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R Boelens

Publications and source records attributed to R Boelens.

At least 127 records · Page 7Linked to original sources
PubMed1989

The amino-terminal domain of LexA repressor is alpha-helical but differs from canonical helix-turn-helix proteins: a two-dimensional 1H NMR study.

The amino-terminal DNA binding domain of LexA repressor consisting of 84 amino acid residues has been studied by two-dimensional 1H NMR. Sequence-specific 1H resonance assignments were made for the first 60 amino acid residues. The secondary structure of this part of the protein contains three alpha-helices in the peptide segments 8-20, 28-35, and 41-54. The last helix has a distortion around residues 47-48. The peptide segment 28-47 shows weak homology with other helix-turn-helix proteins. To investigate the spatial structure of this region of the molecule distance-geometry calculations were performed based on proton-proton distance constraints from nuclear Overhauser effects. The resulting structure shows that the segment 28-47 contains two helices with a loop region between them. The relative orientation of the two helices is similar to that found in helix-turn-helix proteins, but the helices are further apart, with the phenyl ring of Phe-37 located between them. The Brookhaven Protein Data Bank was searched for structurally homologous peptide segments in other proteins. The result of this search was that the two-helical structure of LexA is not more closely related to the canonical helix-turn-helix motif than it is to similar substructures found in other classes of proteins.

Amino Acid Sequence↗
PubMed1988

Photo-CIDNP study of the interaction between lac repressor headpiece and lac operator DNA.

Lac repressor headpiece (HP) and intact lac repressor have been studied using the photo-CIDNP method. At neutral pH histidine 29, tyrosines 7, 12 and 17 and methionine 1 are polarised. His-29 polarizations are weaker and broader in HP59 than in HP51 indicating that the C-terminal octapeptide in HP59 adopts a conformation that allows an interaction with His-29. The photo-CIDNP spectra of intact lac repressor and HP51 are very similar, showing that the same residues are accessible to the photo-excited flavin. An equimolar mixture of HP51 and a 14 base pair lac operator fragment strongly suppresses the photo-CIDNP effect of tyrosines 7 and 17 and abolishes the His-29 polarizations. The results are compared with earlier photo-CIDNP measurements on a complex of headpiece with poly[d(AT)] and with a model derived from a 2D NMR study on a lac headpiece-operator complex.

DNA, Bacterial↗
PubMed1988

Secondary structure and hydrogen bonding of crambin in solution. A two-dimensional NMR study.

The secondary structure of crambin in solution has been determined using two-dimensional NMR and is found to be essentially identical to that of the crystal structure. The H-D exchange of most amide protons can be accounted for in terms of the hydrogen bonds found in the X-ray structure. Exceptions are the amide protons of Cys-4 and Ser-6, which exchange more slowly than expected, and of Asn-46 for which the exchange is faster. These results might be explained by a slightly different conformation of the C-terminal region of the protein in solution. The slow exchange of the amides of Cys-32 and Glu-23 might be due to aggregation involving an extremely hydrophobic part of the protein in solution.

Hydrogen Bonding↗
PubMed1988

The interaction of lac repressor headpiece with its operator: an NMR view.

Analysis of nuclear Overhauser enhancements in two-dimensional NMR spectra of the complex of lac repressor headpiece with a 14 base pair lac operator fragment shows that the second helix of the headpiece binds in the major groove of DNA, as has been suggested. The orientation of this helix is approximately 180 degrees different from the proposed models and from that found in the X-ray structure of the 434 repressor-operator complex. The model of the lac headpiece-operator complex provides a good explanation for a large amount of biochemical, genetic and physical data.

Amino Acid Sequence↗
PubMed1987

1H NMR study of the exchangeable protons of the duplex d(GCGTTGCG).d(CGCAACGC) containing a thymine photodimer.

A comparison is presented of the imino proton NMR spectra of the double stranded octamer d(GCGTTGCG).d(CGCAACGC) and the same octamer in which the two central thymine residues occur as a cis-syn thymine dimer. Except for the terminal base pairs all imino protons were detected and assigned in the NMR spectrum. The spectra show that in the thymine dimer duplex, contrary to common belief, all base pairs occur in a hydrogen bonded form, although the hydrogen bonds of the two central AT base pairs are substantially weakened. The melting temperature decreases about 13 degrees C on thymine dimer formation.

Imines↗
PubMed1987

Complex of lac repressor headpiece with a 14 base-pair lac operator fragment studied by two-dimensional nuclear magnetic resonance.

Two-dimensional nuclear Overhauser enhancement spectra are presented of the complex of lac repressor headpiece with a 14 base-pair lac operator fragment. Analysis of nuclear Overhauser enhancements observed between protein and DNA shows that the second helix of the headpiece ("the recognition helix") binds in the major groove of DNA as has been suggested, but that the orientation of this helix is approximately 180 degrees different from the proposed models.

Bacterial Proteins↗
PubMed1987

Conformational changes in the oligonucleotide duplex d(GCGTTGCG) x d(CGCAACGC) induced by formation of a cis-syn thymine dimer. A two-dimensional NMR study.

In order to obtain insight into the repair mechanism of DNA containing thymine photo-dimer, the conformation of the duplex d(GCGTTGCG) x d(CGCAACGC) with a thymine dimer incorporated has been studied by proton NMR and the results are compared with NMR data of the parent octamer. Two-dimensional nuclear Overhauser enhancement (2D NOE) spectroscopy and two-dimensional homonuclear Hartmann-Hahn spectroscopy have been applied to assign all the non-exchangeable base protons and most of the deoxyribose protons of both duplexes. From these experiments it is clear that indeed a cis-syn cyclobutane-type thymine photodimer is formed by the irradiation of this oligonucleotide with ultraviolet light. Comparison of 2D NOE spectra and the 1H chemical shifts of the damaged and the intact DNA duplexes reveals that formation of a thymine dimer induces small distortions of the B-DNA structure, the main conformational change occurring at the site of the thymine dimer.

Binding Sites↗
PubMed1987

Two-dimensional 1H NMR study of two cyclobutane type photodimers of thymidylyl-(3'----5')-thymidine.

2D NMR spectroscopy and J coupling constant analysis are applied to resolve the structure of two photoproducts of thymidylyl-(3'----5')-thymidine. These products are cyclobutane type thymine dimers possessing the cis-syn (the predominant one) and trans-syn geometry. The cis-syn is formed in an ANTI-ANTI conformation about the N-glycosyl linkages and resembles the normal base-stacked configuration. The glycosidic conformation in solution of the 5' terminal fragment differs from the crystal in which the less common SYN conformation is observed. In this isomer only the sugar pucker of the 3' terminal fragment is changed substantially with respect to the dinucleotide. The trans-syn isomer is formed in a SYN-ANTI glycosidic conformation. In this isomer the sugar puckers of both deoxyribose rings are affected and a preference for a pure 2'-endo conformation is observed.

Cyclobutanes↗
PubMed1987

Conformational analysis of a cyclic thymopoietin-analogue by 1H n.m.r. spectroscopy and restrained molecular dynamics simulations.

The internuclear distances of the cyclic thymopoietin derivative c[D-Val-Tyr-Arg-Lys-Glu] have been determined using two-dimensional nuclear Overhauser n.m.r. spectroscopy. These distances are used as constraints for a restrained Molecular Dynamics (MD) simulation. The two starting structures used for the calculations consist of a beta and gamma turn for model 1 and two gamma turns for model 2. The rms difference in atomic positions of the two conformations is 0.242 nm. They converge during the restrained MD simulation to the same final structure. The positional rms difference of the time averaged (5-14 ps) conformations is 0.011 nm. The hydrogen bond pattern is similar to that of model 1, but in addition we find three more gamma turns. The vicinal NH-C alpha H couplings agree well with those calculated from the time averaged structures.

Magnetic Resonance Spectroscopy↗
PubMed1986

Kringle 4 from human plasminogen: a proton magnetic resonance study via two-dimensional photochemically induced dynamic nuclear polarization spectroscopy.

Two-dimensional (2D) proton magnetic resonance techniques used in conjunction with laser photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy have been applied to studying the kringle 4 domain from human plasminogen at 360 MHz. Out of 11 potential CIDNP-sensitive aromatic side chains, only 5 (His3, Tyr41, Tyr50, Trp72, and Tyr74) appear to be accessible to 3-(carboxymethyl)lumiflavin, the dye used to photogenerate spin polarization. Of these, Trp72 and Tyr74 are known to be at, or near, the lysine-binding site. The spin-spin scalar (J) and phase-sensitive dipolar (Overhauser) connectivities in the 2D experiments yield absolute assignments for the aromatic signals stemming from the exposed tyrosyl and tryptophanyl rings. Moreover, a number of side-chain H beta resonances can be identified and assigned to specific types of aromatic amino acid residues.

Amino Acid Sequence↗
PubMed1986

Photochemically induced dynamic nuclear polarisation NMR study of the aromatic residues of sea-anemone polypeptide cardiac stimulants.

One-dimensional and two-dimensional photochemically induced dynamic nuclear polarisation (photo-CIDNP) nuclear magnetic resonance spectra have been recorded for the sea-anemone polypeptide cardiac stimulants anthopleurin-A and Anemonia sulcata toxins I and II. In anthopleurin-A and toxin II, all three Trp residues are accessible to the flavin dye, although Trp-23 in anthopleurin-A shows a weaker photo-CIDNP response than Trp-33 and Trp-45. Tyr-25 in anthopleurin-A also shows a strong response. In toxin I, Trp-23, Trp-33 and Tyr-45 (which replaces Trp in this molecule) are accessible to the dye. The pH dependences of the photo-CIDNP spectra of all three polypeptides have been examined. The response of Trp-33 increases significantly with pH. The two His residues of anthopleurin-A and toxin II display a response in their imidazole forms, but not their imidazolium forms. The surface accessibilities of Trp-23 and Trp-33 are discussed in relation to the interaction of these polypeptides with the Na+ channel.

Animals↗
PubMed1985

A protein structure from nuclear magnetic resonance data. lac repressor headpiece.

A procedure is described to determine the three-dimensional structure of biomolecules from nuclear magnetic resonance data. This procedure combines model building with a restrained molecular dynamics algorithm, in which distance information from nuclear Overhauser effects is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or headpiece (amino acid residues 1 to 51) of the lac repressor from Escherichia coli, for which no crystal structure is available. The relative orientation of the three helices of the headpiece is similar to that of the three homologous helices found in the cI repressor of bacteriophage lambda.

Amino Acid Sequence↗
PubMed1985

Hydrogen exchange of individual amide protons in the E. coli lac repressor DNA-binding domain: a nuclear magnetic resonance study.

Proton exchange in lac repressor headpiece was studied by COSY and 2D NOE spectroscopy. The exchange rates of amide protons, stabilized by the hydrogen bonds of the three alpha-helices of the headpiece, could be determined quantitatively. The exchange rates in these helices showed repetitive patterns of about three to four residues. A correlation with the position of the amide proton in the interior or the exterior of the alpha-helix of the protein was found. The exchange data strongly support the validity of the three-dimensional structure, as determined recently (Kaptein, R. et al., J. Mol. Biol. 182, 179-182 (1985)).

DNA-Binding Proteins↗
PubMed1984

Spatial arrangement of the three alpha helices in the solution conformation of E. coli lac repressor DNA-binding domain.

The relative orientations of the 3 helices in the DNA-binding domain ('headpiece') of lac repressor have been determined using distance constraints obtained from 2-dimensional 1H nuclear Overhauser enhancement spectra. The relative orientations of its helices is similar to that of the central 3 helices in the DNA-binding domain of the lambda repressor of the bacteriophage lambda.

DNA, Bacterial↗
PubMed1984

The use of two-dimensional nuclear-magnetic-resonance spectroscopy and two-dimensional difference spectra in the elucidation of the active center of Megasphaera elsdenii flavodoxin.

1H-1H 'through bond' correlated (COSY) and 1H-1H 'through space' (NOESY) two-dimensional NMR techniques were applied to study the structure of Megasphaera elsdenii flavodoxin in the oxidized and reduced state. It is shown that two-dimensional NOESY difference spectra between spectra of flavodoxin in the reduced and semiquinone state are sensitive to the active center of the fully reduced state. The sphere of the active center observed in the difference spectra can be varied easily by changing the relative amount of flavodoxin semiquinone in the second sample. The difference NOESY spectra simplified the analysis of the complex spectra. Resonances could be assigned to Ala-56, Tyr-89 and Trp-91, which are located in the direct vicinity of the protein-bound flavin. The relative positions and side-chain dihedral angles of these residues are compared for the two redox states. Ala-56 and Tyr-89 show identical relative positions and dihedral angles in the two redox states, although the rotational motion of Tyr-89 is enhanced in the oxidized state. In both redox states Trp-91 is immobilized and extremely close to the prosthetic group. However, a small displacement of Trp-91 towards the (N(5) atom of the flavin occurs upon reduction. The results obtained for Trp-91 are in excellent agreement with crystallographic results of the related flavodoxin from Clostridium MP. However, the latter studies showed a somewhat different position of the tyrosine residue compared with our results.

Amino Acids↗
PubMed1984

The cytochrome c oxidase-azide-nitric oxide complex as a model for the oxygen-binding site.

The complex of cytochrome c oxidase with NO and azide has been studied by EPR at 9.2 and 35 GHz. This complex which shows delta ms = 2 EPR triplet and strong anisotropic signals, due to the interaction of cytochrome a2+3 X NO (S = 1/2) and Cu2+B (S = 1/2), is photodissociable . Its action spectrum is similar to that of cytochrome a2+3 X NO with bands at 430, 560 and 595 nm, but shows an additional band in the near ultraviolet region. The quantum yield of the photodissociation process of cytochrome a2+3 X NO in the metal pair appears to depend on the redox state of CuB. When the photolysed sample was warmed to 77 K, a complex was observed with the EPR parameters of cytochrome a3+3 - N-3 - Cu1 +B (S = 1/2). This process of electron and ligand transfer can be reversed by heating the sample to 220 K. It is suggested that in the triplet species azide is bound to Cu2+B whereas NO is bridged between Cu2+B and the haem iron of the cytochrome a2+3. The complex has a triplet ground state and a singlet excited state with an exchange interaction J = -7.1 cm-1 between both spins. The anisotropy in the EPR spectra is mainly due to a magnetic dipole-dipole interaction between cytochrome a2+3 X NO and Cu2+B. From simulations of the triplet EPR spectra obtained at 9 and 35 GHz, a value for the distance between the nitroxide radical and Cu2+B of 0.33 nm was found. A model of the NO binding in the cytochrome a3-Cu pair shows a distance between the haem iron of cytochrome a3 and CuB of 0.45 nm. It is concluded that the cytochrome a3-CuB pair forms a cage in which the dioxygen molecule is bidentate coordinated to the two metals during the catalytic reaction.

Azides↗