Search

Most studies that focus on heating-related greenhouse gas (GHG) emissions do so with either a focus on technical aspects or a focus on heating-related attitudes and behavior. Either way, they assig...

This chapter enhances the previous understanding of Energy Lifestyles by identifying groups with distinct patterns of energy behavior across six areas of life. In contrast to most previous studies, the identification of groups is exclusively conducted on the basis of behavior-related data, whereas the characterization of the groups follows in a second step using psychological and socio-demographic variables. This chapter explicitly considers the multidimensionality of behavior and provides a comprehensive overview of different Energy Lifestyles and their potential roles in energy transition. The finding that there are almost no “average users” points out that policy designs must go beyond average figures based on the national emission figures and need to focus on different Energy Lifestyles.

This chapter provides an overview of the theoretical approaches to environmental attitudes and behaviors. It includes a discussion of different scales and surveys used in other programs with a focus on this topic. Scales measuring general environmental behavior, just like items in surveys, tend to focus on behavioral intentions and are correlated with environmentally friendly attitudes. In contrast, emission-related behavior depends more on context and socio-demographic characteristics and is rarely asked in surveys. Gaps frequently occur between environmental attitudes and general behaviors—the value-action gap—and between environmental behaviors and the actual ecological consequences of actions—the behavior-impact gap. Finally, previous results and problems encountered in the validation of self-reports on environmental behavior are highlighted.

This chapter presents the development of the survey in the underlying study in this book, a description of our sample, and the results of the validation efforts. Our survey includes variations of existing scales which have been used to measure climate-relevant behaviors within the areas of housing, mobility, diet, and consumption. We go beyond these existing questions, however, as we also include various new items and analyze the validity of existing and new questions. Based on our findings we recommend a set of questions for different significant areas of emissions, for example, asking about the distance traveled by car in the previous year, the number of short-haul and long-haul flights, and a question about the frequency of the consumption of particularly energy-intensive foods.

The final chapter of this book summarizes the main insights from previous chapters and tests whether our approach is also applicable at an international level. Our international outlook shows that the five survey items, which capture more than three quarters of Austrian greenhouse gas (GHG) emissions, also work well in other European countries. Similarly, the lifestyles identified in Chap. 10.1007/978-3-030-85796-7_6 are also present in other societies. Furthermore, this chapter shows that the prevalence of these lifestyles as well as the explanatory power of the models correlate with societal factors such as political institutions, affluence, environmental degradation, and socio-demographic characteristics. The final chapter closes with a brief consideration of the impact of the COVID-19 pandemic on environmental attitudes and individual willingness to make sacrifices for the environment.

This chapter starts with an overview of the current national emission figures for Austria. Subsequently, the emissions of our respondents are presented in detail and contrasted with the number of national emissions. Since one goal of the study was to improve survey research in measuring environmentally significant behavior, questions are selected which allow a valid estimation of the total emissions caused by a person. We propose a set of five variables which capture around 77% of an individual’s total emissions. Furthermore, we are able to confirm that socio-demographic variables such as age, income, and residential area have a significant impact on an individual’s emission consumption.

This introduction offers an overview of our research approach, discusses the connection between sociology and climate research, and presents our two research aims. In substantive terms, we address the question of which behaviors are of climate relevance, who is engaging in these behaviors, in which contexts do these behaviors occur, and which individual perceptions and values are related to them. In terms of research methods, we focus on the measurement of climate-relevant behaviors using population surveys. Our goal in this regard is to develop an instrument that allows a valid estimate of an individual’s greenhouse gas (GHG) emissions with as few questions as possible.

This chapter focuses on the question of why environmentally conscious individuals find it difficult to behave in a correspondingly eco-friendly way. This phenomenon is referred to in the literature as the “value-action gap.” In this study, the definition of this gap is further expanded by considering individual intention. The discrepancies between environmental values, intention, and behavior are explored in the context of two environmentally relevant behaviors—mobility and consumption—using qualitative interviews. Firstly, key structural and intrapersonal obstacles to pro-environmental behavior are identified. Secondly, individual needs and requirements for facilitating eco-friendly behavior are addressed. Finally, strategies and potentials for pro-environmental change in society are discussed.

To enjoy a fulfilling life, a person needs six fundamental life requirements to be met. These six requirements or “life-areas” are housing, mobility, consumption of goods (e.g., clothing), diet, other activities (entertainment), and information. In the beginning of this chapter, a top-down estimate of Austrian consumption-based emissions in each life-area is presented. These are organized into segments that may be easily reduced by changing individual behavior and those segments that are fundamental aspects of our society. The remainder of this chapter discusses how to estimate the greenhouse gas (GHG) output. There is a trade-off between accuracy and level of detail, and the need to combine bottom-up survey results with the top-down national emissions inventory. How these trade-offs may be handled is demonstrated using a practical example.

With this opinion we are responding to an article by Martin Kunz (2020), in which he discussed the issue of discrimination of certain kinds of “honey” by certain regulatory works, such as European ...

ZusammenfassungKap. 11 hat gezeigt, dass es derzeit noch große Unsicherheiten darüber gibt, wie hoch tatsächlich der Beitrag des Tourismus zu den nationalen und internationalen Treibhausgasemissionen ist. Es ist jedoch gewiss, dass der Anteil bedeutend ist und, in Zusammenhang mit dem prognostizierten Wachstum des weltweiten Tourismus, wahrscheinlich weiter steigen wird. Daher stellt sich die Frage, wie die Emissionen der touristischen Aktivitäten gesenkt werden könnten. Im vorliegenden Kapitel wird berichtet, welche Verpflichtungen und Strategien es dazu aktuell auf politischer Ebene gibt, beginnend mit internationalen Verträgen, insbesondere dem Pariser Klimaschutzabkommen, und Vorgaben der Europäischen Union, bei denen Österreich sich verpflichtet hat, teilzunehmen. In weiterer Folge richtet sich das Augenmerk auf eine detaillierte Betrachtung und Bewertung der Klimaschutz‑, Klimawandelanpassungs- und Tourismusstrategien auf Bundesebene und in den einzelnen Bundesländern. Im Anschluss daran werden allgemein mögliche Maßnahmen und Potenziale besprochen. Schließlich wird unter dem Schlagwort „Paris Lifestyle“ diskutiert, welche Maßnahmen notwendig sind, um auf individueller Ebene zum Ziel eines CO2-armen bzw. -neutralen Tourismus beitragen zu können. Dazu werden auch Beispiele innovativer, emissionsarmer Angebote von Tourismusbetrieben und Destinationen vorgestellt.

Inhibition of intestinal glucose resorption can serve as an effective strategy for the prevention of an increase in blood glucose levels. We have recently shown that various extracts prepared from guava (Psidium guajava) inhibit sodium-dependent glucose cotransporter 1 (SGLT1)- and glucose transporter 2 (GLUT2)-mediated glucose transport in vitro (Caco-2 cells) and in vivo (C57BL/6N mice). However, the efficacy in humans remains to be confirmed. For this purpose, we conducted a parallelized, randomized clinical study with young healthy adults. Thirty-one volunteers performed an oral glucose tolerance test (OGTT) in which the control group received a glucose solution and the intervention group received a glucose solution containing a guava fruit extract prepared by supercritical CO2 extraction. The exact same extract was used for our previous in vitro and in vivo experiments. Blood samples were collected prior to and up to two hours after glucose consumption to quantitate blood glucose and insulin levels. Our results show that, in comparison to the control group, consumption of guava fruit extract resulted in a significantly reduced increase in postprandial glucose response over the basal fasting plasma glucose levels after 30 min (&Delta, control 2.60 &plusmn, 1.09 mmol/L versus &Delta, intervention 1.96 &plusmn, 0.96 mmol/L, p = 0.039) and 90 min (&Delta, control 0.44 &plusmn, 0.74 mmol/L versus &Delta, intervention &minus, 0.18 &plusmn, 0.88 mmol/L, p = 0.023). In addition, we observed a slightly reduced, but non-significant insulin secretion (&Delta, control 353.82 &plusmn, 183.31 pmol/L versus &Delta, intervention 288.43 &plusmn, 126.19 pmol/L, p = 0.302). Interestingly, storage time and repeated freeze-thawing operations appeared to negatively influence the efficacy of the applied extract. Several analytical methods (HPLC-MS, GC-MS, and NMR) were applied to identify putative bioactive compounds in the CO2 extract used. We could assign several substances at relevant concentrations including kojic acid (0.33 mg/mL) and 5-hydroxymethylfurfural (2.76 mg/mL). Taken together, this clinical trial and previous in vitro and in vivo experiments confirm the efficacy of our guava fruit extract in inhibiting intestinal glucose resorption, possibly in combination with reduced insulin secretion. Based on these findings, the development of food supplements or functional foods containing this extract appears promising for patients with diabetes and for the prevention of insulin resistance. Trial registration: 415-E/2319/15-2018 (Ethics Commissions of Salzburg).

Inhibition of intestinal glucose resorption can serve as an effective strategy for the prevention of an increase in blood glucose levels. We have recently shown that various extracts prepared from guava (Psidium guajava) inhibit sodium-dependent glucose cotransporter 1 (SGLT1)- and glucose transporter 2 (GLUT2)-mediated glucose transport in vitro (Caco-2 cells) and in vivo (C57BL/6N mice). However, the efficacy in humans remains to be confirmed. For this purpose, we conducted a parallelized, randomized clinical study with young healthy adults. Thirty-one volunteers performed an oral glucose tolerance test (OGTT) in which the control group received a glucose solution and the intervention group received a glucose solution containing a guava fruit extract prepared by supercritical CO2 extraction. The exact same extract was used for our previous in vitro and in vivo experiments. Blood samples were collected prior to and up to two hours after glucose consumption to quantitate blood glucose and insulin levels. Our results show that, in comparison to the control group, consumption of guava fruit extract resulted in a significantly reduced increase in postprandial glucose response over the basal fasting plasma glucose levels after 30 min (Δ control 2.60 ± 1.09 mmol/L versus Δ intervention 1.96 ± 0.96 mmol/L; p = 0.039) and 90 min (Δ control 0.44 ± 0.74 mmol/L versus Δ intervention −0.18 ± 0.88 mmol/L; p = 0.023). In addition, we observed a slightly reduced, but non-significant insulin secretion (Δ control 353.82 ± 183.31 pmol/L versus Δ intervention 288.43 ± 126.19 pmol/L, p = 0.302). Interestingly, storage time and repeated freeze-thawing operations appeared to negatively influence the efficacy of the applied extract. Several analytical methods (HPLC-MS, GC-MS, and NMR) were applied to identify putative bioactive compounds in the CO2 extract used. We could assign several substances at relevant concentrations including kojic acid (0.33 mg/mL) and 5-hydroxymethylfurfural (2.76 mg/mL). Taken together, this clinical trial and previous in vitro and in vivo experiments confirm the efficacy of our guava fruit extract in inhibiting intestinal glucose resorption, possibly in combination with reduced insulin secretion. Based on these findings, the development of food supplements or functional foods containing this extract appears promising for patients with diabetes and for the prevention of insulin resistance. Trial registration: 415-E/2319/15-2018 (Ethics Commissions of Salzburg).

Spider dragline fibers are predominantly made out of the major ampullate spidroins (MaSp) 1 and 2. The assembly of dissolved spidroin into a stable fiber is highly controlled for example by dimerization of its amino-terminal domain (NRN) upon acidification, as well as removal of sodium chloride along the spinning duct. Clustered residues D39, E76 and E81 are the most highly conserved residues of the five-helix bundle, and they are hypothesized to be key residues for switching between a monomeric and a dimeric conformation. Simultaneous replacement of these residues by their non-titratable analogues results in variant D39N/E76Q/E81Q, which is supposed to fold into an intermediate conformation between that of the monomeric and the dimeric state at neutral pH. Here we report the resonance assignment of Latrodectus hesperus NRN variant D39N/E76Q/E81Q at pH 7.2 obtained by high-resolution triple resonance NMR spectroscopy.

Der vorliegende Beitrag diskutiert einerseits vorliegende Erkenntnisse uber die Wirksamkeit von Feedback durch „Smart Meters“ und behandelt andererseits am Beispiel von Lebensmitteleinkaufen die Frage, welche Rolle „smarte“ Technologien fur die Transformation etablierten Konsumverhaltens in Richtung einer Auswahl von Produkten mit besserer Klimavertraglichkeit spielen konnten. Aufbauend auf die Frage, welche Informationen hinsichtlich der Klimabilanz verschiedener Produktalternativen Konsumentinnen und Konsumenten zur Verfugung stehen bzw. fur eine gezielte Entscheidung nach Klimaschutz-Kriterien zur Verfugung stehen mussten, werden zunachst verschiedene Faktoren thematisiert, die fur eine Einschatzung des theoretische Emissionsreduktions-Potentials relevant erscheinen. Daran anknupfend wird diskutiert, welche Voraussetzungen aus wissenschaftlich-technischer Sicht, Stichwort „Datenbanken“, erfullt sein mussten, um Nutzerinnen und Nutzern einer Smartphone-App wahrend ihres Einkaufs entsprechende Informationen uber die lebenszyklusbasierte Klimabilanz verschiedener Angebote bereitstellen zu konnen.

The analytical framework presented herein is based on the identification of social groups with distinct patterns in their energy-relevant behaviour. This was achieved by clustering individuals according to their primary energy demands in six main areas of life. Due to the close relationship between energy-relevant behaviour and associated impacts, the suggested approach is considered better suited for the identification of groups with actual differences in their climate and energy-related behaviour than conventional approaches that cluster individuals based on their psychological or sociodemographic characteristics. Moreover, it is assumed that this focus on energy-relevant behaviour leads to a higher measuring equivalence in a country comparison or in a longitudinal setting. From an analytical point of view, the most significant benefit of the presented method over conventional lifestyle typologies is that all psychological, cultural and sociodemographic factors can be used as explanatory variables without resulting in circular reasoning. In terms of required data, the approach was designed around what could be collected by conventional survey methods. Variables such as energy use and emissions were calculated by the means of life cycle assessment (LCA) based on self-reported behaviour and equipment use. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Within the cocoa market (Theobroma cacao L.), quality and prices are often determined by geographical origin, making traceability indispensable. Therefore, to investigate possibilities of tracing by analytical methods, 48 carefully selected cocoa samples from 20 countries have been profiled using a combination of stable isotope-ratio mass spectrometry (IRMS) and proton nuclear magnetic resonance (

World society and decision makers are running out of time to implement measures on climate change mitigation and adaptation. Incomplete knowledge and vast challenges in communicating climate change are crucial factors in this problem. In order to increase people’s awareness of their role in climate change, highly specific communication strategies are necessary. Besides insufficient information on group-specific realities of life, existing strategies are often limited by the absence of quantitative data that could give decision makers the opportunity to estimate the potential and evaluate the success of communication measures. In order to meet these requirements, energy use and corresponding emissions must be analysed in relation to behavioural patterns and technology choices of relevant social groups. This perspective leads to a more detailed understanding of how energy use and the responsibility for greenhouse gas emissions are distributed within society. This paper presents an interdisciplinary approach for providing the required knowledge within a single research process and describes its most relevant features as compared to previous methods. We describe the empirical development of an impact based “Energy Lifestyle” typology for the Austrian society and describe the six identified groups in detail with special focus on the challenges that might evolve in group specific communication. Thereafter, we set the six Energy Lifestyles in context with the name-giving concept “Paris Lifestyle” and discuss its role for evaluating the succession towards the goals set out in the Paris Agreement.

Within the cocoa market (Theobroma cacao L.), quality and prices are often determined by geographical origin, making traceability indispensable. Therefore, to investigate possibilities of tracing by analytical methods, 48 carefully selected cocoa samples from 20 countries have been profiled using a combination of stable isotope-ratio mass spectrometry (IRMS) and proton nuclear magnetic resonance (1H NMR). Chemometric analysis of combined data sets from both, stable isotope data (δ13C, δ15N, δ18O, δ2H, %C, %N, %O, %H) and 1H NMR fingerprints, achieved good separation with increased classification rates compared to classification with data of the isolated methods. IRMS contributed primarily to discrimination between countries, while 1H NMR significantly contributed to separation of varieties, but also the regions within individual countries. This study thus demonstrates that combination of two analytical methods is an effective tool to enhance both, accuracy and precision, in authenticity testing of cocoa.

Fe(II)/α-ketoglutarate-dependent oxygenases are versatile catalysts associated with a number of different biological functions in which they use the oxidizing power of activated dioxygen to convert a variety of substrates. A mononuclear nonheme iron center is used to couple the decarboxylation of the cosubstrate α-ketoglutarate with a two-electron oxidation of the substrate, which is a hydroxylation in most cases. Although Fe(II)/α-ketoglutarate-dependent oxygenases have diverse amino acid sequences and substrate specifity, it is assumed that they share a common mechanism. One representative of this enzyme family is the Fe(II)/α-ketoglutarate-dependent taurine dioxygenase that catalyzes the hydroxylation of taurine yielding sulfite and aminoacetaldehyde. Its mechanism has been studied in detail becoming a model system for the whole enzyme family. However, its oligomeric state and architecture have been disputed. Here, we report the biochemical and kinetic characterization of the Fe(II)/α-ketoglutarate-dependent taurine dioxygenase from Pseudomonas putida KT2440 (TauD(Pp) ). We also present three crystal structures of the apo form of this enzyme. Comparisons with taurine dioxygenase from Escherichia coli (TauD(Ec) ) demonstrate that both enzymes are quite similar regarding their spectra, structure and kinetics, and only minor differences for the accumulation of intermediates during the reaction have been observed. Structural data and analytical gel filtration, as well as sedimentation velocity analytical ultracentrifugation, show that both TauD(Pp) and TauD(Ec) are tetramers in solution and in the crystals, which is in contrast to the earlier description of taurine dioxygenase from E. coli as a dimer. Database The atomic coordinates and structure factors have been deposited with the Brookhaven Protein Data Bank (entry 3PVJ, 3V15, 3V17) Structured digital abstract • tauDpp and tauDpp bind by molecular sieving (View interaction) • tauDpp and tauDpp bind by x-ray crystallography (View interaction) • tauDEc and tauDEc bind by molecular sieving (View interaction).

The characterization of the structural dynamics of proteins, including those that present a substantial degree of disorder, is currently a major scientific challenge. These dynamics are biologically relevant and govern the majority of functional and pathological processes. We exploited a combination of enhanced molecular simulations of metadynamics and NMR measurements to study heterogeneous states of proteins and peptides. In this way, we determined the structural ensemble and free-energy landscape of the highly dynamic helix 1 of the prion protein (PrP-H1), whose misfolding and aggregation are intimately connected to a group of neurodegenerative disorders known as transmissible spongiform encephalopathies. Our combined approach allowed us to dissect the factors that govern the conformational states of PrP-H1 in solution, and the implications of these factors for prion protein misfolding and aggregation. The results underline the importance of adopting novel integrated approaches that take advantage of experiments and theory to achieve a comprehensive characterization of the structure and dynamics of biological macromolecules. © 2012 Biophysical Society.

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRalpha1 ECD, we generated a model of the human GlyRalpha1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRalpha1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.

Tricyclic aromatic compounds (TCA) are promising candidates for treatment of transmissible spongiform encephalopathies. Direct binding to the cellular prion protein (PrP(C)) has been proposed as anti-prion active mechanism. We here show by means of NMR-spectroscopy that binding of TCA occurs with millimolar affinity to motifs consisting of two neighboring aromatic residues (Ar-Ar motif). It is independent of the secondary structure of this motif and of the side chain attached to the TCA and it is not specific to PrP(C). Because biologically inactive 9-aminoacridine (9-aa) binds with similar K(D) as anti-prion active quinacrine, direct interaction with PrP(C) as mechanism of action appears highly unlikely. However, binding of 9-aa to Ar-Ar-motifs in proteins can be used as reporter for biological macromolecule interactions, by measuring changes in T(1)-NMR relaxation times of 9-aa.

We present a detailed investigation of unfolded and partially folded states of a mutant apomyoglobin (apoMb) where the distal histidine has been replaced by phenylalanine (H64F). Previous studies have shown that substitution of His64, located in the E helix of the native protein, stabilizes the equilibrium molten globule and native states and leads to an increase in folding rate and a change in the folding pathway. Analysis of changes in chemical shift and in backbone flexibility, detected via [1H]-15N heteronuclear nuclear Overhauser effect measurements, indicates that the phenylalanine substitution has only minor effects on the conformational ensemble in the acid- and urea-unfolded states, but has a substantial effect on the structure, dynamics, and stability of the equilibrium molten globule intermediate formed near pH 4. In H64F apomyoglobin, additional regions of the polypeptide chain are recruited into the compact core of the molten globule. Since the phenylalanine substitution has negligible effect on the unfolded ensemble, its influence on folding rate and stability comes entirely from interactions within the compact folded or partly folded states. Replacement of His64 with Phe leads to favorable hydrophobic packing between the helix E region and the molten globule core and leads to stabilization of helix E secondary structure and overall thermodynamic stabilization of the molten globule. The secondary structure of the equilibrium molten globule parallels that of the burst phase kinetic intermediate; both intermediates contain significant helical structure in regions of the polypeptide that comprise the A, B, E, G, and H helices of the fully folded protein.

Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif.

A continuous evolutionary pressure of the biotic and abiotic world has led to the development of a diversity of microbial pathways to degrade and biomineralize aromatic and heteroaromatic compounds. The heterogeneity of compounds metabolized by bacteria like Pseudomonas putida indicates the large variety of enzymes necessary to catalyse the required reactions. Quinoline, a N-heterocyclic aromatic compound, can be degraded by microbes along different pathways. For P. putida 86 quinoline degradation by the 8-hydroxycoumarin pathway has been described and several intermediates were identified. To select enzymes catalysing the later stages of the 8-hydroxycoumarin pathway P. putida 86 was grown with quinoline. The FMN-containing enzyme xenobiotic reductase A (XenA) was isolated and analysed for its reactivity with intermediates of the 8-hydroxycoumarin pathway. XenA catalyses the NADPH-dependent reduction of 8-hydroxycoumarin and coumarin to produce 8-hydroxy-3,4-dihydrocoumarin and 3,4-dihydrocoumarin, respectively. Crystallographic analysis of XenA alone and in complex with the two substrates revealed insights into the mechanism. XenA shows a dimeric arrangement of two (beta/alpha)(8) barrel domains each binding one FMN cofactor. High resolution crystal structures of complexes with 8-hydroxycoumarin and coumarin show different modes of binding for these molecules in the active site. While coumarin is ideally positioned for hydride transfer from N-5 of the isoalloxazine ring to C-4 of coumarin, 8-hydroxycoumarin forms a non-productive complex with oxidised XenA. Orientation of 8-hydroxycoumarin in the active site appears to be dependent on the electronic state of the flavin. We postulate that XenA catalyses the last step of the 8-hydroxycoumarin pathway before the heterocyclic ring is hydrolysed to yield 3-(2,3-dihydroxyphenyl)-propionic acid. As XenA is also found in P. putida strains unable to degrade quinoline, it appears to have more than one physiological function and is an example of how enzymes with low substrate specificity can help to explain the variety of degradation pathways possible.

The prion protein is thought to induce prion diseases by changing its conformation from the cellular form, PrP(C), into the infectious Scrapie-form, PrP(Sc). Little is known about the structural and dynamical features of this conformational change. We here introduce a novel concept that involves rare large scale motions between the subdomains beta1-alpha1-beta2 and alpha2-alpha3 in the carboxy-terminal, globular part of PrP. The interface between these two subdomains carries most pathogenic mutations known to be associated with prion diseases. Based on computational simulations as well as experimental results we propose that such a large scale motion subsequently destabilizes large parts of the cellular conformer PrP(C), thus, rendering it prone to structural rearrangements, including aggregation of now partially unfolded parts of the PrP sequence. We hypothesize that such large scale motions occur as a rare event even under equilibrium conditions and that the interaction of such partially destabilized PrP(C)-conformers, which we named PrP(C*), contributes to the formation of pathogenic oligomeric species of the prion protein.

The conversion of prion helix 1 from an alpha-helical into an extended conformation is generally assumed to be an essential step in the conversion of the cellular isoform PrPC of the prion protein to the pathogenic isoform PrPSc. Peptides encompassing helix 1 and flanking sequences were analyzed by nuclear magnetic resonance and circular dichroism. Our results indicate a remarkably high instrinsic helix propensity of the helix 1 region. In particular, these peptides retain significant helicity under a wide range of conditions, such as high salt, pH variation, and presence of organic co-solvents. As evidenced by a data base search, the pattern of charged residues present in helix 1 generally favors helical structures over alternative conformations. Because of its high stability against environmental changes, helix 1 is unlikely to be involved in the initial steps of the pathogenic conformational change. Our results implicate that interconversion of helix 1 is rather representing a barrier than a nucleus for the PrPC-->PrPSc conversion.

Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) α1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR α1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment andcis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/β2–3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR α1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/β2–3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding.

Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.

NMR spectroscopy of 6-acetylmorphine (6-AM), a chloroform-soluble model compound for the hydrophilic, highly potent analgesic drug morphine-6-glucoronide (M6G), in a hydrophobic solvent indicates one hydrogen bonded water molecule per molecule of 6-AM. By analysis of nuclear Overhauser enhancements (NOEs) we find a 6-AM dimer in which the monomers are linked by two water molecules. Molecular modeling studies underscore the stability of such dimeric structures involving water molecules for 6-AM and point out their more lipophilic character allowing penetration of the blood–brain barrier.

We present an evaluation of the accuracy and precision of relaxation rates calculated using a variety of methods, applied to data sets obtained for several very different protein systems. We show that common methods of data evaluation, such as the determination of peak heights and peak volumes, may be subject to bias, giving incorrect values for quantities such as R1 and R2. For example, one common method of peak-height determination, using a search routine to obtain the peak-height maximum in successive spectra, may be a source of significant systematic error in the relaxation rate. The alternative use of peak volumes or of a fixed coordinate position for the peak height in successive spectra gives more accurate results, particularly in cases where the signal/noise is low, but these methods have inherent problems of their own. For example, volumes are difficult to quantitate for overlapped peaks. We show that with any method of sampling the peak intensity, the choice of a 2- or 3-parameter equation to fit the exponential relaxation decay curves can dramatically affect both the accuracy and precision of the calculated relaxation rates. In general, a 2-parameter fit of relaxation decay curves is preferable. However, for very low intensity peaks a 3 parameter fit may be more appropriate.

We report the cloning and expression of micro-myoglobin, a 78-amino-acid fragment containing residues 29-105 of sperm whale myoglobin, and spanning the region from mid-helix B to mid-helix G of the globin fold. In contrast to full-length myoglobin and to mini-myoglobin (residues 32-129), the micro-myoglobin apoprotein is almost unfolded. However, circular dichroism and absorption spectroscopy data indicate that this fragment is capable of folding into a functional heme-binding unit forming a complex with the prosthetic group with characteristics similar to native myoglobin. Therefore, this case represents a new example of cofactor-assisted folding. The experimental data suggest independence between myoglobin subdomains.

Studies of proteins unfolded in acid or chemical denaturant can help in unraveling events during the earliest phases of protein folding. In order for meaningful comparisons to be made of residual structure in unfolded states, it is necessary to use random coil chemical shifts that are valid for the experimental system under study. We present a set of random coil chemical shifts obtained for model peptides under experimental conditions used in studies of denatured proteins. This new set, together with previously published data sets, has been incorporated into a software interface for NMRView, allowing selection of the random coil data set that fits the experimental conditions best.

Enterotoxigenic Escherichia coli (ETEC) infections account for the majority of cases of acute secretory diarrhea. The causative agents are enterotoxins secreted by ETEC, among them is the heat-stable enterotoxin, STh. STh is a 19-amino acid peptide containing three disulfide bonds that stimulates fluid secretion in the bowel by binding to the receptor domain of intestinal guanylyl cyclase C (GC-C). Since GC-C agonists have pharmacologic potential for diagnosis and treatment of disorders such as constipation-predominant irritable bowel syndrome (IBS-C), chronic constipation, and colorectal carcinoma, it is crucial to develop methods for the large-scale production of STh and related peptides. Here, we present a strategy for recombinant expression of STh that relies on the use of the prosequence of human uroguanylin to support proper folding and disulfide bond formation. The chimeric protein CysCys-STh consisting of the propeptide of uroguanylin as N-terminus and the STh peptide as C-terminus was expressed in E. coli, and an efficient purification protocol was developed. Trypsin digestion of this protein released the enterotoxin which could be obtained in high purity. NMR and mass spectrometry confirmed the identity and homogeneity of the toxin, and its biological activity was confirmed by a cell-based in vivo assay. The expression scheme introduced here represents a cost-efficient and scalable way of STh production.