Combined EXAFS and DFT Structure Calculations Provide Structural Insights into the 1:1 Multi‐Histidine Complexes of CuII,CuI, and ZnII with the Tandem Octarepeats of the Mammalian Prion Protein

The metal‐coordinating properties of the prion protein (PrP) have been the subject of intense focus and debate since the first reports of its interaction with copper just before the turn of the century. The picture of metal coordination to PrP has been improved and refined over the past decade, but structural details of the various metal coordination modes have not been fully elucidated in some cases. In the present study, we have employed X‐ray absorption near‐edge spectroscopy as well as extended X‐ray absorption fine structure (EXAFS) spectroscopy to structurally characterize the dominant 1:1 coordination modes for Cu^II, Cu^I, and Zn^II with an N‐terminal fragment of PrP. The PrP fragment corresponds to four tandem repeats representative of the mammalian octarepeat domain, designated as OR₄, which is also the most studied PrP fragment for metal interactions, making our findings applicable to a large body of previous work. Density functional theory (DFT) calculations have provided additional structural and thermodynamic data, and candidate structures have been used to inform EXAFS data analysis. The optimized geometries from DFT calculations have been used to identify potential coordination complexes for multi‐histidine coordination of Cu^II, Cu^I, and Zn^II in an aqueous medium, modelled using 4‐methylimidazole to represent the histidine side chain. Through a combination of in silico coordination chemistry as well as rigorous EXAFS curve‐fitting, using full multiple scattering on candidate structures derived from DFT calculations, we have characterized the predominant coordination modes for the 1:1 complexes of Cu^II, Cu^I, and Zn^II with the OR₄ peptide at pH 7.4 at atomic resolution, which are best represented as square‐planar [Cu^II(His)₄]²⁺, digonal [Cu^I(His)₂]⁺, and tetrahedral [Zn^II(His)₃(OH₂)]²⁺, respectively.     

Interaction of a C-F bond with the π-system of a C═C bond or "head on" with a proximate C-H bond

We describe the synthesis and preliminary study of two molecules, in which a fluorine atom is positioned proximately above the π-orbitals of a C═C bond or else wherein a C-F bond interacts in a "head on" fashion with a proximate C-H bond. The spectroscopic characteristics of these unusual interactions are documented, X-ray crystallographic analyses are reported, and theoretical calculations are employed to support the observed spectroscopy.     

Structure, electronic properties and catalytic behaviour of an activity-enhancing CYP102A1 (P450(BM3)) variant

The substrate-free crystal structure of a five-mutation directed evolution variant of CYP102A1 (P450(BM3)) with generic activity-enhancing properties ("KT2") has been determined to 1.9-? resolution. There is a close resemblance to substrate-bound structures of the wild-type enzyme (WT). The disruption of two salt bridges that link the G- and I-helices in WT causes conformational changes that break several hydrogen bonds and reduce the angle of the kink in the I-helix where dioxygen activation is thought to take place. The side-chain of a key active site residue, Phe87, is rotated in one molecule of the asymmetric unit, and the side-chains of Phe158 and Phe261 cascade into the orientations found in fatty-acid-bound forms of the enzyme. The iron is out of the porphyrin plane, towards the proximal cysteine. Unusually, the axial water ligand to the haem iron is not hydrogen-bonded to Ala264. The first electron transfer from the reductase domain to the haem domain of substrate-free KT2 is almost as fast as in palmitate-bound WT even though the reduction potential of the haem domain is only slightly more oxidising than that of substrate-free WT. However, NADPH is turned over slowly in the absence of substrate, so the catalytic cycle is gated by a step subsequent to the first electron transfer-a contrast to WT. Propylbenzene binding slightly raises the first electron transfer rate in WT but not in KT2. It is proposed that the generic rate accelerating properties of KT2 arise from the substrate-free form being in a catalytically ready conformation, such that substrate-induced changes to the structure play a less significant role in promoting the first electron transfer than in WT.     

Surface partitioning studies of N-methylcarbamate-treated post-harvest crops using SFE-HPLC-postcolumn reaction-fluorescence

The partitioning characteristics of selected carbamate insecticides (carbaryl, aldicarb, bendiocarb and pirimicarb) on five fruit and vegetable types were investigated. Post-harvest samples were surface-saturated with a methanolic-aqueous mixed carbamate spiking solution for a number of time periods. Samples were taken at 3, 7, 10 and 14 d, and extracted using supercritical CO2 at pressure = 300 atm modified with 10% dimethyl sulfoxide. Extracts were analysed by HPLC-postcolumn reaction-fluorescence detection at lambda ex = 330 nm and lambda em = 450 nm for N-methylcarbamates and at lambda ex = 315 nm and lambda em = 380 nm for pirimicarb. The relative partitioning of each insecticide between sample skin and flesh was investigated. This included the determination of both half-life and normalised matrix metabolic rate studies with respect to each carbamate. Multilinear regression (MLR) was applied to a number of insecticide and matrix-based variables to develop regression models for carbamate partitioning for each matrix type studied. Experimentally derived carbamate half-lives ranged from 3.6 d (carbaryl in pear flesh) to 8.0 d (bendiocarb in banana skin). Determinations of normalised metabolic rates were based on calculating the time period from the point of sampling through to the point where carbamate concentration was reduced to 5% of its initial value. These values ranged from 16.2 d (bendiocarb in potato skin) to 34.7 d (bendiocarb in banana skin). Although no practicable MLR partitioning models were obtained, it was found that the models created indicated that carbamate solubility in water (and hence log P) and the number of days in contact with the spiking solution were the most important parameters in model construction.     

Generalised ‘join the shortest queue’ policies for the dynamic routing of jobs to multi-class queues

Jobs or customers arrive and require service that may be provided at one of several different stations. The associated routing problems concern how customers may be assigned to stations in an optimal manner. Much of the classical literature concerns a single class of customers seeking service from a collection of homogeneous stations. We argue that many contemporary application areas call for the analysis of routing problems in which many classes of customer seek service provided at a collection of diverse stations. This paper is the first to consider routing policies in such complex environments which take appropriate account of the degree of congestion at each service station. A simple and intuitive class of policies emerges from a policy improvement approach. In a numerical study, the policies were close to optimal in all cases.     

Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line

Neuronal ubiquitin C-terminal hydrolase (UCH-L1) has been linked to Parkinson's disease (PD), the progression of certain nonneuronal tumors, and neuropathic pain. Certain lung tumor-derived cell lines express UCH-L1 but it is not expressed in normal lung tissue, suggesting that this enzyme plays a role in tumor progression, either as a trigger or as a response. Small-molecule inhibitors of UCH-L1 would be helpful in distinguishing between these scenarios. By utilizing high-throughput screening (HTS) to find inhibitors and traditional medicinal chemistry to optimize their affinity and specificity, we have identified a class of isatin O-acyl oximes that selectively inhibit UCH-L1 as compared to its systemic isoform, UCH-L3. Three representatives of this class (30, 50, 51) have IC(50) values of 0.80-0.94 micro M for UCH-L1 and 17-25 micro M for UCH-L3. The K(i) of 30 toward UCH-L1 is 0.40 micro M and inhibition is reversible, competitive, and active site directed. Two isatin oxime inhibitors increased proliferation of the H1299 lung tumor cell line but had no effect on a lung tumor line that does not express UCH-L1. Inhibition of UCH-L1 expression in the H1299 cell line using RNAi had a similar proproliferative effect, suggesting that the UCH-L1 enzymatic activity is antiproliferative and that UCH-L1 expression may be a response to tumor growth. The molecular mechanism of this response remains to be determined.     

Mass spectrometry of isomeric methyl phenylpentanoates and methyl phenylnonanoates

The mass spectra of methyl 3-, 4- and 5-phenyl pentanoates, and all the straight-chain methyl phenylnonanoates have been recorded. The major fragmentation patterns have been discussed; when the phenyl group is non-terminal, cleavage was observed at the branching position. It is shown that a mixture of methyl 3-, 4- and 5-phenylpentanoates may be analysed both qualitatively and quantitatively by mass spectrometry; in a methyl phenylnonanoate mixture a similar analysis is possible with the exception of the 8- and 9-phenyl isomers.     

A direct stereospecific synthesis of 9,11-etheno- and 9,11-ethano-PGH1 derivatives

The reaction of methyl 10-oxodec-S-ynoate with cyclopentadiene is reported as the key step in the synthesis of 9,11-etheno- and 9,11-ethano-PGH₁ derivatives.     

Quantitative use of the angular variation technique in studies of tin by X-ray photoelectron spectroscopy

The relative photoionisation cross-sections of the tin 3d and 4d electrons are determined and combined with angular variation studies to establ