Chlorophyll Catabolites – Chemical and Structural Footprints of a Fascinating Biological Phenomenon

Twenty years ago, the molecular basis for the seasonal disappearance of chlorophyll was still enigmatic. In the meantime, our knowledge on chlorophyll breakdown has grown considerably. As outlined here, it has been possible to decipher the basic transformations involved in natural chlorophyll breakdown by identification of chlorophyll catabolites in higher plants, and with the help of the synthesis of (putative) catabolic intermediates. In vascular plants, chlorophyll breakdown typically converts the green plant pigments efficiently into colorless and non-fluorescent tetrapyrroles. It involves colored intermediates only fleetingly and in an (elusive) enzyme-bound form. The non-fluorescent chlorophyll catabolites accumulate in the vacuoles of degreened leaves and are considered the products, primarily, of a detoxification process. However, they are effective antioxidants, and may thus also have physiologically beneficial chemical properties.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009

Putative prion protein from Fugu (Takifugu rubripes)

Prion proteins (PrP) of mammals, birds, reptiles and amphibians have been successfully cloned, expressed and purified in sufficient yields to enable 3D structure determination by NMR spectroscopy in solution. More recently, PrP ortholog genes have also been identified in several fish species, based on sequence relationships with tetrapod PrPs. Even though the sequence homology of fish PrPs to tetrapod PrPs is below 25%, structure prediction programs indicate a similar organization of the 3D structure. In this study, we generated recombinant polypeptide constructs that were expected to include the C‐terminal folded domain of Fugu‐PrP1 and analyzed these proteins using biochemical and biophysical methods. Because soluble expression could not be achieved, and refolding from guanidine–HCl did not result in a properly folded protein, we co‐expressed Escherichia coli chaperone proteins in order to obtain the protein in a soluble form. Although CD spectroscopy indicated the presence of some regular secondary structure in the protein thus obtained, there was no evidence for a globular 3D fold in the NMR spectra. We thus conclude that the polypeptide products of the fish genes annotated as corresponding to bona fide prnp genes in non‐fish species cannot be prepared for structural studies when using procedures similar to those that were successfully used with PrPs from mammals, birds, reptiles and amphibians

NMR Structure of the Bank Vole Prion Protein at 20 °C Contains a Structured Loop of Residues 165–171

The recent introduction of bank vole (Clethrionomys glareolus) as an additional laboratory animal for research on prion diseases revealed an important difference when compared to the mouse and the Syrian hamster, since bank voles show a high susceptibility to infection by brain homogenates from a wide range of diseased species such as sheep, goats, and humans. In this context, we determined the NMR structure of the C-terminal globular domain of the recombinant bank vole prion protein (bvPrP) [bvPrP(121–231)] at 20 °C. bvPrP(121–231) has the same overall architecture as other mammalian PrPs, with three α-helices and an antiparallel β-sheet, but it differs from PrP of the mouse and most other mammalian species in that the loop connecting the second β-strand and helix α2 is precisely defined at 20 °C. This is similar to the previously described structures of elk PrP and the designed mouse PrP (mPrP) variant mPrP[S170N,N174T](121–231), whereas Syrian hamster PrP displays a structure that is in-between these limiting cases. Studies with the newly designed variant mPrP[S170N](121–231), which contains the same loop sequence as bvPrP, now also showed that the single-amino-acid substitution S170N in mPrP is sufficient for obtaining a well-defined loop, thus providing the rationale for this local structural feature in bvPrP

NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231)

The recombinant murine prion protein, mPrP(23-231), was expressed in E. coli with uniform 15N-labeling. NMR experiments showed that the previously determined globular three-dimensional structure of the C-terminal domain mPrP(121-231) is preserved in the intact protein, and that the N-terminal polypeptide segment 23-120 is flexibly disordered. This structural information is based on nearly complete sequence-specific assignments for the backbone amide nitrogens, amide protons and alpha-protols of the polypeptide segment of residues 121-231 in mPrP(23-231). Coincidence of corresponding sequential and medium-range nuclear Overhauser effects (NOE) showed that the helical secondary structures previously identified in mPrP(121-231) are also present in mPrP(23-231), and near-identity of corresponding amide nitrogen and amide proton chemical shifts indicates that the three-dimensional fold of mPrP(121-231) is also preserved in the intact protein. The linewidths in heteronuclear 1H-15N correlation spectra and 15N[1H]-NOEs showed that the well structured residues 126-230 have correlation times of several nanoseconds, as is typical for small globular proteins, whereas correlation times shorter than 1 nanosecond were observed for all residues of mPrP(23-231) outside of this domain

Which Combinations Of New Venture Firms Resources Payoff? A Configurational Perspective

Baldauf, Artur; Schweiger, Simone A.; Wuethrich, Adria

NMR structure of the bovine prion protein isolated from healthy calf brains

NMR structures of recombinant prion proteins from various species expressed in Escherichia coli have been solved during the past years, but the fundamental question of the relevancy of these data relative to the naturally occurring forms of the prion protein has not been directly addressed. Here, we present a comparison of the cellular form of the bovine prion protein isolated and purified from healthy calf brains without use of detergents, so that it contains the two carbohydrate moieties and the part of the GPI anchor that is maintained after enzymatic cleavage of the glycerolipid moiety, with the recombinant bovine prion protein expressed in E. coli. We show by circular dichroism and 1H-NMR spectroscopy that the three-dimensional structure and the thermal stability of the natural glycoprotein and the recombinant polypeptide are essentially identical. This result indicates possible functional roles of the glycosylation of prion proteins in healthy organisms, and provides a platform and validation for future work on the structural biology of prion proteins, which will have to rely primarily on the use of recombinant polypeptides

NMR structure of the bovine prion protein isolated from healthy calf brains

NMR structures of recombinant prion proteins from various species expressed in Escherichia coli have been solved during the past years, but the fundamental question of the relevancy of these data relative to the naturally occurring forms of the prion protein has not been directly addressed. Here, we present a comparison of the cellular form of the bovine prion protein isolated and purified from healthy calf brains without use of detergents, so that it contains the two carbohydrate moieties and the part of the GPI anchor that is maintained after enzymatic cleavage of the glycerolipid moiety, with the recombinant bovine prion protein expressed in E. coli. We show by circular dichroism and (1)H-NMR spectroscopy that the three-dimensional structure and the thermal stability of the natural glycoprotein and the recombinant polypeptide are essentially identical. This result indicates possible functional roles of the glycosylation of prion proteins in healthy organisms, and provides a platform and validation for future work on the structural biology of prion proteins, which will have to rely primarily on the use of recombinant polypeptides

Prion Protein mPrP[F175A](121-231): Structure and Stability in Solution

The three-dimensional structures of prion proteins (PrPs) in the cellular form (PrP(C)) include a stacking interaction between the aromatic rings of the residues Y169 and F175, where F175 is conserved in all but two so far analyzed mammalian PrP sequences and where Y169 is strictly conserved. To investigate the structural role of F175, we characterized the variant mouse prion protein mPrP[F175A](121-231). The NMR solution structure represents a typical PrP(C)-fold, and it contains a 3(10)-helical β2-α2 loop conformation, which is well defined because all amide group signals in this loop are observed at 20°C. With this "rigid-loop PrP(C)" behavior, mPrP[F175A](121-231) differs from the previously studied mPrP[Y169A](121-231), which contains a type I β-turn β2-α2 loop structure. When compared to other rigid-loop variants of mPrP(121-231), mPrP[F175A](121-231) is unique in that the thermal unfolding temperature is lowered by 8°C. These observations enable further refined dissection of the effects of different single-residue exchanges on the PrP(C) conformation and their implications for the PrP(C) physiological function

Beyond the matrix-assisted laser desorption ionization (MALDI) biotyping workflow: in search of microorganism-specific tryptic peptides enabling discrimination of subspecies

A well-accepted method for identification of microorganisms uses matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled to analysis software which identifies and classifies the organism according to its ribosomal protein spectral profile. The method, called MALDI biotyping, is widely used in clinical diagnostics and has partly replaced conventional microbiological techniques such as biochemical identification due to its shorter time to result (minutes for MALDI biotyping versus hours or days for classical phenotypic or genotypic identification). Besides its utility for identifying bacteria, MS-based identification has been shown to be applicable also to yeasts and molds. A limitation to this method, however, is that accurate identification is most reliably achieved on the species level on the basis of reference mass spectra, making further phylogenetic classification unreliable. Here, it is shown that combining tryptic digestion of the acid/organic solvent extracted (classical biotyping preparation) and resolubilized proteins, nano-liquid chromatography (nano-LC), and subsequent identification of the peptides by MALDI-tandem TOF (MALDI-TOF/TOF) mass spectrometry increases the discrimination power to the level of subspecies. As a proof of concept, using this targeted proteomics workflow, we have identified subspecies-specific biomarker peptides for three Salmonella subspecies, resulting in an extension of the mass range and type of proteins investigated compared to classical MALDI biotyping. This method therefore offers rapid and cost-effective identification and classification of microorganisms at a deeper taxonomic level