Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP

Stereochemistry of the Conversions of l-Threonine and d-Threonine into 2-Oxobutanoate by the l-Threonine and d-Threonine Dehydratases of Serratia marcescens

dl[3‐ 2 H]Threonine, l‐[3‐ 2 H]threonine, (2RS,3S)‐2‐amino[3‐ 2 H 1 ] butanoic acid, (2RS,3S) 2‐amino[3‐ 3 H 1 ]butanoic acid and dl‐2‐amino[3‐ 14 C]butanoic acid were synthesised. l‐[3‐ 2 ‐H]Threonine was converted into l‐[4‐ 2 H 1 ]isoleucine by Serratia marcescens strain IHr313. (2RS,3S)‐2‐Amino[3‐ 2 ‐H 1 ]butanoic acid was converted into l‐[4‐ 4 ‐H 1 ]isoleucine by S. marcescens strain 149. Analysis by 220‐MHz NMR spectroscopy of the labelled l‐isoleucine produced showed that the same diastereotopic hydrogen at C‐4 was labelled in each experiment, proving that, during the conversion of l‐threonine into 2‐oxobutanoate mediated by biosynthetic l‐threonine dehydratase, the hydroxyl group at C‐3 was replaced by hydrogen with retention of configuration. S. marcescens strain 149 lacks l‐threonine dehydratase but possesses an inducible d‐threonine dehydratase. This strain converted dl‐[3‐ 2 H]threonine into dl‐[4‐ 2 H 1 ]isoleucine with the deuterium located in the diastereotopic C‐4 hydrogen derived from the 3 pro‐R hydrogen of 2‐aminobutanoic acid. This result proved that during the conversion of d‐threonine into 2‐oxobutanoate mediated by d‐threonine dehydratase, the hydroxyl group at C‐3 is replaced by hydrogen with retention of configuration. These results also prove that the protonation at C‐3 of the proposed enamine intermediate in the transformations catalysed by threonine dehydratase is under enzymatic control. The present results, taken in conjunction with the independent assignment of the signals in the 220‐MHz NMR spectrum of l‐isoleucine due to the diastereotopic protons at C‐4, prove that during the ethyl migration step in l‐isoleucine biosynthesis, the configuration at the migrating centre is retained.(undefined)info:eu-repo/semantics/publishedVersio

A compilation of global bio-optical in situ data for ocean-colour satellite applications - Version two

© Authors. A global compilation of in situ data is useful to evaluate the quality of ocean-colour satellite data records. Here we describe the data compiled for the validation of the ocean-colour products from the ESA Ocean Colour Climate Change Initiative (OC-CCI). The data were acquired from several sources (including, inter alia, MOBY, BOUSSOLE, AERONET-OC, SeaBASS, NOMAD, MERMAID, AMT, ICES, HOT and GePCO) and span the period from 1997 to 2018. Observations of the following variables were compiled: spectral remote-sensing reflectances, concentrations of chlorophyll a, spectral inherent optical properties, spectral diffuse attenuation coefficients and total suspended matter. The data were from multi-project archives acquired via open internet services or from individual projects, acquired directly from data providers. Methodologies were implemented for homogenization, quality control and merging of all data. No changes were made to the original data, other than averaging of observations that were close in time and space, elimination of some points after quality control and conversion to a standard format. The final result is a merged table designed for validation of satellite-derived ocean-colour products and available in text format. Metadata of each in situ measurement (original source, cruise or experiment, principal investigator) was propagated throughout the work and made available in the final table. By making the metadata available, provenance is better documented, and it is also possible to analyse each set of data separately. This paper also describes the changes that were made to the compilation in relation to the previous version (Valente et al., 2016). The compiled data are available at https://doi.org/10.1594/PANGAEA.898188 (Valente et al., 2019)