Search PubMed⌕ Search

Biomedical subjects

H Rüterjans

Publications and source records attributed to H Rüterjans.

At least 37 records · Page 2Linked to original sources
PubMed2000

HNCAN pulse sequences for sequential backbone resonance assignment across proline residues in perdeuterated proteins.

A TROSY-based triple-resonance pulse scheme is described which correlates backbone 1H and 15N chemical shifts of an amino acid residue with the 15N chemical shifts of both the sequentially preceding and following residues. The sequence employs 1J(NC alpha) and 2J(NC alpha) couplings in two sequential magnetization transfer steps in an 'out-and-back' manner. As a result, N,N connectivities are obtained irrespective of whether the neighbouring amide nitrogens are protonated or not, which makes the experiment suitable for the assignment of proline resonances. Two different three-dimensional variants of the pulse sequence are presented which differ in sensitivity and resolution to be achieved in one of the nitrogen dimensions. The new method is demonstrated with two uniformly 2H/13C/15N-labelled proteins in the 30-kDa range.

Amino Acid Sequence↗
PubMed2000

Solution structure of ileal lipid binding protein in complex with glycocholate.

Ileal lipid binding protein (ILBP) is a cytosolic lipid-binding protein that binds both bile acids and fatty acids. We have determined the solution structure of porcine ILBP in complex with glycocholate by homonuclear and heteronuclear two-dimensional NMR spectroscopy. The conformation of the protein-ligand complex was determined by restrained energy minimization and simulated annealing calculations after docking the glycocholate ligand into the protein structure. The overall tertiary structure of ILBP is highly analogous to the three-dimensional structures of several other intracellular lipid binding proteins (LBPs). Like the apo-structure, the bile-acid complex of ILBP is composed of 10 anti-parallel beta-strands that form a water-filled clam-shell structure, and two short alpha-helices. Chemical shift data indicated that the bile acid ligand is bound inside the protein cavity. Furthermore, 13C-edited heteronuclear single-quantum correlation-NOESY experiments showed NOE contacts between several aromatic residues located in the proposed bile acid portal region and the 13C-labeled ligand. A single bile acid molecule is bound inside the protein, with the steroid moiety penetrating deep into the water-accessible internal cavity, such that ring A is located right above the plane of the Trp49 indole ring. The carboxylate tail of the ligand is protruding from the proposed bile acid portal into the surrounding aqueous solution. The body of the steroid moiety is oriented with the nonpolar face in contact with the mostly hydrophobic residues of beta-strands C, D and E, while the polar face shows contacts with the side-chains of Tyr97, His99, Glu110 and Arg121 in beta-strands H, I and J. Thus, the conformational arrangement of the ligand complex suggests that the binding affinity of ILBP for bile acid molecules is based mainly on strong hydrophobic interactions inside the protein cavity. Furthermore, this binding mode explains how ILBP can transport unconjugated and conjugated bile acids.

Animals↗
PubMed2000

Solution structure of the functional domain of Paracoccus denitrificans cytochrome c552 in the reduced state.

In order to determine the solution structure of Paracoccus denitrificans cytochrome c552 by NMR, we cloned and isotopically labeled a 10.5-kDa soluble fragment (100 residues) containing the functional domain of the 18.2-kDa membrane-bound protein. Using uniformly 15N-enriched samples of cytochrome c552 in the reduced state, a variety of two-dimensional and three-dimensional heteronuclear double-resonance NMR experiments was employed to achieve complete 1H and 15N assignments. A total of 1893 distance restraints was derived from homonuclear 2D-NOESY and heteronuclear 3D-NOESY spectra; 1486 meaningful restraints were used in the structure calculations. After restrained energy minimization a family of 20 structures was obtained with rmsd values of 0.56 +/- 0. 10 A and 1.09 +/- 0.09 A for the backbone and heavy atoms, respectively. The overall topology is similar to that seen in previously reported models of this class of proteins. The global fold consists of two long helices at the N-terminus and C-terminus and three shorter helices surrounding the heme moiety; the helices are connected by well-defined loops. Comparison with the X-ray structure shows some minor differences in the positions of the Trp57 and Phe65 side-chain rings as well as the heme propionate groups.

Amino Acid Sequence↗
PubMed2000

Flavin-protein interactions in flavocytochrome b2 as studied by NMR after reconstitution of the enzyme with 13C- and 15N-labelled flavin.

A new procedure was devised for reversibly removing the flavin from flavocytochrome b2. It allowed reconstitution with selectively enriched 13C- and 15N-labelled FMN for an NMR analysis of the chemical shifts of the enriched positions as well as that of 31P. From these measurements, it was possible to deduce information about the hydrogen-bonding pattern of FMN in the protein, the hybridization states of the nitrogen atoms and (in part) the pi-electron distribution. The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1) and N(5) form hydrogen bonds to the apoenzyme in both redox states. Nevertheless, according to 15N-chemical shifts, the bond from the protein to N(3) is very weak in both redox states, whereas that to N(5) is strong for the oxidized state, and is weakened upon flavin reduction. On the other hand, the 13C-NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger hydrogen bonds with the enzyme than most other flavoproteins in both redox states. From coupling constant measurements it is shown that the N(3) proton is not solvent accessible. Although no N-H coupling constant could be measured for N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated in flavocytochrome b2 as in all other known flavoproteins. With respect to N(10), it is more sp3-hybridized in the oxidized state than in free FMN, whereas the other nitrogen atoms show a nearly planar structure. In the reduced state, N(5) and N(10) in bound FMN are both more sp3-hybridized than in free FMN, but N(5) exhibits a higher degree of sp3-hybridization than N(10), which is only slightly shifted out of the isoalloxazine plane. In addition, two-electron reduction of the enzyme leads to anion formation on N(1), as indicated by its 15N-chemical shift of N(1) and characteristic upfield shifts of the resonances of C(2), C(4) and C(4a) compared to the oxidized state, as observed for most flavoproteins. 31P-NMR measurements show that the phosphate geometry has changed in enzyme bound FMN compared to the free flavin in water, indicating a strong interaction of the phosphate group with the apoenzyme.

Binding Sites↗
PubMed2000

Bovine beta-lactoglobulin: interaction studies with palmitic acid.

Bovine beta-lactoglobulin (BLG) in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure-function relationship in this protein, the interactions between 13C enriched palmitic acid (PA) and BLG were investigated by means of one-, two-, and three-dimensional NMR spectroscopy in the pH range 8.4-2.1. The NMR spectra revealed that at neutral pH the ligand is bound within the central cavity of BLG, with the methyl end deeply buried within the protein. The analysis of 13C spectra of the holo protein revealed the presence of conformational variability of bound PA carboxyl end in the pH range 8.4-5.9, related to the Tanford transition. The release of PA starts at pH lower than 6.0, and it is nearly complete at acidic pH. This finding is relevant in relation to the widely reported hypothesis that this protein can act as a transporter through the acidic gastric tract. Ligand binding and release is shown to be completely reversible over the entire pH range examined, differently from other fatty acid binding proteins whose behavior is analyzed throughout the paper. The mode of interaction of BLG is compatible with the proposed function of facilitating the digestion of milk fat during the neonatal period of calves.

Animals↗
PubMed1999

Functional and conformational characterization of new mutants of heart fatty acid-binding protein.

In this study we investigated the possible involvement of several amino acids (not located in the ligand-binding centre) in fatty acid binding and conformational stability of heart fatty acid-binding protein (H-FABP). We prepared recombinant human H-FABP proteins with mutations in the hydrophobic patch (Phe(4), Trp(8) and Phe(64)), portal region (Phe(16)), hinge region (Leu(66), Gly(67)), second portal region (Glu(72)) and at the protein surface (Lys(21)) respectively. Oleic acid-binding affinity and conformational stability of human H-FABP are significantly decreased or completely lost by mutation of Trp(8) or Phe(16). NMR spectra confirmed that these residues are important for the stability of the protein fold. Substitution of Phe(4) or Phe(64) resulted in less stability, but oleic acid-binding affinity was not affected. Mutation of Lys(21) had no effect on either structural integrity or fatty acid-binding affinity. Replacement of Leu(66) or Gly(67) did not affect fatty acid binding, but protein stability was reduced. Finally, mutation of Glu(72) to Ser caused no change of affinity, but NMR spectra and urea-denaturation curves showed the extremely poor stability of this mutant. In conclusion, no relationship was observed between fatty acid-binding affinity and conformational stability.

Carrier Proteins↗
PubMed1999

Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase.

A membrane-bound c-type cytochrome, c552, acts as the electron mediator between the cytochrome bc1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans. Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region. Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors. High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.

Cloning, Molecular↗
PubMed1999

Solution structure of a recombinant mouse major urinary protein.

Major urinary proteins (MUPs) form an ensemble of protein isoforms which are expressed and secreted by sexually mature male mice only. They belong to the lipocalin superfamily and share with other members of this family the capacity to bind hydrophobic molecules, some of which are odorants. MUPs, either associated with or free of their natural ligands, play an important role in the reproductive cycle of these rodents by acting as pheromones. In fact, they are able to interact with receptors in the vomeronasal organ of the female mice, inducing hormonal and physiological responses by an as yet unknown mechanism. In order to investigate the structural and dynamical features of these proteins in solution, one of the various wild-type isoforms (rMUP: 162 residues) was cloned and subsequently isotopically labeled. The complete 1H, 13C and 15N resonance assignment of that isoform, achieved by using a variety of multidimensional heteronuclear NMR experiments, has been reported recently. Here, we describe the refined high-resolution three-dimensional solution structure of rMUP in the native state, obtained by a combination of distance geometry and energy minimization calculations based on 2362 NOE-derived distance restraints. A comparison with the crystal structure of the wild-type MUPs reveals, aside from minor differences, a close resemblance in both secondary structure and overall topology. The secondary structure of the protein consists of eight antiparallel beta-strands forming a single beta-sheet and an alpha-helix in the C-terminal region. In addition, there are several helical and hairpin turns distributed throughout the protein sequence, mostly connecting the beta-strands. The tertiary fold of the beta-sheet creates a beta-barrel, common to all members of the lipocalin superfamily. The shape of the beta-barrel resembles a calyx, lined inside by mostly hydrophobic residues that are instrumental for the binding and transport of small nonpolar ligand molecules.

Amino Acid Sequence↗
PubMed1999

A comparative study of the backbone dynamics of two closely related lipid binding proteins: bovine heart fatty acid binding protein and porcine ileal lipid binding protein.

The backbone dynamics of bovine heart fatty acid binding protein (H-FABP) and porcine ileal lipid binding protein (ILBP) were studied by 15N NMR relaxation (T1 and T2) and steady state heteronuclear 15N[1H] NOE measurements. The microdynamic parameters characterizing the backbone mobility were determined using the 'model-free' approach. For H-FABP, the non-terminal backbone amide groups display a rather compact protein structure of low flexibility. In contrast, for ILBP an increased number of backbone amide groups display unusually high internal mobility. Furthermore, the data indicate a higher degree of conformational exchange processes in the microsec-msec time range for ILBP compared to H-FABP. These results suggest significant differences in the conformational stability for these two structurally highly homologous members of the fatty acid binding protein family.

Animals↗
PubMed1998

Solution structure of the recombinant oxidized rabbit uteroglobin using homonuclear and heteronuclear multidimensional NMR.

Rabbit uteroglobin (rab-UG) is a 16-kDa homodimeric secretory protein with potent anti-inflammatory/immunomodulatory properties. Its physiological role is still unclear, although it was observed that several small hydrophobic molecules bind to the oxidized and the reduced uteroglobin. It is suggested that the formation and/or disruption of the two disulphide bridges not only regulates this binding itself, but also the affinity to the ligand. The determination of the solution structure has been started with the assignment of 1H, 15N and 13C resonances of the oxidized rabbit uteroglobin, based on several two-dimensional and three-dimensional homonuclear and heteronuclear double and triple resonance experiments. The assignment was possible with the overproduction of the wild-type as well as of uniformly 15N-labeled and 15N/13C-labeled samples of the recombinant protein. A complete assignment of 1H, 15N and 13C resonances, the secondary-structure elements and the tertiary structure in solution is presented. The tertiary solution structure was found to be in good agreement with the previously determined crystal structure of rab-UG and with the solution structure of human uteroglobin (h-UG). h-UG and rab-UG are extremely stable proteins within a wide range of pH and temperatures. Some of the binding characteristics of ligands of rab-UG and a mutant with all cysteine residues exchanged to serine residues are discussed.

Amino Acid Sequence↗
PubMed1998

Bound water in apo and holo bovine heart fatty-acid-binding protein determined by heteronuclear NMR spectroscopy.

Two- and three-dimensional heteronuclear NMR experiments have been performed to identify internally bound water molecules in the solution structure of bovine heart fatty-acid-binding protein (heart FABP). NOE and rotating-frame NOE (ROE) cross peaks between protein protons and protons of bound water molecules were observed in two-dimensional H2O-ROE/NOE-1H,15N-heteronuclear single quantum coherence spectra recorded from a uniformly 13C/15N-enriched sample of bovine heart FABP. Contacts between water protons and 23 NH protons of the protein backbone were identified. The protein structure consists of 10 antiparallel beta-strands (betaA-betaJ), forming two nearly orthogonal beta-sheets, and a short helix-turn-helix motif connecting beta-strands A and B. The spatial folding resembles a beta-barrel. Most of the water molecules are localized in the gap between beta-strands D and E, and near the two alpha-helices. In the delipidated heart FABP additional contacts between water molecules and NH protons could be observed using a three-dimensional rotating frame Overhauser 1H,15N heteronuclear single quantum coherence experiment obtained with a 15N-labeled sample of apo-heart FABP.

Animals↗
PubMed1998

Hydration water molecules of nucleotide-free RNase T1 studied by NMR spectroscopy in solution.

The hydration of uncomplexed RNase T1 was investigated by NMR spectroscopy at pH 5.5 and 313 K. Two-dimensional heteronuclear NOE and ROE difference experiments were employed to determine the spatial proximity and the residence times of water molecules at distinct sites of the protein. Backbone carbonyl oxygens involved in intermolecular hydrogen bonds to water molecules were identified based on 1J(NC) coupling constants. These coupling constants were determined from 2D-H(CA)CO and 15N-HSQC experiments with selective decoupling of the 13C alpha nuclei during the t1 evolution time. Our results support the existence of a chain of water molecules with increased residence times in the interior of the protein which is observed in several crystal structures with different inhibitor molecules and serves as a space filler between the alpha-helix and the central beta-sheet. The analysis of 1J(NC) coupling constants demonstrates that some of the water molecules seen in crystal structures are not involved in hydrogen bonds to backbone carbonyls as suggested by crystal structures. This is especially true for a water molecule, which is probably hydrogen bonded by the protonated carboxylate group of D76 and the hydroxyl group of T93 in solution, and for a water molecule, which was reported to connect four different amino acid residues in the core of the protein by intermolecular hydrogen bonds.

Amino Acid Sequence↗
PubMed1997

Limits of NMR structure determination using variable target function calculations: ribonuclease T1, a case study.

Limits of NMR structure determination using multidimensional NMR spectroscopy, variable target function calculations and relaxation matrix analysis were explored using the model protein ribonuclease T1 (RNase T1). The enzyme consists of 104 amino acid residues and has a molecular mass of approximately 11 kDa. Primary experimental data comprise 1856 assigned NOE intensities, 493 3J coupling constants and 62 values of amid proton exchange rates. From these data, 2580 distance bounds, 168 allowed ranges for torsional angles and stereospecific assignments for 75% of beta-methylene protons as well as for 80% of diastereotopic methyl groups were derived. Whenever possible, the distance restraints were refined in a relaxation matrix analysis including amid proton exchange data for improvement of lower distance limits. Description of side-chain conformations were based on various models of motional averaging of 3J coupling constants. The final structure ensemble was selected from the starting ensemble comparing the global precision of structures with order parameters derived from 15N relaxation time measurements. Significant differences between the structure of RNase T1 in solution and in the crystal became apparent from a comparison of the two highly resolved structures.

Binding Sites↗