Three-dimensional NMR structure of a self-folding domain of the prion protein PrP(121-231)
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Animals↗
Biomedical subjects
M Billeter
Publications and source records attributed to M Billeter.
Comparison between clinical examination, cw-Doppler ultrasound and colour-duplex sonography in the diagnosis of incompetent perforating veins.
OBJECTIVES: This study was conducted to investigate the accuracy and agreement between clinical examination, continuous wave (cw)-Doppler ultrasound and colour-duplex sonography (CDS) in diagnosing incompetent perforating veins (ICPV). DESIGN: Observational cohort study. MATERIAL AND METHODS: Nineteen patients with chronic venous insufficiency (CVI) were examined clinically, by hand-held cw-Doppler ultrasound in combination with tourniquet compression and CDS. RESULTS: The low accuracy of the clinical examination and the diagnosis of ICPV by cw-Doppler were surprising. The specificity was 15% and sensitivity 29%, when CDS was taken as the "gold standard". Furthermore the results show clearly that the application of a tourniquet cannot provide reliable results. CONCLUSIONS: In patients with CVI, clinical and cw-Doppler cannot accurately diagnose ICPV when compared to CDS.
Adult↗
Structural role of a buried salt bridge in the 434 repressor DNA-binding domain.
The independently folding 63-residue N-terminal DNA-binding domain of the 434 repressor, 434(1-63), contains a buried Arg10-Glu35 salt bridge. A corresponding salt bridge is found in a variety of prokaryotic and eukaryotic DNA-binding proteins with helix-turn-helix motifs. Here, the NMR solution structures of 434(1-63) and the mutant protein 434[R10M](1-63) were determined to investigate the structural role of this salt bridge. Both proteins contain the same type of global fold, with five alpha-helices and a helix-turn-helix motif formed by the helices II and III. The primary structural difference caused by the Arg10 --> Met mutation is a translation of helix I along its axis relative to the helix II-turn-helix III motif. This limited conformational change is paralleled by a 9 kJ M(-1) decrease of the stability of the folded mutant protein in aqueous solution at pH 4.8. It affects the pKa value of Glu19 as well as the population of a hydrogen bond between the backbone amide proton of Asn16 and the side-chain carboxylate group of Glu19. Using the crystal structure of the 434 repressor dimer complexed with the operator DNA as a basis, model building of the DNA complex with the NMR structure of 434[R10M](1-63) shows that Asn16, which is located on the protein surface, makes direct contact with the DNA and indicates that the point mutation Arg10 --> Met should also lead to modifications of the protein-protein contacts in the complex.
Binding Sites↗
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↗
Hydration and DNA recognition by homeodomains.
A 2-nanosecond molecular dynamics (MD) simulation of an Antennapedia homeodomain-DNA complex in explicit solvent water at ambient temperature and pressure was performed to supplement experimental nuclear magnetic resonance (NMR) data on the structure and dynamics of this complex. In addition to direct protein-DNA contacts, the MD trajectory attributes an essential role for specific DNA recognition to hydration water molecules that mediate intermolecular contacts. The simulation provides a detailed description of the pathways of hydration water molecules exchanging in and out of the protein-DNA interface and indicates that the residence times of these "interior" waters are on the nanosecond time scale, near the lower end of the range determined by NMR.
Amino Acids↗
Domains of the measles virus N protein required for binding to P protein and self-assembly.
The nucleocapsid protein (N, 525 amino acids) of measles virus plays a central role in the replication of the viral genomic RNA. Its functions require interactions with itself and with other viral components. The N protein encapsidates genomic RNA, a function reflected in its ability to self-assemble into nucleocapsid-like particles in the absence of other viral proteins. The substrate for the packaging of nascent RNA during RNA replication is a complex between the N and phosphoprotein (P). The domains on the N protein that promote binding to P protein and self-assembly have been identified utilizing a series of N protein deletions. Two noncontiguous regions, amino acids 4-188 and 304-373 of N protein, are required for the formation of the soluble N-P complex, while deletion of amino acids 189-239 did not affect N-P binding. Amino acids 240-303 appear to be necessary for the stability of the protein. The N-terminal 398 amino acids are all required for the formation of organized nucleocapsid-like particles, since deletion of the central region from amino acids 189-373 completely abolished N-N interaction, and deletion of amino acids 4-188 and 374-492 caused the formation of unstructured aggregates.
Animals↗
The new program OPAL for molecular dynamics simulations and energy refinements of biological macromolecules.
A new program for molecular dynamics (MD) simulation and energy refinement of biological macromolecules, OPAL, is introduced. Combined with the supporting program TRAJEC for the analysis of MD trajectories, OPAL affords high efficiency and flexibility for work with different force fields, and offers a user-friendly interface and extensive trajectory analysis capabilities. Salient features are computational speeds of up to 1.5 GFlops on vector supercomputers such as the NEC SX-3, ellipsoidal boundaries to reduce the system size for studies in explicit solvents, and natural treatment of the hydrostatic pressure. Practical applications of OPAL are illustrated with MD simulations of pure water, energy minimization of the NMR structure of the mixed disulfide of a mutant E. coli glutaredoxin with glutathione in different solvent models, and MD simulations of a small protein, pheromone Er-2, using either instantaneous or time-averaged NMR restraints, or no restraints.
Computer Simulation↗
MOLMOL: a program for display and analysis of macromolecular structures.
MOLMOL is a molecular graphics program for display, analysis, and manipulation of three-dimensional structures of biological macromolecules, with special emphasis on nuclear magnetic resonance (NMR) solution structures of proteins and nucleic acids. MOLMOL has a graphical user interface with menus, dialog boxes, and on-line help. The display possibilities include conventional presentation, as well as novel schematic drawings, with the option of combining different presentations in one view of a molecule. Covalent molecular structures can be modified by addition or removal of individual atoms and bonds, and three-dimensional structures can be manipulated by interactive rotation about individual bonds. Special efforts were made to allow for appropriate display and analysis of the sets of typically 20-40 conformers that are conventionally used to represent the result of an NMR structure determination, using functions for superimposing sets of conformers, calculation of root mean square distance (RMSD) values, identification of hydrogen bonds, checking and displaying violations of NMR constraints, and identification and listing of short distances between pairs of hydrogen atoms.
Antennapedia Homeodomain Protein↗
Homeodomain-type DNA recognition.
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Animals↗
Messenger ribonucleic acid kinetics in human oocytes--effects of in vitro culture and nuclear maturational status.
OBJECTIVE: To study the effects of different nuclear maturational status (prophase I [PI] versus metaphase II [MII]) and in vitro culture on the kinetics of maternal messenger ribonucleic acid (mRNA) in human oocytes. DESIGN: Molecular biology on excess oocytes obtained from our clinical IVF program. INTERVENTIONS: The oocytes, classified as either PI or MII at collection, were used as such or cultured in vitro for an additional 24 hours. The relative levels of c-mos and cyclin-B1 were measured using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: The mean levels of c-mos and cyclin-B1 transcripts were indistinguishable between the PI, MII, PI oocytes matured in vitro, PI oocytes failing to mature, and MII oocytes cultured for additional 24 hours. The variability in the levels of these transcripts increased during the vitro culture. CONCLUSIONS: The level of c-mos and cyclin-B1 transcripts were not different in PI versus MII oocytes, therefore, differences seen in the clinical outcome of PI and MII oocytes may be unrelated to levels of these gene products. C-mos and cyclin B1 mRNA were maintained in vitro, thus degradation of maternal RNA is not activated in excess during the 24-hour culture.
Base Sequence↗
[Color duplex ultrasonography in the diagnosis of deficient perforating veins in comparison to cw-Doppler ultrasonography and clinical examination].
The study was conducted to investigate the accuracy and agreement between clinical examination, cw-Doppler ultrasound and color-duplex sonography in diagnosing incompetent perforating veins. Nineteen patients with chronic venous insufficiency (CVI) were examined clinically, by hand-held cw-Doppler ultrasound in combination with tourniquet compression and color-coded duplex sonography CDS. The accuracy of the clinical examination and the diagnosis of ICPV by cw-Doppler were surprisingly low. The specificity was 15% and sensitivity 29%, when CDS was taken as gold standard. Furthermore the results show clearly that the application of a tourniquet cannot provide reliable results. It is concluded that in patients with CVI clinical and cw-Doppler examinations are not sufficient for an accurate diagnosis of incompetent perforating veins. Color-duplex provides a new noninvasive approach for accurate anatomical and functional diagnosis, which is of great importance prior to surgical interventions and/or sclerotherapy.
Adult↗
NMR studies of DNA duplexes singly cross-linked by different synthetic linkers.
Molecular modelling studies resulted in the design of a variety of non-nucleotidic covalent linkers to bridge the 3'-end of the (+)-strand and the 5'-end of the (-)-strand in DNA duplexes. Three of these linkers were synthesized and used to prepare singly cross-linked duplexes d(GTGGAATTC)-linker-d(GAATTCCAC). Linker I is an assembly of a propylene-, a phosphate- and a second propylene-group and is thought to mimic the backbone of two nucleotides. Linkers II and III consist of five and six ethyleneglycol units, respectively. The melting temperatures of the cross-linked duplexes are 65 degrees C for I and 73 degrees C for II and III, as compared with 36 degrees C for the corresponding non-linked nonadeoxynucleotide duplex. The three cross-linked duplexes were structurally characterized by nuclear magnetic resonance spectroscopy. The 1H and 31P resonance assignments in the DNA stem were obtained using standard methods. For the resonance assignment of the linker protons, two-dimensional 1H-31P heteronuclear COSY and two-quantum-experiments were used. Distance geometry calculations with NOE-derived distance constraints were performed and the resulting structures were energy-minimized. In duplex I, the nucleotides flanking the propylene-phosphate-propylene-linker do not form a Watson-Crick base pair, whereas in duplexes II and III the entire DNA stem is in a B-type double helix conformation.
Base Sequence↗
The sequence and conformation of human pancreatic procarboxypeptidase A2. cDNA cloning, sequence analysis, and three-dimensional model.
A full-length cDNA clone coding for human pancreatic preprocarboxypeptidase A2 has been isolated from a lambda gt 11 human pancreatic library. Expression clones were identified by specific interaction with antisera raised against the native protein. The open reading frame of the polynucleotide sequence is 1254 base pairs in length and encodes a protein of 417 amino acids. This cDNA includes a short leader signal peptide of 16 amino acids and a 94-amino acid-long activation segment. The amino acid sequence shows 89% identity to that of rat procarboxypeptidase A2, the only A2 form sequenced so far, and 64% identity to that of human procarboxypeptidase A1. The newly determined sequence was modeled to the three-dimensional crystal structures of both bovine carboxypeptidase A and porcine procarboxypeptidase A1 by a novel distance geometry approach. Biases in the modeling were avoided by relying exclusively on automatic procedures and by using random structures as starting points. Information taken from the known homologous structures refers only to the backbone since no explicit data describing the conformation of side chains were transferred. Ten structures of human carboxypeptidase A2 were determined on the basis of each of the two known crystal structures. The root-mean-square distance for the backbone atoms between the 10 structures and their mean for 237 selected residues is 0.7 A when starting from the bovine protein and 0.8 A for 251 selected residues when starting from the porcine protein. The 94 residue-long activation segment was also determined in the modeling based on the porcine zymogen; its structure is well defined but not its orientation with respect to the enzyme moiety. The model obtained for human procarboxypeptidase A2 is discussed with respect to the specificity and activation of the enzyme.
Amino Acid Sequence↗
NMR solution structure of the recombinant tick anticoagulant protein (rTAP), a factor Xa inhibitor from the tick Ornithodoros moubata.
The solution structure of the recombinant tick anticoagulant protein (rTAP) was determined by 1H nuclear magnetic resonance (NMR) spectroscopy in aqueous solution at pH 3.6 and 36 degrees C. rTAP is a 60-residue protein functioning as a highly specific inhibitor of the coagulation protease factor Xa, which was originally isolated from the tick Ornithodoros moubata. Its regular secondary structure consists of a two-stranded antiparallel beta-sheet with residues 22-28 and 32-38, and an alpha-helix with residues 51-60. The relative orientation of these regular secondary structure elements has nearly identical counterparts in the bovine pancreatic trypsin inhibitor (BPTI). In contrast, the loop between the beta-sheet and the C-terminal alpha-helix as well as the N-terminal 20-residue segment preceding the beta-sheet adopt different three-dimensional folds in the two proteins. These observations are discussed with regard to the implication of different mechanisms of protease inhibition by rTAP and by Kunitz-type protein proteinase inhibitors.
Amino Acid Sequence↗
Homeodomain-DNA recognition.
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Amino Acid Sequence↗
The nuclear magnetic resonance solution structure of the mixed disulfide between Escherichia coli glutaredoxin(C14S) and glutathione.
The determination of the nuclear magnetic resonance (NMR) solution structured of the mixed disulfide between the mutant Escherichia coli glutaredoxin Grx(C14S) and glutathione (GSH), Grx(C14S)-SG, is described, the binding site for GSH on Grx(C14S) is located, and the non-bonding interactions between -SG and the protein are characterized. Based on nearly complete sequence-specific NMR assignments, 1010 nuclear Overhauser enhancement upper distance constraints and 116 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by energy minimization in a waterbath with the AMBER force field in the program OPAL. The -SG moiety was found to be localized on the surface of the protein in a cleft bounded by the amino acid residues Y13, T58, V59, Y72, T73 and D74. Hydrogen bonds have been identified between -SG and the residues V59 and T73 of Grx(C14S), and the formation of an additional hydrogen bond with Y72 and electrostatic interactions with the side-chains of D74 and K45 are also compatible with the NMR conformational constraints. Comparison of the reduced and oxidized forms of Grx with Grx(C14S)-SG shows that the mixed disulfide more closely resembles the oxidized form of the protein. Functional implications of this observation are discussed. Comparisons are also made with the related proteins bacteriophage T4 glutaredoxin and glutathione S-transferase.
Amino Acid Sequence↗
Inflammatory aneurysm of the splenic artery.
The case of a middle-aged man is described, who was admitted because of intermittent back pain and a high sedimentation rate. Abdominal sonography and arteriography showed a large aneurysm of the splenic artery, but failed to recognize the aneurysm as of inflammatory origin. However, the inflammatory nature of the aneurysm was evident on computer tomography scan. Intraoperatively the inflammatory origin of the aneurysm was confirmed. A saphenous vein graft was implanted and marsupialization of the aneurysm performed. Histology could clearly verify the diagnosis of an inflammatory aneurysm. This report indicates the possibility of inflammatory changes occurring in connection with a visceral artery rather than solely with the abdominal aorta.
Aneurysm↗
Nuclear magnetic resonance spectroscopy of a DNA complex with the uniformly 13C-labeled Antennapedia homeodomain and structure determination of the DNA-bound homeodomain.
A 1:1 complex formed by the mutant Antennapedia(C39S) homeodomain and a 14 base-pair DNA duplex (molecular weight approximately 18,000) was prepared in which the protein was uniformly 13C-labeled. Using two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy with 13C(omega 1, omega 2)-double-half-filter and three-dimensional 13C-correlated NOE spectroscopy, nearly complete sequence-specific resonance assignments were obtained for both the protein and the DNA in the complex. On this basis conformational constraints needed for a three-dimensional structure determination were collected. Using 855 intramolecular distance constraints as input, the structure of the DNA-bound Antp(C39S) homeodomain was calculated with the program DIANA, followed by restrained energy minimization with the program OPAL. A group of 20 conformers characterizes a well-defined structure for residues 8 to 56, with an average of 0.5 A of the pairwise root-mean-square deviations calculated for the backbone atoms of the individual conformers relative to the mean coordinates. The quality of the resulting structure is comparable to the one for the free protein, and the global fold of the free Antp(C39S) homeodomain was found to be conserved in the DNA complex. The structure of the DNA-bound protein was the starting point for the subsequent structure determination of the complete Antp(C39S) homeodomain-DNA complex in solution.
Amino Acid Sequence↗