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Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein

JOURNAL OF MOLECULAR BIOLOGY 1999年第5期289卷 1163-1178页

作者： Wopfner, F Weidenhöfer, G Schneider, R von Brunn, A Gilch, S Schwarz, TF Werner, T Schätzl, HM Univ Munich Max Von Pettenkofer Inst Hyg & Med Microbiol Dept Virol Genectr D-81377 Munich Germany GSF Forschungszentrum Umwelt & Gesundheit Inst Mammalian Genet D-85758 Oberschleissheim Germany Stiftung Juliusspital Cent Lab D-97070 Wurzburg Germany

Prion diseases are fatal neurodegenerative disorders in man and animal associated with conformational conversion of a cellular prion protein (PrPc) into the pathologic isoform (PrPSc). The function of PrPc and the tertiary structure of PrPSc are unclear. Various data indicate which parts of PrP might control the species barrier in prion diseases and the binding of putative factors to PrP. To elucidate these features, we analyzed the evolutionary conservation of the prion protein. Here, we add the primary PrP structures of 20 ungulates, three rodents, three carnivores, one maritime mammal, and nine birds. Within mammals and birds we found a high level of amino acid sequence identity, whereas between birds and mammals the overall homology was low. Various structural elements were conserved between mammals and birds. Using the CONRAD space-scale alignment, which predicts conserved and variable blocks, we observed similar patterns in avian and mammalian PrPs, although 130 million years of separate evolution lie in between. Our data support the suggestion that the repeat elements might have expanded differently within the various classes of vertebrates. Of note is the N-terminal part of PrP (amino acid residues 23-90), which harbors insertions and deletions, whereas in the C-terminal portion (91-231) mainly point mutations are found. Strikingly, we found a high level of conservation of sequences that are not part of the structured segment 121-231 of PrPc and of the structural elements therein, e.g. the N-terminal region from amino acid residue 23-90 and the regions located upstream of alpha-helices 1 and 3. (C) 1999 Academic Press.

关键词： prion protein genes non-mammalian prion proteins structure-function relationships species barrier prion disease

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Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding

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PROTEIN ENGINEERING 1999年第4期12卷 297-303页

作者： Gill, R Verma, C Wallach, B Urso, B Pitts, J Wollmer, A De Meyts, P Wood, S Univ Klinikum Southampton Dept Biochem Sch Biol Sci Southampton SO16 7PX Hants England Univ York Dept Chem York UO1 5DD N Yorkshire England Hagedorn Res Inst DK-2820 Gentofte Denmark Univ London Birkbeck Coll Dept Crystallog London WC1E 7HX England Rhein Westfal TH Aachen Inst Biochem D-52057 Aachen Germany

Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities;native IGF-1 [18-61,6-48,47-52] and IGF-1 swap [18-61,6-47, 48-52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae, IGF-1 swap had a similar to 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1,The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-I swap for the insulin receptor was similar to 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region a-helix and a rearrangement in the relative orientation of the A- and B-regions, The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region a-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.

关键词： insulin insulin-like growth factor-1 molecular dynamics receptor binding structure-function relationships

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Indispensability of transmembrane domains of Golgi UDP-galactose transporter as revealed by analysis of genetic defects in UDP-galactose transporter-deficient murine Had-1 mutant cell lines and construction of deletion mutants

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JOURNAL OF BIOCHEMISTRY 1999年第6期126卷 1107-1117页

作者： Ishida, N Yoshioka, S Iida, M Sudo, K Miura, N Aoki, K Kawakita, M Tokyo Metropolitan Inst Med Sci Dept Physiol Chem Bunkyo Ku Tokyo 1138613 Japan

UDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galactosyltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters, We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations, Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein.

关键词： FISH green fluorescent protein nucleotide-sugar transporter structure-function relationships UDP-galactose transporter

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Influence of substituents on the solution conformation of the exopolysaccharide produced by Pseudomonas 'gingeri' strain Pf9

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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 1999年第4期26卷 249-253页

作者： Gianni, R Cescutti, P Bosco, M Fett, WF Rizzo, R Univ Trieste Dipartimento Biochim Biofis & Chim Macromol I-34127 Trieste Italy POLYTech SCARL I-34012 Trieste Italy USDA ARS Eastern Reg Res Ctr Wyndmoor PA 19038 USA

The influence of pyruvate ketals and acetyl groups on the conformational behaviour of the exopolysaccharide produced by Pseudomonas 'gingeri' strain Pf9 has been investigated experimentally through studies of intrinsic viscosity and circular dichroism experiments. A conformational variation was detected as a function of the ionic strength. Measurements carried out on the native polymer, as well as on both de-pyruvated and de-acetylated samples, suggested a critical role for the acetyl group on the solution conformation of the polysaccharide. Molecular mechanics calculations indicated the possibility of intramolecular hydrogen bonding between acetyl substituents on the mannose and the C(2)OH group of the preceding saccharidic unit. NMR linewidth measurements, carried out as a function of temperature, on the low molecular weight de-pyruvated sample indicated different polymeric backbone dynamics in aqueous solutions with respect to that observed in 0.3 M NaCl solutions. (C) 1999 Elsevier Science B.V. All rights reserved.

关键词： microbial polysaccharides Pseudomonas structure-function relationships conformational properties acetyl and pyruvate groups

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Influence of substituents on the solution conformation of the exopolysaccharide produced by Pseudomonas 'gingeri' strain Pf9

Influence of substituents on the solution conformation of th...

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19th International Carbohydrate Symposium

关键词： microbial polysaccharides Pseudomonas structure-function relationships conformational properties acetyl and pyruvate groups

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Computational analysis of chain flexibility and fluctuations in Rhizomucor miehei lipase

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PROTEIN ENGINEERING 1999年第9期12卷 747-754页

作者： Peters, GH Bywater, RP Novo Nordisk AS Biostruct Grp DK-2760 Malov Denmark Tech Univ Denmark Dept Chem DK-2800 Lyngby Denmark

We have performed molecular dynamics simulation of Rhizomucor miehei lipase (Rml) with explicit water molecules present. The simulation was carried out in periodic boundary conditions and conducted for 1.2 ns in order to determine the concerted protein dynamics and to examine how well the essential motions are preserved along the trajectory. Protein motions are extracted by means of the essential dynamics analysis method for different lengths of the trajectory. Motions described by eigenvector 1 converge after approximately 200 ps and only small changes are observed with increasing simulation time. Protein dynamics along eigenvectors with larger indices, however, change with simulation time and generally, with increasing eigenvector index, longer simulation times are required for observing similar protein motions (along a particular eigenvector). Several regions in the protein show relatively large fluctuations and in particular motions in the active site lid and the segments Thr57-Asn63 and the active site hinge region Pro101-Gly104 are seen along several eigenvectors. These motions are generally associated with glycine residues, while no direct correlations are observed between these fluctuations and the positioning of prolines in the protein structure. The partial opening/closing of the lid is an example of induced fit mechanisms seen in other enzymes and could be a general mechanism for the activation of Rml.

关键词： enzyme essential dynamics molecular modelling protein stability structure-function relationships site-directed mutagenesis

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Molecular basis of film formation from a soybean protein: comparison between the conformation of glycinin in aqueous solution and in films

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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 1998年第4期23卷 241-249页

作者： Subirade, M Kelly, I Gueguen, J Pezolet, M Univ Laval Ctr Rech Sci & Ingn Macromol Dept Chim Laval PQ G1K 7P4 Canada INRA Lab Biochim & Technol Prot F-44316 Nantes 03 France

Fourier transform infrared spectroscopy has been used to investigate the conformational changes of glycinin, a major storage protein of soybean seeds, upon film-forming. The results show that the secondary structure of glycinin is mainly composed of a beta-sheet (48%) and unordered (49%) structures. The amide I band of glycinin in film-forming conditions, i.e. in alkaline media and in the presence of plasticizing agent, reveals the conversion of 18% of the secondary structure of the protein from the beta-sheet (6%) and random coil (12%) to the alpha-helical conformation due to the helicogenic effect of the ethylene glycol used as the plasticizing agent. Conformational changes also occur upon the him-forming process leading to the formation of intermolecular hydrogen-bonded beta-sheet structures. Results obtained from other plant families indicate that, whatever the origin and conformation of protein, formation of films leads to the appearance of intermolecular hydrogen-bonded beta-sheet structures, suggesting that this type of structure might be essential for the network formation in films. Thus, it is hypothesized that, in the him state, intermolecular hydrogen bonding between segments of beta-sheet may act as junction zones in the film network. This study reveals for the first time that there is a close relationship between the conformation of proteins and the mechanical properties of films. (C) 1998 Elsevier Science B.V. All rights reserved.

关键词： film glycinin structure Fourier transform infrared spectroscopy vegetable proteins structure-function relationships

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Morphological differences between fast and slowly adapting lingual afferent terminations in the principal and oral nuclei in the cat

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JOURNAL OF COMPARATIVE NEUROLOGY 1998年第1期396卷 64-83页

作者： Moritani, M Yoshida, A Honma, S Nagase, Y Takemura, M Shigenaga, Y Osaka Univ Fac Dent Dept Oral Anat Suita Osaka 565 Japan

Previous studies indicated that fast-adapting (FA) and slowly adapting (SA) mechanoreceptive afferents innervating the facial or intraoral structures give rise to morphologically distinct terminal arbors in the individual subdivisions of the trigeminal sensory nuclear complex. The present study examined the collateral morphologies of lingual afferents in the nuclei principalis (Vp) and oralis (Vo) of the cat. Seven FA and six SA lingual afferents were physiologically characterized and stained by the intra-axonal horseradish peroxidase (HRP) injection technique, The two types of afferents established terminal arbors in the dorsomedial subdivision (Vpd) of the Vp, and the rostrodorsomedial (Vo.r) and dorsomedial subdivisions (***) of the Vo, but the collateral morphologies are different between the two types. The FA afferents gave rise to mediolaterally extended oblong arbors in each subdivision, but the arbors were better developed in the Vo.r than in the Vpd and ***. The number of collaterals, intercollateral distance, number of boutons per collateral, and bouton size were also different among the subdivisions. The SA afferents were divided into two subtypes;one had a preferential projection into the Vpd or the Vo.r and ***, and others lacked a selected projection. Although the shape of their arbors varied from a stringy form to a roundish form, the general profile was denser, better developed, and rounder than that of FA afferents in each subdivision. The intercollateral distance and bouton size were different among the subdivisions. The number of boutons per collateral, bouton density, and bouton size were larger in SA than FA afferents in each subdivision. The present study demonstrated that two functionally distinct lingual afferents manifest unique morphological differences in the Vpd and Vo. (C) 1998 Wiley-Liss, Inc.

关键词： mechanoreceptors structure-function relationships somatotopy intra-axonal HRP

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Amino acids within residues 181-200 of the nicotinic acetylcholine receptor α1 subunit involved in nicotine binding

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BIOCHEMICAL PHARMACOLOGY 1998年第3期55卷 341-347页

作者： Lentz, TL Chaturvedi, V Conti-Fine, BM Yale Univ Sch Med Dept Cell Biol New Haven CT 06520 USA Univ Minnesota Coll Biol Sci Dept Biochem St Paul MN 55108 USA

Structural determinants of L-[H-3]nicotine binding to the sequence flanking Cys 192 and Cys 193 of the Torpedo acetylcholine receptor alpha 1 subunit were investigated using synthetic peptides (residues 181-200) and fusion proteins (residues 166-211). Nicotine binding at a single concentration (30 nM) was compared with 71 peptides and fusion proteins in which individual amino acids at positions 181-200 were substituted. Substitution of Lys 185, Tyr 190, Cys 192, Cys 193, Thr 196, and Tyr 198 resulted in the greatest reduction in nicotine binding. Equilibrium binding of [H-3]nicotine to peptide 181-200 revealed a binding component with an apparent K-D of 1.2 mu M. Substitution of Lys 185 (with Glu), His 186, Tyr 190, Cys 192, Cys 193, and Tyr 198 resulted in a significant reduction in affinity. Affinity was not affected significantly by substitution of Arg 182, Lys 185 (with Gly or Arg), Val 188, Tyr 189, Pro 194, Asp 195, Thr 196, and Asp 200. It is concluded that Lys 185, His 186, Tyr 190, Cys 192, Cys 193, and Tyr 198 play the greatest role in nicotine binding to residues 181-200 of the alpha 1 subunit. Previous studies have implicated Tyr 190, Cys 192, Cys 193, and Tyr 198 in agonist binding to the acetylcholine receptor. These results confirm a role for these residues and also demonstrate a function for Lys 185 and His 186 in nicotine binding. (C) 1998 Elsevier Science Inc.

关键词： acetylcholine receptor amino acids fusion proteins nicotine structure-function relationships synthetic peptides

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Insights into the molecular basis for fatty acyl specificities of lipases from Geotrichum candidum and Candida rugosa

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CHEMISTRY AND PHYSICS OF LIPIDS 1998年第1-2期93卷 57-66页

作者： Holmquist, M Royal Inst Technol Dept Biochem & Biotechnol SE-10044 Stockholm Sweden

Despite immense progress in our comprehension of lipase structure and function during the past decade, the basis for lipase acyl specificities has remained poorly understood. This review summarizes some recent advances in the understanding at the molecular-level of substrate acyl recognition by two members in a group of large (M-w similar to 60 kDa) microbial lipases. Two aspects of acyl specificity will be focused upon. (1) The unique preference of a fungal Geotrichum candidum lipase for long-chain cis (Delta-9) unsaturated fatty acid moieties in the substrate. Mutational analysis of this lipase identified residues essential for its anomalous acyl preference. This information highlighted for the first time parts in the lipase molecule involved in substrate acyl differentiation. These results are discussed in the context of the 3D-structure of a G. candidum lipase isoenzyme and structures of the related Candida rugosa lipase in complex with inhibitors. (ii) The mechanism by which the yeast C. rugosa lipase discriminates between enantiomers of a substrate with a chiral acyl moiety. Molecular modeling in combination with substrate engineering and kinetic analyses, identified two alternative substrate binding modes. This allowed for the proposal of a molecular mechanism explaining how long-chain alcohols can act as enantioselective inhibitors of this enzyme. A picture is thus beginning to emerge of the interplay between lipase structure and fatty acyl specificity. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

关键词： structure-function relationships interfacial activation protein engineering molecular modeling chiral recognition enantioselectivity

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