Search PubMed⌕ Search

Biomedical subjects

M Billeter

Publications and source records attributed to M Billeter.

At least 55 records · Page 3Linked to original sources
PubMed1993

Determination of the nuclear magnetic resonance solution structure of an Antennapedia homeodomain-DNA complex.

The nuclear magnetic resonance (NMR) solution structure of a complex formed by the mutant Antennapedia homeodomain with Cys39 replaced by Ser, Antp(C39S), and a 14 base-pair DNA duplex containing the BS2 operator sequence was determined using uniform 13C and 15N-labeling of the protein. Two-dimensional nuclear Overhauser enhancement spectroscopy ([1H,1H]NOESY) with 15N(omega 2)-half-filter and 13C(omega 1, omega 2)-double-half-filter, and three-dimensional heteronuclear-correlated [1H,1H]NOESY yielded a total of 855 intramolecular NOE upper distance constraints in the homeodomain, 151 upper distance constraints within the DNA duplex, and 39 intermolecular protein-DNA upper distance constraints. These data were used as the input for the structure calculation with simulated annealing followed by molecular dynamics in a water bath and energy refinement. A group of 16 conformers was thus generated which represent the solution structure of the Antp(C39S) homeodomain-DNA complex. The new structure determination confirms the salient features reported previously from a preliminary investigation of the same complex, in particular the location of the recognition helix in the major groove with the turn of the helix-turn-helix motif outside the contact area with the DNA, and the N-terminal arm of the homeodomain contacting the minor groove of the DNA. In addition, distinct amino acid side-chain-DNA contacts could be identified, and evidence was found that the invariant residue Asn51 (and possibly also Gln50) is in a slow dynamic equilibrium between two or several different DNA contact sites. The molecular dynamics calculations in a water bath yielded structures with hydration water molecules in the protein-DNA interface, which coincides with direct NMR observations of hydration waters. In the Appendix the experimental data obtained with the Antp(C39S) homeodomain-DNA complex and the techniques used for the structure calculation are evaluated using a simulated input data set derived from the X-ray crystal structure of a DNA complex with a homologous homeodomain. This study indicates that a nearly complete set of NOE upper distance constraints for the Antp(C39S) homeodomain and the protein-DNA interface was presently obtained. It further shows that the structure calculation used here yields a precise reproduction of the crystal structure from the simulated input data, and also results in hydration of the protein-DNA interface in the recalculated complex.

Animals↗
PubMed1993

Determination of the NMR solution structure of the Hoechst 33258-d(GTGGAATTCCAC)2 complex and comparison with the X-ray crystal structure.

BACKGROUND: The chromosomal stain, Hoechst 33258, binds to the minor groove of the DNA double helix and specifically recognizes a run of four A-T base pairs. Extensive biochemical and biophysical studies have been aimed at understanding the binding of the dye to DNA at the atomic level. Among these studies there have been several crystal structure determinations and some preliminary structural studies by NMR. RESULTS: On the basis of our own previously reported NMR data, we have now determined the three-dimensional solution structure of the 1:1 complex between Hoechst 33258 and the self-complementary DNA duplex d(GTGGAATTCCAC)2. Two coexisting families of con formers, which exhibit differences in their intermolecular hydrogen bonding pattern, were found and the two terminal rings of the dye displayed greater internal mobility than the rest of the molecule. CONCLUSIONS: The observed multiple ligand-binding modes in the complex between Hoechst 33258 and DNA and differential internal mobility along the bound ligand provide a novel, dynamic picture of the specific inter actions between ligands that bind in the minor groove and DNA. The dynamic state revealed by these studies may account for some of the significant differences previously observed between different crystal structures of Hoechst 33258 complexed with a different DNA duplex, d(CGCGAATTCGCG)2.

Bisbenzimidazole↗
PubMed1992

NMR determination of residual structure in a urea-denatured protein, the 434-repressor.

A nuclear magnetic resonance (NMR) structure determination is reported for the polypeptide chain of a globular protein in strongly denaturing solution. Nuclear Overhauser effect (NOE) measurements with a 7 molar urea solution of the amino-terminal 63-residue domain of the 434-repressor and distance geometry calculations showed that the polypeptide segment 54 to 59 forms a hydrophobic cluster containing the side chains of Val54, Val56, Trp58, and Leu59. This residual structure in the urea-unfolded protein is related to the corresponding region of the native, folded protein by simple rearrangements of the residues 58 to 60. Based on these observations a model for the early phase of refolding of the 434-repressor(1-63) is proposed.

Amino Acid Sequence↗
PubMed1992

Determination of the nuclear magnetic resonance solution structure of the DNA-binding domain (residues 1 to 69) of the 434 repressor and comparison with the X-ray crystal structure.

The DNA-binding domain of the phage 434 repressor consisting of N-terminal residues 1 to 69 (434 repressor(1-69)), was expressed in Escherichia coli with natural isotope abundance, uniform 15N-labeling and biosynthetically directed fractional 13C-labeling in extent of about 10%. With these protein preparations the three-dimensional structure was determined in solution. The techniques used were nuclear magnetic resonance (n.m.r.) spectroscopy for the collection of conformational constraints, calculation of the protein structure from the n.m.r. data with the program DIANA and structure refinements by restrained energy minimization with a modified version of the program AMBER. A group of 20 conformers characterizes a well-defined structure for residues 1 to 63, with an average of 0-6 A for the root-mean-square deviations (RMSD) calculated for the backbone atoms of the individual conformers relative to the mean co-ordinates. The spatial structure of C-terminal residues 64 to 69 is not defined by the n.m.r. data. The molecular architecture of the 434 repressor(1-69) in solution includes five alpha-helices extending from residues 2 to 13, 17 to 24, 28 to 35, 45 to 52 and 56 to 60, which enclose a well-defined hydrophobic core. The n.m.r. structure is closely similar to the reported crystal structure of the 434 repressor(1-69), with an RMSD value of 1.1 A for the backbone atoms of residues 1 to 63. Small differences between the two structures in regions of the first helix and the loop between helices 3 and 4 were analyzed relative to possible correlations with protein-protein contacts in the crystal lattice and the different milieus of pH and ionic strength in the crystals and n.m.r. samples. Further systematic comparisons of local conformational features indicated that there are correlations between amino acid types, local precision of the structure determination by both techniques and local differences between the structures in the crystals and in solution. Overall, hydrophobic residues are most precisely characterized and agree most closely in the two environments.

Amides↗
PubMed1992

NMR structure of oxidized Escherichia coli glutaredoxin: comparison with reduced E. coli glutaredoxin and functionally related proteins.

The determination of the NMR structure of oxidized Escherichia coli glutaredoxin in aqueous solution is described, and comparisons of this structure with that of reduced E. coli glutaredoxin and the related proteins E. coli thioredoxin and T4 glutaredoxin are presented. Based on nearly complete sequence-specific 1H-NMR assignments, 804 nuclear Overhauser enhancement distance constraints and 74 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR. The molecular architecture of oxidized glutaredoxin is made up of three helices and a four-stranded beta-sheet. The three-dimensional structures of oxidized and the recently described reduced glutaredoxin are very similar. Quantitative analysis of the exchange rates of 34 slowly exchanging amide protons from corresponding series of two-dimensional [15N,1H]-correlated spectra of oxidized and reduced glutaredoxin showed close agreement, indicating almost identical hydrogen-bonding patterns. Nonetheless, differences in local dynamics involving residues near the active site and the C-terminal alpha-helix were clearly manifested. Comparison of the structure of E. coli glutaredoxin with those of T4 glutaredoxin and E. coli thioredoxin showed that all three proteins have a similar overall polypeptide fold. An area of the protein surface at the active site containing Arg 8, Cys 11, Pro 12, Tyr 13, Ile 38, Thr 58, Val 59, Pro 60, Gly 71, Tyr 72, and Thr 73 is proposed as a possible site for interaction with other proteins, in particular ribonucleotide reductase. It was found that this area corresponds to previously proposed interaction sites in T4 glutaredoxin and E. coli thioredoxin. The solvent-accessible surface area at the active site of E. coli glutaredoxin showed a general trend to increase upon reduction. Only the sulfhydryl group of Cys 11 is exposed to the solvent, whereas that of Cys 14 is buried and solvent inaccessible.

Amino Acid Sequence↗
PubMed1992

Precise vicinal coupling constants 3JHN alpha in proteins from nonlinear fits of J-modulated [15N,1H]-COSY experiments.

Improved experimental schemes for the recently introduced J-modulated [15N,1H]-correlation experiment for measurements of the homonuclear amide proton-C alpha proton vicinal coupling constants, 3JHN alpha, in uniformly 15N-labeled proteins are described, and a nonlinear fit procedure is presented for quantitative evaluation of 3JHN alpha. The method was first tested with the N-terminal DNA-binding domain of the 434 repressor (M = 7.3 kDa), where at 13 degrees C precise values of 3JHN alpha in the range 2.0-9.5 Hz were obtained for all residues with resolved 15N-1H cross peaks. It was then applied to the Antennapedia homeodomain complexed to a synthetic 14-base pair DNA fragment (molecular weight of the complex approximately 18 kDa). The 3JHN alpha values measured were found to be in excellent agreement with those predicted from the secondary structure of this protein in the complex.

Amino Acid Sequence↗
PubMed1992

Comparison of the NMR solution structure with the X-ray crystal structure of the activation domain from procarboxypeptidase B.

The NMR solution structure of the activation domain isolated from porcine procarboxypeptidase B is compared with the X-ray crystal structure of the corresponding segment in the intact proenzyme. For the region of the polypeptide chain that has a well-defined three-dimensional structure in solution, i.e., the backbone atoms of residues 11-76 and 25 amino acid side chains in this segment that form a hydrophobic core in the activation domain, the root-mean-square distance between the two structures is 1.1.A. There are no significant differences in average atom positions between the two structures, but only the NMR structure shows increased structural disorder in three outlying loops located along the same edge of the activation domain. These regions of increased structural disorder in the free domain coincide only partially with the interface to the enzyme domain in the proenzyme.

Amino Acid Sequence↗
PubMed1992

Pancreatic procarboxypeptidases: their activation processes related to the structural features of the zymogens and activation segments.

The molecular events leading to the complete activation of pancreatic procarboxypeptidases A and B have been investigated. For both proteins the activation process follows a similar general scheme: trypsin is responsible for the first cleavage that separates the active enzyme from the activation segment, the degradation of the activation segment proceeds only from its C-terminal end, and activity release can be correlated with the disappearance of the long forms of the activation segment. In both systems, trypsin and the released carboxypeptidase participate in the trimming of the severed activation regions. However, the rate of enzymatic activation is much faster in the case of procarboxypeptidase B. This phenomenon may be explained by some structural differences in the connecting region which acts as a linker between the globular domain of the activation segment and the N-terminal end of carboxypeptidases and also by the higher efficiency of carboxypeptidase B for the digestion of its own activation segment. It is not due to unfolding of the activation domain, since the isolated activation domain retains its globular conformation in solution.

Amino Acid Sequence↗
PubMed1991

Sequence-specific 1H n.m.r. assignments and determination of the three-dimensional structure of reduced Escherichia coli glutaredoxin.

The determination of the nuclear magnetic resonance structure of reduced E. coli glutaredoxin in aqueous solution is described. Based on nearly complete, sequence-specific resonance assignments, 813 nuclear Overhauser effect distance constraints and 191 dihedral angle constraints were employed as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR. The molecular architecture of reduced glutaredoxin is made up of three helices and four-stranded beta-sheet. The first strand of the beta-sheet (residues 2 to 7) runs parallel to the second strand (32 to 37) and antiparallel to the third strand (61 to 64), and the sheet is extended in an antiparallel fashion with a fourth strand (67 to 69). The first helix with residues 13 to 28 and the last helix (71 to 83) run parallel to each other on one side of the beta-sheet, with their direction opposite to that of the two parallel beta-strands, and the helix formed by residues 44 to 53 fills space available due to the twist of the beta-sheet and the reduced length of the last two beta-strands. The active site Cys11-Pro-Tyr-Cys14 is located after the first beta-strand and occupies the latter part of the loop connecting this strand with the first helix.

Amino Acid Sequence↗
PubMed1991

[Swelling of the knee, fatigue].

A 43 year old man was admitted because of fatigue and swelling of the knees. Swelling of both knees, acropachy and turtle-back nails were registered during clinical investigation and laboratory tests showed leucocytosis, increased blood-sedimentation rate and alkaline phosphatase. Leucine aminopeptidase was normal. X-ray showed symmetric metaphyseal periosteal reactions on femora and tibiae of both sides compatible with hypertrophic osteoarthropathy. A parahilar round tumor measuring 5 x 7 cm infiltrating the right upper lobe was detected on chest x-ray suggestive of lung cancer. Pierre-Marie-Bamberger syndrome was diagnosed (hypertrophic osteoarthropathy associated with cancer of the lung). Under radiotherapy to the tumor the osteoarthropathy subsided and alkaline phosphatase returned to normal.

Adult↗
PubMed1991

The NMR structure of the activation domain isolated from porcine procarboxypeptidase B.

The three-dimensional structure of the activation domain isolated from porcine pancreatic procarboxypeptidase B was determined using 1H NMR spectroscopy. A group of 20 conformers is used to describe the solution structure of this 81 residue polypeptide chain, which has a well-defined backbone fold from residues 11-76 with an average root mean square distance for the backbone atoms of 1.0 +/- 0.1 A relative to the mean of the 20 conformers. The molecular architecture contains a four-stranded beta-sheet with the polypeptide segments 11-17, 36-39, 50-56 and 75-76, two well defined alpha-helices from residues 20-30 and 60-70, and a 3(10) helix from residues 43-46. The three helices are oriented almost exactly antiparallel to each other, are all on the same side of the beta-sheet, and the helix axes from an angle of approximately 45 degrees relative to the direction of the beta-strands. Three segments linking beta-strands and helical secondary structures, with residues 32-35, 39-43 and 56-61, are significantly less well ordered than the rest of the molecule. In the three-dimensional structure two of these loops (residues 32-35 and 56-61) are located close to each other near the protein surface, forming a continuous region of increased mobility, and the third disordered loop is separated from this region only by the peripheral beta-strand 36-39 and precedes the short 3(10) helix.

Amino Acid Sequence↗
PubMed1991

Efficient analysis of protein 2D NMR spectra using the software package EASY.

The program EASY supports the spectral analysis of biomacromolecular two-dimensional (2D) nuclear magnetic resonance (NMR) data. It provides a user-friendly, window-based environment in which to view spectra for interactive interpretation. In addition, it includes a number of automated routines for peak-picking, spin-system identification, sequential resonance assignment in polypeptide chains, and cross peak integration. In this uniform environment, all resulting parameter lists can be recorded on disk, so that the paper plots and handwritten notes which normally accompany manual assignment of spectra can be largely eliminated. For example, in a protein structure determination by 2D 1H NMR, EASY accepts the frequency domain datasets as input, and after combined use of the automated and interactive routines it can yield a listing of conformational constraints in the format required as input for the calculation of the 3D structure. The program was extensively tested with current protein structure determinations in our laboratory. In this paper, its main features are illustrated with data on the protein basic pancreatic trypsin inhibitor.

Algorithms↗
PubMed1991

Expression of measles virus RNA in peripheral blood mononuclear cells of patients with measles, SSPE, and autoimmune diseases.

In order to characterize measles virus (MV) infection in peripheral blood mononuclear cells (PBMCs), RNA was isolated from PBMCs after PHA-stimulation for 72 hr of 9 patients with acute measles, 16 patients with subacute sclerosing panencephalitis (SSPE), 13 patients with various autoimmune diseases, and 16 healthy control donors. The RNA obtained was screened for the presence of MV N (nucleocapsid) gene specific transcripts of either positive or negative orientation in a S1 nuclease protection assay. The sensitivity of this assay allowed us to detect one infected cell in 20,000 PBMCs or 0.1 to 0.05 copies of MV-specific RNA per cell. Using single-stranded DNA or RNA probes expression of MV genomic RNA of negative polarity could be detected in only one case of acute measles and one healthy control donor. Conversely, N-specific transcripts of positive polarity, indicating active transcription, could only be detected in patients with acute measles. In addition, in infected PBMCs and in a persistently MV-infected B cell line positive stranded N-specific transcripts containing leader usually present at very low frequency have been found in relatively increased amounts in comparison with transcripts lacking leader. Whereas the ratio of these RNA species during lytic infection with MV in Vero cells is about 1:50, the ratio found here ranges from 1:3 to 1:10. This altered ratio indicates a specific regulation of MV specific transcription in cells of lymphoid origin that has not been found in any other cell system analyzed.

Animals↗
PubMed1991

Choice of antibiotic in nonelective cesarean section.

The use of antibiotics for prophylaxis against infection among women undergoing nonelective cesarean section has become the standard of care in the United States. Many different antibiotics have been used successfully. Single-dose regimens administered after the cord is clamped have proven just as effective as multiple-dose regimens. Although the most frequently used class of antibiotics is the cephalosporin family, the single best agent has not been determined. This study was a double-blind, randomized trial in which we compared a narrow-spectrum cephalosporin (cefazolin; n = 63) with an expanded-spectrum cephamycin (cefoxitin; n = 66) and with a broad-spectrum cephalosporin (cefotaxime; n = 60) used as a single-dose prophylaxis in patients undergoing a nonelective cesarean section. Of the 194 patients enrolled in the study, 189 were evaluable. There was no significant difference between the groups in mean age, gravidity, parity, duration of labor, duration of ruptured membranes, number of vaginal examinations, or socioeconomic status (socioeconomic status was defined by third-party coverage). There was no significant difference among the antibiotics in the incidence of immediate or delayed postoperative infections. These data indicate that a less expensive, narrow-spectrum cephalosporin is as effective as more expensive, broader-spectrum cephamycins and cephalosporins as prophylaxis for patients undergoing nonelective cesarean section.

Adult↗
PubMed1991

Intravenous ciprofloxacin versus ceftazidime for treatment of nosocomial pneumonia and urinary tract infection.

Intravenous ciprofloxacin and ceftazidime were compared for efficacy in the treatment of nosocomial pneumonia and urinary tract infection (UTI). Patients with nosocomial pneumonia were randomized to receive ciprofloxacin (as the lactate salt) 300 mg i.v. every 12 hours or ceftazidime (with sodium carbonate) 2 g i.v. every eight hours. Patients with UTI were randomized to receive ciprofloxacin 200 mg i.v. every 12 hours or ceftazidime 1 g i.v. every eight hours. Sputum and urine specimens were collected before, during, and after therapy. For patients with pneumonia, the organisms most frequently isolated before treatment began were Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, and Proteus mirabilis. Of the 17 pneumonia patients who completed ciprofloxacin treatment, 15 (88%) had resolution of signs and symptoms or improvement. Of the 15 ceftazidime-treated pneumonia patients, 13 (87%) had resolution or improvement. Staphylococcus aureus, Streptococcus species, Acinetobacter species, and K. pneumoniae infections persisted for the ciprofloxacin treatment failures. Infections by Enterobacter cloacae and Acinetobacter species persisted for the ceftazidime treatment failures. For UTI patients, E. coli was the organism most frequently isolated before treatment. All 14 UTI patients who completed treatment showed resolution or improvement. In the ciprofloxacin group two patients were superinfected by Enterococcus species, and in the ceftazidime group there were two superinfections by Enterococcus species and one by Enterobacter cloacae. Intravenous ciprofloxacin was as effective as ceftazidime in the treatment of nosocomial pneumonia and urinary tract infection. Caution should be exercised when treating serious infections by streptococci or staphylococci.

Adult↗