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W Saenger

Publications and source records attributed to W Saenger.

At least 55 records · Page 3Linked to original sources
PubMed1999

Interaction kinetics of tetramethylrhodamine transferrin with human transferrin receptor studied by fluorescence correlation spectroscopy.

We applied fluorescence correlation spectroscopy (FCS) to characterize the interaction dynamics of fluorescence-labeled transferrin with transferrin receptor (hTfR) associates isolated from human placenta. The dissociation constant for the equilibrium binding of TMR-labeled ferri-transferrin to hTfR in detergent free solution was determined to be 7 +/- 3 nM. Binding curves were compatible with equal and independent binding sites present on the hTfR associates. Under pseudo-first-order conditions, with respect to transferrin, complex formation is monophasic. From these curves, association and dissociation rate constants for a reversible bimolecular binding reaction were determined, with (1.1 +/- 0.1) x 10(4) M-1 s-1 for the former and (6 +/- 4) x 10(-)4 s-1 for the latter. In dissociation exchange experiments, biphasic curves and concentration-independent reciprocal relaxation times were determined. From isothermal titration calorimetry experiments, we obtained an enthalpy change of -44.4 kJ/mol associated with the reaction. We thus conclude that the reaction is mainly enthalpy driven.

Calorimetry↗
PubMed1999

V-Amylose at atomic resolution: X-ray structure of a cycloamylose with 26 glucose residues (cyclomaltohexaicosaose).

The amylose fraction of starch occurs in double-helical A- and B-amyloses and the single-helical V-amylose. The latter contains a channel-like central cavity that is able to include molecules, "iodine's blue" being the best-known representative. Molecular models of these amylose forms have been deduced by solid state 13C cross-polarization/magic angle spinning NMR and by x-ray fiber and electron diffraction combined with computer-aided modeling. They remain uncertain, however, as no structure at atomic resolution is available. We report here the crystal structure of a hydrated cycloamylose containing 26 glucose residues (cyclomaltohexaicosaose, CA26), which has been determined by real/reciprocal space recycling starting from randomly positioned atoms or from an oriented diglucose fragment. This structure provides conclusive evidence for the structure of V-amylose, as the macrocycle of CA26 is folded into two short left-handed V-amylose helices in antiparallel arrangement and related by twofold rotational pseudosymmetry. In the V-helices, all glucose residues are in syn orientation, forming systematic interglucose O(3)n...O(2)(n+l) and O(6)n...O(2)(n+6)/O(3)(n+6) hydrogen bonds; the central cavities of the V-helices are filled by disordered water molecules. The folding of the CA26 macrocycle is characterized by typical "band-flips" in which diametrically opposed glucose residues are in anti rather than in the common syn orientation, this conformation being stabilized by interglucose three-center hydrogen bonds with O(3)n as donor and O(5)(n+l), O(6)(n+l) as acceptors. The structure of CA26 permitted construction of an idealized V-amylose helix, and the band-flip motif explains why V-amylose crystallizes readily and may be packed tightly in seeds.

Carbohydrate Conformation↗
PubMed1999

Photosystem I, an improved model of the stromal subunits PsaC, PsaD, and PsaE.

An improved electron density map of photosystem I (PSI) calculated at 4-A resolution yields a more detailed structural model of the stromal subunits PsaC, PsaD, and PsaE than previously reported. The NMR structure of the subunit PsaE of PSI from Synechococcus sp. PCC7002 (Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062) has been used as a model to interpret the region of the electron density map corresponding to this subunit. The spatial orientation with respect to other subunits is described as well as the possible interactions between the stromal subunits. A first model of PsaD consisting of a four-stranded beta-sheet and an alpha-helix is suggested, indicating that this subunit partly shields PsaC from the stromal side. In addition to the improvements on the stromal subunits, the structural model of the membrane-integral region of PSI is also extended. The current electron density map allows the identification of the N and C termini of the subunits PsaA and PsaB. The 11-transmembrane alpha-helices of these subunits can now be assigned uniquely to the hydrophobic segments identified by hydrophobicity analyses.

Amino Acid Sequence↗
PubMed1999

Localization of two phylloquinones, QK and QK', in an improved electron density map of photosystem I at 4-A resolution.

An improved electron density map of photosystem I from Synechococcus elongatus calculated at 4-A resolution for the first time reveals a second phylloquinone molecule and thereby completes the set of cofactors constituting the electron transfer system of this iron-sulfur type photosynthetic reaction center: six chlorophyll a, two phylloquinones, and three Fe4S4 clusters. The location of the newly identified phylloquinone pair, the individual plane orientations of these molecules, and the resulting distances to other cofactors of the electron transfer system are discussed and compared with those determined by magnetic resonance techniques.

Cyanobacteria↗
PubMed1999

Identification of an RcsA/RcsB recognition motif in the promoters of exopolysaccharide biosynthetic operons from Erwinia amylovora and Pantoea stewartii subspecies stewartii.

The regulation of capsule synthesis (Rcs) regulatory network is responsible for the induction of exopolysaccharide biosynthesis in many enterobacterial species. We have previously shown that two transcriptional regulators, RcsA and RcsB, do bind as a heterodimer to the promoter of amsG, the first reading frame in the operon for amylovoran biosynthesis in the plant pathogenic bacterium Erwinia amylovora. We now identified a 23-base pair fragment from position -555 to -533 upstream of the translational start site of amsG as sufficient for the specific binding of the Rcs proteins. In addition, we could detect an RcsA/RcsB-binding site in a corresponding region of the promoter of cpsA, the homologous counterpart to the E. amylovora amsG gene in the operon for stewartan biosynthesis of Pantoea stewartii. The specificity and characteristic parameters of the protein-DNA interaction were analyzed by DNA retardation, protein-DNA cross-linking, and directed mutagenesis. The central core motif TRVGAAWAWTSYG of the amsG promoter was found to be most important for the specific interaction with RcsA/RcsB, as evaluated by mutational analysis and an in vitro selection approach. The wild type P. stewartii Rcs binding motif is degenerated in two positions and an up-mutation according to our consensus motif resulted in about a 5-fold increased affinity of the RcsA/RcsB proteins.

Bacterial Proteins↗
PubMed1999

Crystal structure of the tet repressor in complex with a novel tetracycline, 9-(N,N-dimethylglycylamido)- 6-demethyl-6-deoxy-tetracycline.

The tetracycline analog 9-(N, N-dimethylglycylamido)-6-demethyl-6-deoxy-tetracycline (9glyTc) belongs to a new group of tetracyclines called glycylcyclines. They are strong antibiotics showing reduced sensitivity against the major tetracycline resistance mechanisms. We have determined the crystal structure of 9glyTc in complex with Tet repressor class D, TetR(D), at 2.4 A resolution. Sterical hindrance at the entrance of the tetracycline binding tunnel of TetR by the bulky and charged glycyl amido substituent interferes with conformational changes required for the mechanism of induction, and leads to decreased induction efficiency as observed for point mutations of amino acid residues located in the neighbourhood to the glycylamido moiety of bound 9glyTc.

Crystallography, X-Ray↗
PubMed1999

Decamer-like conformation of a nona-peptide bound to HLA-B*3501 due to non-standard positioning of the C terminus.

The N and C termini of peptides presented by major histocompatibility complex (MHC) class I molecules are held within the peptide binding groove by a network of hydrogen bonds to conserved MHC residues. However, the published structure of the human allele HLA-B*3501 complexed with the nef octa-peptide VPLRPMTY, revealed non-standard positioning for both peptide termini. To investigate whether these deviations are indeed related to the length of the nef-peptide, we have determined the structure of HLA-B*3501 presenting a nona-peptide to 2.5 A resolution. A comparison of HLA-B*3501/peptide complexes with structures of other HLA molecules exhibits allele-specific properties of HLA-B*3501, as well as peptide-induced structural changes. Independent of the length of the bound peptide, HLA-B*3501 positions the peptide C terminus significantly closer to the alpha1-helix and nearer to the A pocket than observed for other HLA class I/peptide complexes. This reorientation is accompanied by a shift within the N-terminal part of the alpha2-helix towards the middle of the binding groove. Due to the short distance between the N and C termini, the nona-peptide is compressed and forced to zig-zag vertically within the binding groove. Its conformation rather resembles that of a deca-peptide than of other nona-peptides bound to class I molecules. Superposition of both HLA-B*3501/peptide complexes additionally reveals a significant, peptide-dependent deviation between the N-terminal parts of the alpha1-helices which might be due to different positioning of the peptide N termini. Taken together, these data illustrate the strong interdependence between the HLA class I molecule and the bound peptide.

Alleles↗
PubMed1999

Tetracycline-chelated Mg2+ ion initiates helix unwinding in Tet repressor induction.

The homodimeric tetracycline repressor (TetR) regulates resistance to the antibiotic tetracycline at the transcriptional level. TetR binds in the absence of Tc to palindromic operator sequences utilizing two helix-turn-helix (HTH) motifs. If the tetracycline-Mg2+ complex [MgTc]+ enters two identical binding tunnels buried within the TetR homodimer, a conformational change takes place, and the induced [TetR/[MgTc]+]2 complex releases operator DNA. To demonstrate the contribution of Mg2+ to [MgTc]+ binding and TetR induction, the Mg2+ concentration in the induced TetR homodimer was progressively reduced by addition of EDTA, resulting in two X-ray crystal structures of Mg2+-free and half-occupied TetR(D). Tc remains bound to the [MgTc]+-binding sites, despite the complete or partial absence of Mg2+. Together with inducer-free TetR(D), the structures were refined to between 2.2 and 2.7 A resolution and compared with fully induced TetR(D) in complex with two [MgTc]+. Each inducer binding tunnel has three constituent parts, one hydrophobic and two hydrophilic ones. One of the hydrophilic contact areas binds Tc by hydrogen bonding; the hydrophobic region correctly positions Tc and partially closes the entrance to the binding tunnel; the second hydrophilic region coordinates Mg2+, transduces the induction signal, and completes the process of closing the tunnel entrance. Tc confers binding specificity to TetR while Mg2+ is primarily responsible for induction: After binding to the imidazole Nepsilon of His100, Mg2+ is octahedrally coordinated to the 1,3-ketoenolate group of Tc and to three water molecules. One of these waters forms a hydrogen bond to the hydroxyl group Ogamma of Thr103. The induced 2.5 A movement of Thr103 results in the partial unwinding of helix alpha6, associated with a lateral shift of helices alpha4 and alpha9. They simultaneously close the tunnel entrance and cause the DNA-binding domains to adopt a nonbinding conformation, leading to release of operator DNA and expression of the genes responsible for resistance.

Chelating Agents↗
PubMed1999

Thermally induced aggregation of human transferrin receptor studied by light-scattering techniques.

The thermal stability of transferrin receptor isolated from human placenta in detergent-free solution has been investigated by static light-scattering and photon correlation spectroscopy. In detergent-free solution at 293.2 K, human transferrin receptor (hTfR) forms stable associates with a hydrodynamic radius of 16 nm. With increasing temperature the particles get more compact, above 340 K a phase transition takes, place and spontaneous aggregation of the receptor occurs. Under these conditions large clusters are formed that lead to fractal aggregates, coexisting with dendritic crystalline structures. The experimental findings are compatible with a model, which involves a reaction limited cluster-cluster aggregation mechanism in conjunction with a nucleation process. The molar enthalpy change associated with the phase transition was determined to be (1860 +/- 150) kJ/mol(-1) at a transition temperature of (341.3 +/- 0.2) K.

Biophysical Phenomena↗
PubMed1999

Crystallization and preliminary X-ray diffraction studies of Streptococcus pyogenes plasmid pSM19035-encoded omega transcriptional repressor.

The transcriptional repressor, omega protein, from the Streptococcus pyogenes broad-host-range plasmid pSM19035 was crystallized at pH 7. 5 and 8.5 by the vapour-diffusion method using PEG 4000 as precipitant. Two crystal forms were obtained; the first belongs to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 and the second to the hexagonal space group P6(1) or P6(5). The crystals are most likely to contain one omega protein in the asymmetric unit, with V(m) values of 3.2 and 3.5 A(3) Da(-1), respectively. The crystals diffract X-rays to 2.4 and 2.9 A resolution for the tetragonal and hexagonal systems, -respectively.

Bacterial Proteins↗
PubMed1999

Light scattering studies on supersaturated protein solutions.

The difficulties associated with protein crystallization is a major obstacle in modern structural biology. The increasing demand from the protein crystal structure community for quick and non-invasive experimental techniques as well as the rapid progress of modern optics and electronics have led during the past decade to a considerable expansion of the laser light scattering techniques. The latter are now very often employed to elucidate the aggregation kinetics of supersaturated protein solutions and the mechanism(s) underlying the early nucleation stages. The experimental verification of the nucleation processes, the prediction of the effective interaction potentials and the development of appropriate diagnostics schemes are discussed.

Crystallization↗
PubMed1998

Accessing lysozyme nucleation with a novel dynamic light scattering detector.

Dynamic light scattering was employed to investigate the behaviour of nucleating hen egg-white lysozyme solutions. For these studies we employed a novel fiber-optic based microprobe that suppresses multiple light scattering and contributions from large clusters in the spectra by backscattering detection. The time evolution of small lysozyme clusters is found to obey classical nucleation at the initial stages of the reaction.

Animals↗
PubMed1998

Crystallization and preliminary X-ray studies of purine nucleoside phosphorylase from Cellulomonas sp.

The commercially available enzyme purine nucleoside phosphorylase (PNP) from Cellulomonas sp. was purified by ion--exchange chromatography, partially sequenced and crystallized in two different crystal forms using the hanging-drop vapour-diffusion technique. Crystal form A grows as polyeders and/or cubes in the cubic space group P4232 with unit-cell dimension a = 162.5 A. Crystal form B appears as thick plates in the space group P212121 with unit-cell dimensions a = 63.2, b = 108.3 and c = 117.4 A. Both crystal forms contain three monomers (one trimer) in the asymmetric unit.

Bacterial Proteins↗
PubMed1998

Glycosylation of bovine pulmonary angiotensin-converting enzyme modulates its catalytic properties.

To study the role of the oligosaccharide moiety in the catalytic properties of angiotensin-converting enzyme (ACE), we obtained asialo- and partially deglycosylated ACE by enzymatic treatment of two-domain somatic enzyme from bovine lung. Treated enzymes demonstrated appreciable, but different changes of catalytic properties in the reaction of the hydrolysis of N-substituted tripeptides, C-terminal analogs of angiotensin I and bradykinin among them, compared to those for native enzyme. Deglycosylation also altered the catalytic properties of a single N domain of bovine ACE. So, various patterns of glycosylation modulate substrate specificity of somatic ACE and may be the reason for functional heterogeneity of the enzyme.

Animals↗
PubMed1998

A common ancestor for oxygenic and anoxygenic photosynthetic systems: a comparison based on the structural model of photosystem I.

The 4 A structural model of photosystem I (PSI) has elucidated essential features of this protein complex. Inter alia, it demonstrates that the core proteins of PSI, PsaA and PsaB each consist of an N-terminal antenna-binding domain, and a C-terminal reaction center (RC)-domain. A comparison of the RC-domain of PSI and the photosynthetic RC of purple bacteria (PbRC), reveals significantly analogous structures. This provides the structural support for the hypothesis that the two RC-types (I and II) share a common evolutionary origin. Apart from a similar set of constituent cofactors of the electron transfer system, the analogous features include a comparable cofactor arrangement and a corresponding secondary structure motif of the RC-cores. Despite these analogies, significant differences are evident, particularly as regards the distances between and the orientation of individual cofactors, and the length and orientation of alpha-helices. Inferred roles of conserved amino acids are discussed for PSI, photosystem II (PSII), photosystem C (PSC, green sulfur bacteria) and photosystem H (PSH, heliobacteria). Significant sequence homology between the N-terminal, antenna-binding domains of the core proteins of type-I RCs, PsaA, PsaB, PscA and PshA (of PSI, PSC and PSH respectively) with the antenna-binding subunits CP43 and CP47 of PSII indicate that PSII has a modular structure comparable to that of PSI.

Amino Acid Sequence↗
PubMed1998

Crystal structure of the ternary complex of E. coli purine nucleoside phosphorylase with formycin B, a structural analogue of the substrate inosine, and phosphate (Sulphate) at 2.1 A resolution.

The ternary complex of purine nucleoside phosphorylase from E. coli with formycin B and a sulphate or phosphate ion crystallized in the hexagonal space group P6122 with unit cell dimensions a=123.11, c=241.22 A and three monomers per asymmetric unit. The biologically active hexamer is formed through 2-fold crystallographic symmetry, constituting a trimer of dimers. High-resolution X-ray diffraction data were collected using synchrotron radiation (Daresbury, England). The crystal structure was determined by molecular replacement and refined at 2.1 A resolution to an R-value of 0.196. There is one active centre per monomer, composed of residues belonging to two subunits of one dimer. The phosphate binding site is strongly positively charged and consists of three arginine residues (Arg24, Arg87 and Arg43 from a neighbouring subunit), Ser90 and Gly20. It is occupied by a sulphate or phosphate anion, each oxygen atom of which accepts at least two hydrogen bonds or salt-bridges. The sulphate or phosphate anion is also in direct contact with the ribose moiety of formycin B. The ribose binding site is composed of Ser90, Met180, Glu181 and His4, the latter belonging to the neighbouring subunit. The base binding site is exposed to solvent, and the base is unspecifically bound through a chain of water molecules and aromatic-aromatic interactions. In all monomers the nucleosides are in the high syn conformation about the glycosidic bonds with chi in the range 100 to 130 degrees. The architecture of the active centre is in line with the known broad specificity and the kinetic properties of E. coli PNP.

Animals↗
PubMed1998

Conformational changes of the Tet repressor induced by tetracycline trapping.

The X-ray crystal structure analysis of inducer-free Tet repressor, TetR, at 2.4 A resolution identifies one of two openings of the tunnel-like binding site as the entrance for the inducer tetracycline-Mg2+, [Mg Tc]+. Recognition and binding of the inducer unleashes conformational changes leading to the induced state of TetR. In the first step, the C-terminal turn of alpha-helix 6 unwinds, thereby altering the orientation of alpha-helix 4. This different orientation of alpha-helix 4 is stabilized by a series of hydrogen bonds mediated through a chain of eight water molecules. The alpha-helix 4 connects the DNA-binding domain (alpha-helices 1 to 3) to the rigid TetR core, and thus regulates gene expression through its respective orientations.

Amino Acid Sequence↗