prone regions, leading to subsequent errors in the folding pathway. Sites involved in this first stage can be deemed switch regions of the protein, and can represent perfect binding targets for drugs to block the abnormal folding pathway and prevent pathogenic conformational changes. In this study, a prediction algorithm for the switch regions responsible for the start of pathogenic structural changes is introduced. With an accuracy of 94%, this algorithm can successfully find short segments covering sites significant in triggering conformational diseases (CDs) and is the first that can predict switch regions for various CDs. To illustrate its effectiveness in dealing with urgent public health problems, the reason of the increased pathogenicity of H5N1 influenza virus is analyzed; the mechanisms of the pandemic swine-origin 2009 A(H1N1) influenza virus in overcoming species barriers and in infecting large number of potential patients are also suggested. It is shown that the algorithm is a potential tool useful in the study of the pathology of CDs because: (1) it can identify the origin of pathogenic structural conversion with high sensitivity and specificity, and (2) it provides an ideal target for clinical treatment.

Investigating changes in global tropical cyclone storm frequency and intensity

Understanding fluctuations in tropical cyclone activity along United States shores and abroad becomes increasingly important as coastal managers and planners seek to save lives, mitigate damage, and plan for resilience in the face of changing storminess and sea-level rise. Tropical cyclone activity has long been of concern to coastal areas as they bring strong winds, heavy rains, and high seas. Given projections of a warming climate, current estimates suggest that not only will tropical cyclones increase in frequency, but also in intensity (maximum sustained winds and minimum central pressures). An understanding of what has happened historically is an important step in identifying potential future changes in tropical cyclone frequency and intensity. The ability to detect such changes depends on a consistent and reliable global tropical cyclone dataset. Until recently no central repository for historical tropical cyclone data existed. To fill this need, the International Best Track Archive for Climate Stewardship (IBTrACS) dataset was developed to collect all known global historical tropical cyclone data into a single source for dissemination. With this dataset, a global examination of changes in tropical cyclone frequency and intensity can be performed. Caveats apply to any historical tropical cyclone analysis however, as the data contributed to the IBTrACS archive from various tropical cyclone warning centers is still replete with biases that may stem from operational changes, inhomogeneous monitoring programs, and time discontinuities. A detailed discussion of the difficulties in detecting trends using tropical cyclone data can be found in Landsea et al. 2006. The following sections use the IBTrACS dataset to show the global spatial variability of tropical cyclone frequency and intensity. Analyses will show where the strongest storms typically occur, the regions with the highest number of tropical cyclones per decade, and the locations of highest average maximum wind speeds. (PDF contains 3 pages)

Preparation and characterization of an intramolecular electron transfer in a pentaammineruthenium derivative of Candida krisei cytochrome c

A semisynthetic binuclear metalloprotein has been prepared by appending the pentaammineruthenium moiety to histidine 39 of the cytochrome c from the yeast Candida krusei. The site of ruthenium binding was identified by peptide mapping. Spectroscopic and electrochemical properties of the derivative indicate the protein conformation is unperturbed by the modification. A preliminary (minimum) rate constant of 170s^(-1) has been determined for the intramolecular electron transfer from ruthenium(II) to iron(III), which occurs over a distance of at least 13Å (barring major conformational changes). Electrochemical studies indicate that this reaction should proceed with a driving force of ~170mV. The rate constant is an order of magnitude faster than that observed in horse heart cytochrome c for intramolecular electron transfer from pentaammineruthenium(II)(histidine 33) to iron(III) (over a similar distance, and with a similar driving force), suggesting a medium or orientation effect makes the Candida intramolecular electron transfer more favorable.

Bis(thiophenolate)pyridine pincer ligands and trivalent zirconocene complexes relevant to early transition metal polymerization catalysts

Two major topics are covered: the first chapter is focused on the development of post-metallocene complexes for propylene polymerization. The second and third chapters investigate the consequences of diisobutylaluminum hydride (HAlⁱBu₂) additives in zirconocene based polymerization systems.

The synthesis, structure, and solution behavior of early metal complexes with a new tridentate LX₂ type ligand, bis(thiophenolate)pyridine ((SNS) = (2-C₆H₄S)₂-2,6-C₅H₃N) are investigated. SNS complexes of Ti, Zr, and Ta having dialkylamido coligands were synthesized and structurally characterized. The zirconium complex, (SNS)Zr(NMe₂)₂, displays C₂ symmetry in the solid state. Solid-state structures of tantalum complexes (SNS)Ta(NMe₂)₃ and (SNS)TaCl(NEt₂)₂ also display pronounced C₂ twisting of the SNS ligand. 1D and 2D NMR experiments show that (SNS)Ta(NMe₂)₃ is fluxional with rotation about the Ta N(amide) bonds occurring on the NMR timescale. The fluxional behavior of (SNS)TaCl(NEt₂)₂ in solution was also studied by variable temperature ¹H NMR. Observation of separate signals for the diastereotopic protons of the methylene unit of the diethylamide indicates that the complex remains locked on the NMR timescale in one diastereomeric conformation at temperatures below -50 °C.

Reduction of Zr(IV) metallocenium cations with sodium amalgam (NaHg) produces EPR signals assignable to Zr(III) metallocene complexes. Thus, chloro-bridged heterobinuclear ansa-zirconocenium cation [((SBI))Zr(μ-Cl)₂AlMe₂]⁺B(C₆F₅)_4¯ (SBI = rac-dimethylsilylbis(1-indenyl)), gives rise to an EPR signal assignable to the complex (SBI)Zr^III(μ-Cl)₂AlMe₂, while (SBI)Zr^III-Me and (SBI)Zr^III(-H)2AlⁱBu₂ are formed by reduction of [(SBI)Zr(μ-Me)₂AlMe₂]⁺B(C₆F₅)_4¯ and [(SBI)Zr(μ-H)₃(AlⁱBu₂)₂]⁺B(C₆F₅)4¯, respectively. These products are also formed, along with (SBI)Zr^III-ⁱBu and [(SBI)Zr^III]⁺ AlR4¯ when (SBI)ZrMe₂ reacts with HAlⁱBu₂, eliminating isobutane en route to the Zr(III) complex. Studies concerning the interconversion reactions between these and other (SBI)Zr(III) complexes and reaction mechanisms involved in their formation are also reported.

The addition of HAlⁱBu₂ to precatalyst [(SBI)Zr(µ-H)₃(AlⁱBu₂)₂]⁺ significantly slows the polymerization of propylene and changes the kinetics of polymerization from 1st to 2nd order with respect to propylene. This is likely due to competitive inhibition by HAlⁱBu₂. When the same reaction is investigated using [(ⁿBuCp)₂Zr(μ-H)₃(AlⁱBu₂)₂]⁺, hydroalumination between propylene and HAlⁱBu₂ is observed instead of propylene polymerization.

Sequence specific complexation of b DNA at sites containing g,c base pairs

A series of eight related analogs of distamycin A has been synthesized. Footprinting and affinity cleaving reveal that only two of the analogs, pyridine-2- car box amide-netropsin (2-Py N) and 1-methylimidazole-2-carboxamide-netrops in (2-ImN), bind to DNA with a specificity different from that of the parent compound. A new class of sites, represented by a TGACT sequence, is a strong site for 2-PyN binding, and the major recognition site for 2-ImN on DNA. Both compounds recognize the G•C bp specifically, although A's and T's in the site may be interchanged without penalty. Additional A•T bp outside the binding site increase the binding affinity. The compounds bind in the minor groove of the DNA sequence, but protect both grooves from dimethylsulfate. The binding evidence suggests that 2-PyN or 2-ImN binding induces a DNA conformational change.

In order to understand this sequence specific complexation better, the Ackers quantitative footprinting method for measuring individual site affinity constants has been extended to small molecules. MPE•Fe(II) cleavage reactions over a 10^5 range of free ligand concentrations are analyzed by gel electrophoresis. The decrease in cleavage is calculated by densitometry of a gel autoradiogram. The apparent fraction of DNA bound is then calculated from the amount of cleavage protection. The data is fitted to a theoretical curve using non-linear least squares techniques. Affinity constants at four individual sites are determined simultaneously. The distamycin A analog binds solely at A•T rich sites. Affinities range from 10^(6)- 10^(7)M^(-1) The data for parent compound D fit closely to a monomeric binding curve. 2-PyN binds both A•T sites and the TGTCA site with an apparent affinity constant of 10^(5) M^(-1). 2-ImN binds A•T sites with affinities less than 5 x 10^(4) M^(-1). The affinity of 2-ImN for the TGTCA site does not change significantly from the 2-PyN value. At the TGTCA site, the experimental data fit a dimeric binding curve better than a monomeric curve. Both 2-PyN and 2-ImN have substantially lower DNA affinities than closely related compounds.

In order to probe the requirements of this new binding site, fourteen other derivatives have been synthesized and tested. All compounds that recognize the TGTCA site have a heterocyclic aromatic nitrogen ortho to the N or C-terminal amide of the netropsin subunit. Specificity is strongly affected by the overall length of the small molecule. Only compounds that consist of at least three aromatic rings linked by amides exhibit TGTCA site binding. Specificity is only weakly altered by substitution on the pyridine ring, which correlates best with steric factors. A model is proposed for TGTCA site binding that has as its key feature hydrogen bonding to both G's by the small molecule. The specificity is determined by the sequence dependence of the distance between G's.

One derivative of 2-PyN exhibits pH dependent sequence specificity. At low pH, 4-dimethylaminopyridine-2-carboxamide-netropsin binds tightly to A•T sites. At high pH, 4-Me_(2)NPyN binds most tightly to the TGTCA site. In aqueous solution, this compound protonates at the pyridine nitrogen at pH 6. Thus presence of the protonated form correlates with A•T specificity.

The binding site of a class of eukaryotic transcriptional activators typified by yeast protein GCN4 and the mammalian oncogene Jun contains a strong 2-ImN binding site. Specificity requirements for the protein and small molecule are similar. GCN4 and 2-lmN bind simultaneously to the same binding site. GCN4 alters the cleavage pattern of 2-ImN-EDTA derivative at only one of its binding sites. The details of the interaction suggest that GCN4 alters the conformation of an AAAAAAA sequence adjacent to its binding site. The presence of a yeast counterpart to Jun partially blocks 2-lmN binding. The differences do not appear to be caused by direct interactions between 2-lmN and the proteins, but by induced conformational changes in the DNA protein complex. It is likely that the observed differences in complexation are involved in the varying sequence specificity of these proteins.

Intramolecular conflict : conformation and self-assembly of architecturally complex macromolecules in solution

The solution behavior of linear polymer chains is well understood, having been the subject of intense study throughout the previous century. As plastics have become ubiquitous in everyday life, polymer science has grown into a major field of study. The conformation of a polymer in solution depends on the molecular architecture and its interactions with the surroundings. Developments in synthetic techniques have led to the creation of precision-tailored polymeric materials with varied topologies and functionalities. In order to design materials with the desired properties, it is imperative to understand the relationships between polymer architecture and their conformation and behavior. To meet that need, this thesis investigates the conformation and self-assembly of three architecturally complex macromolecular systems with rich and varied behaviors driven by the resolution of intramolecular conflicts. First we describe the development of a robust and facile synthetic approach to reproducible bottlebrush polymers (Chapter 2). The method was used to produce homologous series of bottlebrush polymers with polynorbornene backbones, which revealed the effect of side-chain and backbone length on the overall conformation in both good and theta solvent conditions (Chapter 3). The side-chain conformation was obtained from a series of SANS experiments and determined to be indistinguishable from the behavior of free linear polymer chains. Using deuterium-labeled bottlebrushes, we were able for the first time to directly observe the backbone conformation of a bottlebrush polymer which showed self-avoiding walk behavior. Secondly, a series of SANS experiments was conducted on a homologous series of Side Group Liquid Crystalline Polymers (SGLCPs) in a perdeuterated small molecule liquid crystal (5CB). Monodomain, aligned, dilute samples of SGLCP-b-PS block copolymers were seen to self-assemble into complex micellar structures with mutually orthogonally oriented anisotropies at different length scales (Chapter 4). Finally, we present the results from the first scattering experiments on a set of fuel-soluble, associating telechelic polymers. We observed the formation of supramolecular aggregates in dilute (≤0.5wt%) solutions of telechelic polymers and determined that the choice of solvent has a significant effect on the strength of association and the size of the supramolecules (Chapter 5). A method was developed for the direct estimation of supramolecular aggregation number from SANS data. The insight into structure-property relationships obtained from this work will enable the more targeted development of these molecular architectures for their respective applications.

Phospholipids and a protein-conducting channel regulate cotranslational protein targeting

Signal recognition particle (SRP) and signal recognition particle receptor (SR) are evolutionarily conserved GTPases that deliver secretory and membrane proteins to the protein-conducting channel Sec61 complex in the lipid bilayer of the endoplasmic reticulum in eukaryotes or the SecYEG complex in the inner membrane of bacteria. Unlike the canonical Ras-type GTPases, SRP and SR are activated via nucleotide-dependent heterodimerization. Upon formation of the SR•SRP targeting complex, SRP and SR undergo a series of discrete conformational changes that culminate in their reciprocal activation and hydrolysis of GTP. How the SR•SRP GTPase cycle is regulated and coupled to the delivery of the cargo protein to the protein-conducting channel at the target membrane is not well-understood. Here we examine the role of the lipid bilayer and SecYEG in regulation of the SRP-mediated protein targeting pathway and show that they serve as important biological cues that spatially control the targeting reaction.

In the first chapter, we show that anionic phospholipids of the inner membrane activate the bacterial SR, FtsY, and favor the late conformational states of the targeting complex conducive to efficient unloading of the cargo. The results of our studies suggest that the lipid bilayer acts as a spatial cue that weakens the interaction of the cargo protein with SRP and primes the complex for unloading its cargo onto SecYEG.

In the second chapter, we focus on the effect of SecYEG on the conformational states and activity of the targeting complex. While phospholipids prime the complex for unloading its cargo, they are insufficient to trigger hydrolysis of GTP and the release of the cargo from the complex. SecYEG modulates the conformation of the targeting complex and triggers the GTP hydrolysis from the complex, thus driving the targeting reaction to completion. The results of this study suggest that SecYEG is not a passive recipient of the cargo protein; rather, it actively releases the cargo from the targeting complex. Together, anionic phospholipids and SecYEG serve distinct yet complementary roles. They spatially control the targeting reaction in a sequential manner, ensuring efficient delivery and unloading of the cargo protein.

In the third chapter, we reconstitute the transfer reaction in vitro and visualize it in real time. We show that the ribosome-nascent chain complex is transferred to SecYEG via a stepwise mechanism with gradual dissolution and formation of the contacts with SRP and SecYEG, respectively, explaining how the cargo is kept tethered to the membrane during the transfer and how its loss to the cytosol is avoided.

In the fourth chapter, we examine interaction of SecYEG with secretory and membrane proteins and attempt to address the role of a novel insertase YidC in this interaction. We show that detergent-solubilized SecYEG is capable of discriminating between the nascent chains of various lengths and engages a signal sequence in a well-defined conformation in the absence of accessory factors. Further, YidC alters the conformation of the signal peptide bound to SecYEG. The results described in this chapter show that YidC affects the SecYEG-nascent chain interaction at early stages of translocation/insertion and suggest a YidC-facilitated mechanism for lateral exit of transmembrane domains from SecYEG into the lipid bilayer.

1976 step in the Pacific climate: forty environmental changes between 1968-1975 and 1977-1984

Examination of 40 time series of multidisciplinary environmental variables from the Pacific Ocean and the Americas, collected in 1968 to 1984, demonstrated the remarkable consistency of a major climate-related, step-like change in 1976. To combine the 40 variables (e.g., air and water temperatures, Southern Oscillation, chlorophyll, geese, salmon, crabs, glaciers, atmospheric dust, coral, carbon dioxide, winds, ice cover, Bering Strait transport) into a single time series, standard variants of individual annual values (subtracting the mean and dividing by a standard deviation) were averaged. Analysis of the resulting time series showed that the single step in 1976, separating the 1968-1975 period from the 1977-1984 period, accounted for 89% of variance within the composite time series. Apparently, one of the Earth's large ecosystems occasionally undergoes large abrupt shifts.

Rapid changes in the vegetation of a shallow pond in Epping Forest, related to recent droughts

Over much of Britain, 1995 and 1996 have been perceived as drought years. To evaluate the impact that local climatic conditions are having upon successional changes in higher vegetation (macrophytes), Speakmans Pond in Epping Forest was surveyed and mapped in 1996. The results are related to previous vegetation surveys carried out in 1989 and 1991. In 1989 the dominant marginal vegetation was floating sweet-grass Glyceria fluitans, which also covered a major part of the main body of the pond. Other abundant species included soft rush Juncus effusus, reed mace Typha latifolia and yellow flag Iris pseudocorus. A small (central) area of open water contained bladderwort Utricularia vulgaris and white water-lily Nymphaea alba. A similar plant coverage was found in 1991, with a dominance of floating sweet-grass along the shallow eastern edge. A marked change in the pond was found during the 1996 survey of vegetation in July, when the pool was dry. The major plant cover now consisted of creeping bent Agrostis stolonifera, with isolated clumps of Yorkshire fog Holcus lanatus around the edges; both are terrestrial grasses found on land surrounding the pond. Rushes (Juncus) had increased their distribution round the margins of the pond, and the patch of yellow flag noted in 1989 and 1991 was not found in 1996. The deeper trenches were also dry, but a small patch of white water-lily remained adjacent to one of the trenches.

"Rapid and dramatic changes in vegetation" of a small pond on Holy Island (Northumbria): Chaotic dynamics?

This note describes changes to the relative extent of four structurally dominant submerged macrophytes in a pond on Holy Island National Nature Reserve, Northumbria, between 1991 and 1998. The estimated extent of the four submerged macrophytes and bare substratum between 1991 and 1998 showed dramatic changes with no obvious pattern or periodicity, as well as no identifiable natural or anthropogenic causes. Chaotic variation may be an important character of submerged pond plant populations, so that surveys taken in a single year may give an unreliable picture of plant populations.

The impact of year-to-year changes in the weather on the seasonal dynamics of lakes

The methods currently used to monitor and model lakes were developed when weather conditions were very different to what they are today. Most are based on samples collected at weekly or fortnightly intervals and cannot quantify the effects of short-term, more extreme, variations in the weather. In this article, the author presents some examples to show the importance of developing new monitoring methods using case studies from a number of lakes in the English Lake District. The impact of year-to-year changes and short-term changes on the dynamics of of lakes are highlighted.