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R Riek

Publications and source records attributed to R Riek.

32 records · Page 2Linked to original sources
PubMed1998

Prion protein NMR structure and familial human spongiform encephalopathies.

The refined NMR structure of the mouse prion protein domain mPrP(121-231) and the recently reported NMR structure of the complete 208-residue polypeptide chain of mPrP are used to investigate the structural basis of inherited human transmissible spongiform encephalopathies. In the cellular form of mPrP no spatial clustering of mutation sites is observed that would indicate the existence of disease-specific subdomains. A hydrogen bond between residues 128 and 178 provides a structural basis for the observed highly specific influence of a polymorphism in position 129 in human PrP on the disease phenotype that segregates with the mutation Asp-178-Asn. Overall, the NMR structure implies that only part of the disease-related amino acid replacements lead to reduced stability of the cellular form of PrP, indicating that subtle structural differences in the mutant proteins may affect intermolecular signaling in a variety of different ways.

Animals↗
PubMed1998

Prion protein structural features indicate possible relations to signal peptidases.

Transmissible spongiform encephalopathies (TSEs) in mammalian species are believed to be caused by an oligomeric isoform, PrP(Sc), of the cellular prion protein, PrP(C). One of the key questions in TSE research is how the observed accumulation of PrP(Sc), or possibly the concomitant depletion of PrP(C) can cause fatal brain damage. Elucidation of the so far unknown function of PrP(C) is therefore of crucial importance. PrP(C) is a membrane-anchored cell surface protein that possesses a so far unique three-dimensional structure. While the N-terminal segment 23-120 of PrP(C) is flexibly disordered, its C-terminal residues 121-231 form a globular domain with three alpha-helices and a two-stranded beta-sheet. Here we report the observation of structural similarities between the domain of PrP(121-231) and the soluble domains of membrane-anchored signal peptidases. At the level of the primary structure we find 23% identity and 41% similarity between residues 121-217 of the C-terminal domain of murine PrP and a catalytic domain of the rat signal peptidase. The invariant PrP residues Tyr-128 and His-177 align with the two presumed active-site residues of signal peptidases and are in close spatial proximity in the three-dimensional structure of PrP(121-231).

Animals↗
PubMed1997

Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution.

Fast transverse relaxation of 1H, 15N, and 13C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. Transverse relaxation-optimized spectroscopy (TROSY) is an approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional 1H,15N-correlation experiment with a uniformly 15N-labeled protein in a DNA complex of molecular mass 17 kDa at a 1H frequency of 750 MHz showed that 15N relaxation during 15N chemical shift evolution and 1HN relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional 1H,15N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for 15N and 15 Hz for 1HN at 4 degrees C. Because the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a 15N-labeled protein of 150 kDa at 750 MHz and 20 degrees C one predicts residual linewidths of 10 Hz for 15N and 45 Hz for 1HN, and for the corresponding uniformly 15N,2H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.

Anisotropy↗
PubMed1997

Prion (PrPSc)-specific epitope defined by a monoclonal antibody.

Prions are infectious particles causing transmissible spongiform encephalopathies (TSEs). They consist, at least in part, of an isoform (PrPSc) of the ubiquitous cellular prion protein (PrPC). Conformational differences between PrPC and PrPSc are evident from increased beta-sheet content and protease resistance in PrPSc. Here we describe a monoclonal antibody, 15B3, that can discriminate between the normal and disease-specific forms of PrP. Such an antibody has been long sought as it should be invaluable for characterizing the infectious particle as well as for diagnosis of TSEs such as bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD) in humans. 15B3 specifically precipitates bovine, murine or human PrPSc, but not PrPC, suggesting that it recognizes an epitope common to prions from different species. Using immobilized synthetic peptides, we mapped three polypeptide segments in PrP as the 15B3 epitope. In the NMR structure of recombinant mouse PrP, segments 2 and 3 of the 15B3 epitope are near neighbours in space, and segment 1 is located in a different part of the molecule. We discuss models for the PrPSc-specific epitope that ensure close spatial proximity of all three 15B3 segments, either by intermolecular contacts in oligomeric forms of the prion protein or by intramolecular rearrangement.

Amino Acid Sequence↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1997

Recombinant full-length murine prion protein, mPrP(23-231): purification and spectroscopic characterization.

The cellular prion protein of the mouse, mPrP(C), consists of 208 amino acids (residues 23-231). It contains a carboxy-terminal domain, mPrP(121-231), which represents an autonomous folding unit with three alpha-helices and a two-stranded antiparallel beta-sheet. We expressed the complete amino acid sequence of the prion protein, mPrP(23-231), in the cytoplasm of Escherichia coli. mPrP(23-231) was solubilized from inclusion bodies by 8 M urea, oxidatively refolded and purified to homogeneity by conventional chromatographic techniques. Comparison of near-UV circular dichroism, fluorescence and one-dimensional 1H-NMR spectra of mPrP(23-231) and mPrP(121-231) shows that the amino-terminal segment 23-120, which includes the five characteristic octapeptide repeats, does not contribute measurably to the manifestation of three-dimensional structure as detected by these techniques, indicating that the residues 121-231 might be the only polypeptide segment of PrP(C) with a defined three-dimensional structure.

Amino Acid Sequence↗
PubMed1997

Prion protein NMR structure and species barrier for prion diseases.

The structural basis of species specificity of transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy or "mad cow disease" and Creutzfeldt-Jakob disease in humans, has been investigated using the refined NMR structure of the C-terminal domain of the mouse prion protein with residues 121-231. A database search for mammalian prion proteins yielded 23 different sequences for the fragment 124-226, which display a high degree of sequence identity and show relevant amino acid substitutions in only 18 of the 103 positions. Except for a unique isolated negative surface charge in the bovine protein, the amino acid differences are clustered in three distinct regions of the three-dimensional structure of the cellular form of the prion protein. Two of these regions represent potential species-dependent surface recognition sites for protein-protein interactions, which have independently been implicated from in vitro and in vivo studies of prion protein transformation. The third region consists of a cluster of interior hydrophobic side chains that may affect prion protein transformation at later stages, after initial conformational changes in the cellular protein.

Amino Acid Sequence↗
PubMed1996

NMR structure of the mouse prion protein domain PrP(121-231).

The 'protein only' hypothesis states that a modified form of normal prion protein triggers infectious neurodegenerative diseases, such as bovine spongiform encephalopathy (BSE), or Creutzfeldt-Jakob disease (CJD) in humans. Prion proteins are thought to exist in two different conformations: the 'benign' PrPcform, and the infectious 'scrapie form', PrPsc. Knowledge of the three-dimensional structure of PrPc is essential for understanding the transition to PrPsc. The nuclear magnetic resonance (NMR) structure of the autonomously folding PrP domain comprising residues 121-231 (ref. 6) contains a two-stranded antiparallel beta-sheet and three alpha-helices. This domain contains most of the point-mutation sites that have been linked, in human PrP, to the occurrence of familial prion diseases. The NMR structure shows that these mutations occur within, or directly adjacent to, regular secondary structures. The presence of a beta-sheet in PrP(121-231) is in contrast with model predictions of an all-helical structure of PrPc (ref. 8), and may be important for the initiation of the transition from PrPc to PrPsc.

Amino Acid Sequence↗
PubMed1993

Investigation of reconstitution of the Na, K-ATPase in lipid vesicles.

Vesicles containing Na,K-ATPase were prepared by a dialysis method in buffers with various concentrations of K+ and Na+ ions. Ion-exchange chromatography has been used to separate proteoliposomes into protein-depleted and protein-rich fractions. The pumping activity of reconstituted ion pumps has been determined in the different fractions of the vesicle preparation using voltage-dependent fluorescence dyes. This method allowed to characterise vesicle fractions by a quantity which is proportional to the average number of pumps per vesicle with an active (inside-out) orientation. It could be shown that both, the amount of enzymatic active protein and the orientation of Na,K-ATPase in the vesicle lipid bilayer, is partially controlled by the Na+ and K+ concentration in the buffer during vesicle formation. High Na+ concentrations preferentially maintain the E1 conformation of the enzyme, which is less stable against denaturation during the dialysis, but displays a higher percentage of inside-out orientation of the transport-active protein. High K+ concentrations maintain the E2 conformation of the enzyme, which is stable against denaturation during the dialysis, but leads to a random orientation of the pump during dialysis.

Animals↗
PubMed1988

Reconstitution of Na+/K+-ATPase into phosphatidylcholine vesicles by dialysis of nonionic alkyl maltoside detergents.

The reconstitution of Na+/K+-ATPase from outer medulla of rabbit kidney into large unilamellar liposomes was achieved through detergent removal by dialysis of mixed micellar solutions of synthetic dioleoyl phosphatidylcholine/octyl glucoside and Na+/K+-ATPase/decyl maltoside or decenyl maltoside. Tight, transport-active liposomes were formed when the lipid and the enzyme were solubilized separately in the nonionic detergents and mixed immediately before starting the dialysis. The two maltoside detergents with different structures of the hydrophobic part of the molecule proved to be well suited for the solubilization of Na+/K+-ATPase with high retention of enzyme activity; the inactivation of enzyme being evidently slower with the unsaturated decenyl maltoside. The diameters of the proteoliposomes, 110 and 170 nm, respectively, were also dependent on the structure of the maltoside detergent, the saturated decyl maltoside producing the bigger liposomes. After freeze-fracture, both preparations exhibited intramembranous particles as structural indicators of successful reconstitution. The electrogenic activity of the reconstituted enzyme was determined by fluorescence measurements with Oxonol VI and by tracer-flux measurements with 22Na+.

Animals↗
PubMed1986

Simultaneous vaccination of cattle against foot-and-mouth disease and rinderpest.

A total of 118 local cattle in the Sultanate of Oman were divided into three groups, one which received a trivalent inactivated foot-and-mouth disease vaccine, another which received attenuated tissue culture rinderpest vaccine and a third which received both vaccines simultaneously at separate sites on opposite sides of the neck. The serological response to vaccination was monitored at day 0, 21 and 42 in virus neutralisation tests. The simultaneous administration of the two vaccines produced no ill effects and the serological responses did not differ significantly from the response to either vaccine given separately.

Aging↗