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R Kaptein

Publications and source records attributed to R Kaptein.

195 records · Page 11Linked to original sources
PubMed1981

31P-Nuclear magnetic resonance and freeze-fracture electron microscopic studies on reconstituted bacteriorhodopsin vesicles.

Bacteriorhodopsin has been reconstituted into egg-phosphatidylcholine vesicles by various methods. The resulting preparations have been analyzed on density gradients and by freeze-fracture electron microscopy. The homogeneity of the vesicle preparations and the light-induced intravesicular pH changes have been studied by 31P-NMR, using glucose 6-phosphate as pH probe. It is concluded that bacteriorhodopsin is incorporated in the inside-out mode in vesicles up to about 100 nm. Above this diameter, more or less random insertion takes place.

Bacteriorhodopsins↗
PubMed1980

1H-NMR and photochemically-induced dynamic nuclear polarization studies on bovine pancreatic phospholipase A2.

Proton-NMR resonances of trytophan 3 and tyrosine 69 in bovine pancreatic phospholipase A2, its pro-enzyme and in Ala1-transaminated protein were assigned using photochemically-induced dynamic nuclear polarization (photo-CIDNP) as such or in combination with spin-echo measurements. In addition assignments were made by suppression of cross-relaxation effects using short (0.1 s) high-power laser pulses.

Animals↗
PubMed1980

Photo-CIDNP studies of the influence of ligand binding on the surface accessibility of aromatic residues in dihydrofolate reductase.

The surface accessibility of the histidine, tyrosine, and tryptophan residues of Lactobacillus casei dihydrofolate reductase has been determined from 360-MHz 1H photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR experiments. In the absence of ligands, four (or perhaps five) of the seven histidine residues and at least one of the four tryptophan residues are accessible to a flavin dye molecule. One of the five tyrosine residues is also slightly accessible. Of the accessible histidine residues, one becomes inaccessible on the binding of NADP+ and one on the binding of p-aminobenzoyl glutamate. These have been assigned to residues which interact directly with these two ligands. One histidine residue (probably His-22) shows an increase in accessibility on addition of folate or methotrexate to the enzyme . NADP+ complex. In addition, the binding of several ligands, notably trimethoprim, leads to an increase in the accessibility of a tryptophan residue. This is clear evidence for ligand-induced conformational changes in dihydrofolate reductase and allows us to identify some of the residues involved.

Folic Acid↗
PubMed1980

Laser photo-CIDNP detection of surface aromatic residues in dissociating bovine alpha-lactalbumin at submillimolar concentrations.

Surface aromatic residues of bovine alpha-lactalbumin were detected by laser photo-CIDNP NMR spectroscopy in the submillimolar (100 micro M) range. The association behavior of this protein was observed in this range (Kd approximately equal to 100 micro M) by the variation in absolute NMR peak intensities for surface polarized residues as the monomer-monomer association step lowered surface residue accessibilities. This example was also the first case reported for laser photo-CIDNP studies of proteins where all three polarizable aromatic residues (tyrosine, tryptophan, and histidine) were observed.

Animals↗
PubMed1980

Photochemically induced dynamic nuclear polarization investigation of complex formation of the NH2-terminal DNA-binding domain of lac repressor with poly[d(AT)].

The interaction of the NH2-terminal DNA-binding domain of lac repressor with synthetic oligo[d(AT)] was studied by a photo-CIDNP technique (CIDNP is chemically induced dynamic nuclear polarization). Three of the four tyrosines of the NH2-terminal region were found to be accessible to the photosensitive dye. The corresponding ring proton resonances were enhanced in the photo-CIDNP 1H NMR spectrum, and the only histidine (histidine 29) was located at the surface of the domain, which is supposed to be linked to the core protein of lac repressor by a flexible hinge region. After complex formation of the NH2-terminal region with oligo[d(AT)], two of the three tyrosine residues were no longer accessible to solvent or to photosensitive dye, which is strong evidence that the two tyrosines are part of the contact region.

Binding Sites↗
PubMed1979

The aromatic residues of bovine pancreatic ribonuclease studied by 1H nuclear magnetic resonance.

1. The aromatic proton resonances in the 360-MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessibly to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180 degrees about their C beta--C gamma bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.

Animals↗
PubMed1978

Light-induced pH changes inside bacteriorhodopsin vesicles as measured by 31 P NMR.

31P NMR has been used to measure light-induced pH changes inside bacteriorhodopsin vesicles containing entrapped sodium glucose-6-phosphate. Reversible light-induced pH changes were observed at various pH values. The results indicate that our vesicle preparations were not homogeneous with respect to the generation of pH gradients.

Bacteriorhodopsins↗
PubMed1978

A photo-CIDNP study of the interaction of oligonucleotides with gene-5 protein of bacteriophage M13.

It is shown that photo-CIDNP effects (CIDNP, chemically induced dynamic nuclear polarization) can be generated in the 360-MHz proton NMR spectrum of gene-5 protein from bacteriophage M13. This technique is used to determine the number of tyrosyl residues at the surface of the protein and to assign the resonances from the 3,5-ring protons of these residues. The DNA-binding site of the protein is investigated by formation of complexes with oligonucleotides. Complex formation leads to shifting and/or quenching of the photo-CIDNP emission signals of the surface tyrosines, implying that they are involved in DNA-protein interaction. These experiments are complemented by studying the complex formation of Lys-Tyr-Lys to poly(A).

Binding Sites↗
PubMedDate not supplied

NMR studies of interactions between inhibitors and porcine pancreatic phospholipase A2.

Two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2, bound to a micellar lipid-water interface of fully deuterated dodecylphosphocholine, with competitive inhibitors derived from the following general structure: [formula: see text] X and Y are alkyl chains with various 'reporter groups'. The interactions between the inhibitor and the enzyme were localized by comparison of 2-D nuclear Overhauser effect spectra using protonated and selectively deuterated inhibitors, and inhibitors with groups having easily identifiable chemical shifts. These experiments led us to the following conclusions for the phospholipase A2/inhibitor/micelle complex: i) the His48 C2 ring proton is in close proximity to both the amide proton and the methylene protons at the sn-1 position of the glycerol skeleton of the inhibitor, ii) the acyl chain of the inhibitor at the sn-2 position makes hydrophobic contacts near Phe5, Ile9, Phe22 and Phe106; iii) no interactions between the acyl chain at the sn-1 position and the protein could be identified. Comparison of our results on the enzyme/inhibitor/micelle ternary complex with the crystal structure of the enzyme-inhibitor complex shows that the mode of inhibitor binding is similar. However, in several cases we found indications that the hydrophobic chains of the inhibitors can have multiple conformations.

Animals↗
PubMedDate not supplied

Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: the lac repressor DNA binding domain.

A procedure is described to determine from NMR data the three-dimensional structure of biomolecules. This procedure combines model building with a restrained Molecular Dynamics algorithm, in which distance information from NOEs is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or "headpiece" (amino acids 1-51) of the lac repressor from E. coli, for which no crystal structure is available. The spatial structure of the headpiece is discussed in terms of known physical and biochemical data and of its DNA binding properties.

Binding Sites↗