Stephan Schwarzinger

Random coil chemical shifts in acidic 8 M urea: implementation of random coil shift data in NMRView.

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.

CD and NMR Studies of Prion Protein (PrP) Helix 1 NOVEL IMPLICATIONS FOR ITS ROLE IN THE PrPC→ PrPSc CONVERSION PROCESS

The conversion of prion helix 1 from an α-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.

Convergent solid-phase synthesis of N-glycopeptides facilitated by pseudoprolines at consensus-sequence Ser/Thr residues.

N-Glycosylation is an important posttranslational modification of proteins. A carbohydrate is transferred to an asparagine within an Asn-X-Ser/Thr consensus sequence. The study of the biological aspects of N-glycosylation often requires the synthesis of N-glycopeptides, which are accessible by two main approaches. In the sequential mode glycosylamino acid cassettes are used for peptide elongation. After incorporation of larger oligosaccharides the solubility and reactivity of the peptide is affected and side reactions, for example, involving free OH groups complicate further elongation. In the convergent mode (Lansbury aspartylation) the sugar is connected to an aspartate after complete assembly of the peptide (Scheme 1). The main drawback of the convergent mode is the formation of cyclic aspartimides during peptide elongation and sugar coupling; this formation depends on the peptide sequence, and the coupling conditions. We found that a pseudoproline (Ypro) at the consensus-sequence Ser/Thr residue (AsnX-Ser/Thr(Ypro)) efficiently suppresses the formation of aspartimides in the convergent synthesis of N-glycopeptides on the solid phase. The lack of pure N-glycoproteins for biological studies has stimulated research into their synthesis; these syntheses were carried out mainly by ligation techniques. The required glycopeptides and their thioesters are difficult to obtain as the sugar component interferes with the peptide synthesis. The syntheses of longer glycopeptide thioesters are particularly difficult as they require, for example, additional ligation steps 10] or segment couplings. For longer N-glycopeptides the convergent approach provides advantages over the sequential approach, especially as the Lansbury aspartylation has been shown to be efficient also on the solid phase. The undesired aspartimide formation can be avoided by peptide backbone (NH) protection, however, this approach is tedious for residues other than glycine, and causes racemization when coupling backbone-protected dipeptides. Aspartimide formation during peptide elongation can be reduced by using bulky groups to protect the Asp side chain, for example, the 2-phenylisopropylester (PhiPr); however, trityl anchors are also cleaved under the reaction conditions for PhiPr removal. Dmab-protected Asp residues are compatible with trityl anchors, but the backbone protection of the neighboring amino acid is required. To provide complex N-glycopeptide thioesters by solidphase Lansbury aspartylation we compared three Asp-sidechain protecting groups for the Fmoc-SPPS of an interleukin6 (IL-6) 43–48 hexapeptide. The allyl-protected peptide 6a showed the highest percentage of aspartimide 7ai formation (15 %), followed by the Dmab peptide 6b (8%), and the PhiPr peptide 6c (< 1%; Scheme 2). As well as the protecting group many factors are known to contribute to aspartimide formation for example, the steric bulk of the neighboring amino acid, the basicity of the reaction media, and also the overall conformation of the peptide C-terminal relative to the Asp moiety. We reasoned that constraining the Scheme 1. Lansbury aspartylation leads to glycopeptide 3, Asn peptide 3Asn and aspartimide side product 4ai. PG=protecting group.

Potential bias in NMR relaxation data introduced by peak intensity analysis and curve fitting methods

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.

Energy Landscape of the Prion Protein Helix 1 Probed by Metadynamics and NMR

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.

Putative aggregation initiation sites in prion protein

Misfolded prion protein, PrPSc, is believed to be the pathogenic agens in transmissible spongiform encephalopathies. Little is known about the autocatalytic misfolding process. Looking at the intrinsic properties of short sequence stretches, such as conformational flexibility and the tendency to populate extended conformers, we have examined the aggregation behaviour of various peptides within the region 106–157 of the sequence of human prion protein. We observed fast aggregation for the peptide containing residues I138‐I‐H‐F141. This sequence, which is presented at the surface of cellular prion protein, PrPC, in an almost β‐sheet‐like conformation, is therefore an ideal anchor‐point for initial intermolecular contacts leading to oligomerization. We further report that the aggregation propensity of the neurotoxic peptide 106–126 appears to be centred in its termini and not in the central, alanine‐rich sequence (A113‐G‐AAAA‐G‐A120).

Binding of TCA to the Prion Protein: Mechanism, Implication for Therapy, and Application as Probe for Complex Formation of Bio-macromolecules

Abstract Tricyclic aromatic compounds (TCA) are promising candidates for treatment of transmissible spongiform encephalopathies. Direct binding to the cellular prion protein (PrPC) 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 PrPC. Because biologically inactive 9-aminoacridine (9-aa) binds with similar K D as anti-prion active quinacrine, direct interaction with PrPC 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.

The Fe(II)/α‐ketoglutarate‐dependent taurine dioxygenases from Pseudomonas putida and Escherichia coli are tetramers

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 (TauDPp). We also present three crystal structures of the apo form of this enzyme. Comparisons with taurine dioxygenase from Escherichia coli (TauDEc) 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 TauDPp and TauDEc 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.

Formation of transient dimers by a retroviral protease.

Retroviral proteases have been shown previously to be only active as homodimers. They are essential to form the separate and active proteins from the viral precursors. Spumaretroviruses produce separate precursors for Gag and Pol, rather than a Gag and a Gag-Pol precursor. Nevertheless, processing of Pol into a PR (protease)-RT (reverse transcriptase) and integrase is essential in order to obtain infectious viral particles. We showed recently that the PR-RT from a simian foamy virus, as well as the separate PRshort (protease) domain, exhibit proteolytic activities, although only monomeric forms could be detected. In the present study, we demonstrate that PRshort and PR-RT can be inhibited by the putative dimerization inhibitor cholic acid. Various other inhibitors, including darunavir and tipranavir, known to prevent HIV-1 PR dimerization in cells, had no effect on foamy virus protease in vitro. 1H-15N HSQC (heteronuclear single quantum coherence) NMR analysis of PRshort indicates that cholic acid binds in the proposed PRshort dimerization interface and appears to impair formation of the correct dimer. NMR analysis by paramagnetic relaxation enhancement resulted in elevated transverse relaxation rates of those amino acids predicted to participate in dimer formation. Our results suggest transient PRshort homodimers are formed under native conditions but are only present as a minor transient species, which is not detectable by traditional methods.

Novel Regulatory Site within the TM3–4 Loop of Human Recombinant α3 Glycine Receptors Determines Channel Gating and Domain Structure

Glycine receptors are Cys loop ligand-gated ion channels that mediate fast inhibitory synaptic transmission in the mammalian central nervous system. The functionally distinct splice variants α3L and α3K of the human glycine receptor differ by a 15-amino acid insert within the long intracellular TM3–4 loop, a region of high intersubunit diversity. In a mutational study, effects of the insert on ion channel function and secondary structure of the TM3–4 loop were investigated. Whole cell current responses and protein surface expression data indicated that the major effect of mutations within the insert was on channel gating. Changes in channel gating correlated with the distribution of charged residues about the splice region. Analysis of complex molecular weight indicated that recombinant TM3–4 loops of α3L and α3K associated into oligomers of different stoichiometry. Secondary structure analysis suggested that the insert stabilized the overall fold of the large cytoplasmic domain of α3L subunits. The absence of the insert resulted in a channel that was still functional, but the TM3–4 cytoplasmic domain appeared not stably folded. Thus, our data identified the spliced insert within the large TM 3–4 loop of α3 Gly receptors as a novel regulatory motif that serves a 2-fold role: (i) the presence of the insert stabilizes the overall spatial structure of the domain, and (ii) the insert presents a control unit that regulates gating of the receptor ion channel.