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.

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.

Surface accessibility of aromatic residues in human lysozyme using photochemically induced dynamic nuclear polarization NMR spectroscopy.

Resonances in the photo-CIDNP spectrum of human lysozyme have been assigned to specific spin systems despite extensive spectral overlap using the two-dimensional photo-CIDNP COSY experiment. Five of the 12 tyrosine, tryptophan and histidine residues of human lysozyme are found to be accessible to flavin dye in solution. This result is in good agreement with surface accessibility calculations carried out on the human lysozyme crystal structure.

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.

Interaction of beta-endorphin with sodium dodecyl sulfate in aqueous solution. 1H-NMR investigation.

1H-NMR spectra at 600 MHz and 270 MHz and photo-chemically induced dynamic nuclear polarization (photo-CIDNP) spectra at 360 MHz of beta-endorphin in the presence of sodium dodecyl sulfate (SDS) micelles are reported and discussed in terms of structural changes and immobilization upon binding. On addition of micelles several NH protons show slow H-D exchange rate in 2H2O at p2H 4.6, 25 degrees C, which indicates that some regions of the polypeptide are buried and shielded from the direct interaction with the solvent.

Side-chain cleavage of C27-3-oxo-4-ene sterols.

In this study various C27 sterols with a 3-oxo-4-ene structure were incubated with adrenal cortex mitochondrial preparations. (22R)-22-Hydroxy-4-cholesten-3-one and (20R,22R)-20,22-dihydroxy-4-cholesten-3-one were found to be converted into progesterone. This suggests the existence of a pathway for adrenal progesterone formation analogous to the normal 3 beta-hydroxy-5-ene pathways.

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.

Sequential resonance assignments in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy.

A sequential assignment procedure is outlined, based on two-dimensional NOE ( NOESY ) and two-dimensional J-correlated spectroscopy ( COSY ), for assigning the nonexchangeable proton resonances in NMR spectra of oligonucleotides. As presented here the method is generally applicable to right-handed helical oligonucleotides of intermediate size. We applied it to a lac operator DNA fragment consisting of d( TGAGCGG ) and d( CCGCTCA ) and obtained complete assignments for the adenine H8, guanine H8, cytosine H6 and H5, thymine H6 and 5-methyl, and the deoxyribose H1', H2', H2", H3', and H4' resonances, as well as some H5', H5" (pairwise) assignments.

Determination of the solvent accessibility of specific aromatic residues in gamma-crystallin by photo-CIDNP NMR measurements.

The surface or solvent accessibility of certain individual aromatic residues of calf-gamma II crystallin in solution (1 mM) were measured by the dramatic intensity enhancements of NMR lines generated by the interactions of cyclic radical pair formation of the 3-N-carboxymethyl lumiflavin (flavin I) dye excited (488nm) by an argon laser (5 watts) with the protein. This effect is called photo-chemically induced dynamic nuclear polarization: photo-CIDNP. The "light" and "dark" NMR spectra were taken in alternating scans in the pulsed Fourier transform mode on a Bruker 360 MHz instrument.

Sequence-specific resonance assignments in the 1H nuclear-magnetic-resonance spectrum of the lac repressor DNA-binding domain 1-51 from Escherichia coli by two-dimensional spectroscopy.

The assignment of the 1H nuclear magnetic resonance (NMR) spectrum of the DNA-binding domain 1-51 of lac repressor from Escherichia coli is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional NMR experiments at 360 MHz and 500 MHz. Individual assignments were obtained at 18 degrees C for the backbone protons of 44 out of the total of 51 amino acids residues, the exceptions being Met-1, Lys-2, Tyr-7, Arg-35, Glu-36, Lys-37 and Ile-48.

Secondary structure of the lac repressor DNA-binding domain by two-dimensional 1H nuclear magnetic resonance in solution.

A recently proposed approach for spatial structure determination in noncrystalline proteins by nuclear magnetic resonance was applied to the lac repressor DNA-binding domain. On the basis of sequence-specific 1H NMR assignments, the location of alpha-helices in the amino acid sequence was determined from nuclear Overhauser enhancement data and from amide proton exchange studies. These investigations provide detailed experimental data on the structure of a noncrystalline DNA-binding protein.

In vivo 31P and 13C nuclear magnetic resonance studies of acetate metabolism in Chromatium vinosum.

31P and 13C nuclear magnetic resonance (NMR) experiments were performed on suspensions of the phototrophic bacterium Chromatium vinosum incubated anaerobically in the dark. 31P NMR spectra revealed that during prolonged dark incubation high ATP levels are maintained. This phenomenon was independent of the presence of the energy reserves polyglucose and polyphosphate.

Investigation by photochemically-induced dynamic nuclear polarization and nuclear Overhauser enhancement 1H-NMR of the interaction between beta-endorphin and phospholipid micelles.

The photochemically-induced dynamic nuclear polarization technique has been used to investigate the access of a photoexcited flavin dye to tyrosyl and histidyl residues in [Met]enkephalin and human and camel beta-endorphins, both alone and in the presence of n-dodecylphosphorylcholine micelles. The results indicate that the mode of binding of Tyr-1, but not of residue 27, is similar in the two endorphins and differs from that of Tyr-1 in [Met]enkephalin. In human beta-endorphin, accessibility and mobility of Tyr-27 are strongly reduced in the presence of lipid at physiological pH, whereas in camel beta-endorphin His-27 becomes immobilized only at high pH.

NMR study of the interaction between the lac repressor and the lac operator.

Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy. Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated. The signals of their hydrogen-bonded imino protons were well resolved in the 500-MHz NMR spectra.

Photo-CIDNP 1H-NMR studies of bovine pancreatic phospholipase A2 and its zymogen.

Bovine pancreatic phospholipase A2 and its zymogen were studied by laser photo-CIDNP 1H-NMR. Resonances of Trp3 and Tyr69 protons of the two proteins were assigned. By varying the delay between a short light pulse and the observation pulse, time dependencies of the CIDNP signals were obtained from which effective T1 values could be derived.

Proton nuclear magnetic resonance assignments and surface accessibility of Tryptophan residues in lysozyme using photochemically induced dynamic nuclear polarization spectroscopy.

Tryptophan resonances in the 360-MHz 1H photochemically induced dynamic nuclear polarization spectrum of hen egg white lysozyme are investigated in detail. All resonances of one tryptophan and six of another are identified and assigned to their respective protons. The methods employed, all involving nuclear spin polarization, include the study of cross-relaxation effects and the use of selective radio-frequency irradiation, Gd3+ as a paramagnetic probe, and riboflavin as the chemically induced dynamic nuclear polarization generating dye.

lac Repressor headpiece binds specifically to half of the lac operator: a proton nuclear magnetic resonance study.

The complex formation of the N-terminal domain (headpiece) of the Escherichia coli lac repressor and a synthetic 14-base-pair lac operator fragment has been investigated by 1H NMR. Titration shifts in the imino-proton region of the DNA spectrum and in the aromatic region of the headpiece spectrum are examined in detail and interpreted where possible. The assignment of the resonances in the complex follows in part from the titration data and is completed by nuclear Overhauser measurements.

A photo-CIDNP study of the active sites of Megasphaera elsdenii and Clostridium MP flavodoxins.

Megasphaera elsdenii and Clostridium MP flavodoxins have been investigated by photo-CIDNP techniques. Using time-resolved spectroscopy and external dyes carrying different charges it was possible to assign unambiguously the resonance lines in the NMR-spectra to tyrosine, tryptophan and methionine residues in the two proteins. The results show that Trp-91 in M.

Carbon-13 nuclear magnetic resonance studies of acetate metabolism in intact cells of rhodopseudomonas sphaeroides.

13C-nuclear magnetic resonance was used to study the metabolism of [2-(13)C]acetate in suspensions of Rhodopseudomonas sphaeroides. In the dark, in logarithmic-phase cells the 13C label appeared first in butyrate C-2 and C-4 and subsequently in glutamate C-4 and succinate C-2 and C-3. In the light, synthesis of poly(beta-hydroxybutyrate) (PHB) takes place.

Limited trypsinolysis of porcine and equine colipases. Spectroscopic and kinetic studies.

Porcine and equine colipases have been submitted to mild tryptic digestion. Proteolysis occurs at the Arg5-Gly6 bond with the loss of the N-terminal pentapeptide. Studies of native and trypsin-treated colipases by circular dichroism and laser chemically induced dynamic nuclear polarization indicate that proteolysis induces conformational changes in the region of the tyrosine cluster.

1H NMR studies of lac-operator DNA fragments.

The hydrogen-bonded imino protons of a 14 base pair double-stranded DNA fragment comprising one half of the lac operator of E. coli were investigated by 360 MHz H NMR. From combined melting studies of this synthetic 14 b.

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.