Two-dimensional 1H-nmr studies on the lac repressor DNA binding domain: further resonance assignments and identification of nuclear Overhauser enhancements.
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DNA↗
Biomedical subjects
R M Scheek
Publications and source records attributed to R M Scheek.
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↗
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↗
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↗
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. These assignments are required for the analysis of two-dimensional NOE and J-coupling data in terms of the solution structure of oligonucleotides.
Hydrogen Bonding↗
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. The shift of the His-29 C-2 resonance has been used to assess the binding strength of the complex. Evidence is presented for the presence of a high-affinity site on the lac operator fragment (KD less than or equal to 2 X 10(-5) M), which shows features in common with one of the specific binding sites on the complete lac operator, and for the presence of a second, nonspecific binding site with lower affinity. The influence of this second site on the interpretation of the binding data is discussed.
Escherichia coli↗
Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle.
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Ammonium Sulfate↗
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.p. fragment and its two constituent 7 b.p. fragments a nearly complete assignment for the low-field proton resonances was obtained. The experimental spectra are compared with calculated spectra and with the spectrum of a 51 b.p. DNA restriction fragment from E. coli containing the complete lac operator. Structural information on these oligonucleotides is presented. This study is a prerequisite for future 1H NMR investigations of the interaction of the lac operator with the lac repressor.
DNA, Bacterial↗
Subunit interactions in glyceraldehyde-3-phosphate dehydrogenases. Their involvement in nucleotide binding and cooperativity.
1. The hybridization of rabbit muscle and yeast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) was used to study the involvement of subunit interactions in NAD+ and NADH binding by these enzymes. 2. In the presence of 1 mM NAD+ or NADH no hybrid formation was observed with our preparations of the two enzymes. 3. The inhibition by NADH of the hybrid formation is shown to be a consequence of an unfavourable equilibrium of the hybridization process in the presence of NADH. 4. The inhibition by NAD+ of the hybrid formation is shown to be a consequence of both a shift in the equilibrium, as in the case of NADH, and a decrease in the rate of the dissociation of the enzymes. 5. The dimer of the yeast enzyme binds NAD+ or NADH with equal affinity irrespective of whether it is combined with another yeast dimer in the yeast tetramer or with a rabbit muscle dimer in the hybrid. 6. The binding of NAD+ and NADH to the dimer of the rabbit muscle enzyme is stronger in the rabbit muscle tetramer than in the hybrid; this explains the shift in the equilibrium of the hybridization process caused by these nucleotides. 7. Alkylation of the rabbit muscle enzyme with iodoacetate does not influence the hydridization process in the absence of nucleotides. 8. After alkylation of the rabbit muscle enzyme NADH cannot cause a large shift in the equilibrium of the hybridization process. 9. In accordance with this it was found that the binding of NADH (and NAD+) to the rabbit muscle enzyme is weakened by alkylation, whereas the binding of NADH to the alkylated rabbit muscle subunits is not affected strongly by the hydridization. 10. An attempt is made to combine the effects of nucleotides on the hybridization properties of the yeast enzyme and the alkylated or unalkylated rabbit muscle enzymes with the binding properties of all tetrameric species involved in the hybridization processes in a thermodynamic description of nucleotide binding and subunit interactions.
Animals↗
Resolution of specific histidine resonances in the 360 MHz 1H NMR spectrum of glyceraldehyde-3-phosphate dehydrogenase, a 145 000 molecular weight protein, by photo-CIDNP.
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Affinity Labels↗
Subunit interactions in rabbit-muscle glyceraldehyde-phosphate dehydrogenase, as measured by NAD+ and NADH binding.
1. The binding parameters for NADH and NAD+ to rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been measured by quenching of the flourescence of the protein and the NADH. 2. The fact that the degree of protein fluorescence quenching by bound NAD+ or NADH, excited at 285 nm and measured at 340 nm ('blue' tryptophans), is not linearly related to the saturation functions of these nucleotides, leads to a slight overestimation of the interaction energy and an underestimation of the concentration of sites, if linearity is assumed. 3. This is also the case for NADH, but not for NAD+, when the protein fluorescence is excited at 305 nm and measured at 390 nm ('red' tryptophans). 4. The binding of NAD+ can be described by a model in which the binding of NAD+, via negative interactions within the dimer, induces weaker binding sites, with the result that the microscopic dissociation constant is 0.08 microM at low saturation and 0.18 microM for the holoenzyme. 5. The binding of NADH can be described on the basis of the same model, the dissociation constant at low saturation being 0.5 microM and of the holoenzyme 1.0 microM. 6. The fluorescence of bound NADH is not sensitive to the conformational changes that cause the decrease in affinity of bound NAD+ or NADH. 7. The binding of NAD+ to the 3-phosphoglyceroyl enzyme can be described by a dissociation constant that is at least two orders of magnitude greater than the dissociation constants of the unacylated enzyme. The affinity of NAD+ to this form of the enzyme is in agreement with the Ki calculated from product inhibition by NAD+ of the reductive dephosphorylation of 1,3-diphosphoglycerate.
Acylation↗
Preparation and properties of rabbit-muscle glyceraldehyde-phosphate dehydrogenase with equal binding parameters for the third and fourth NAD+ molecules.
1. A method of preparing rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) is described which yields a preparation differing in important respects from those previously described and resembling the enzyme isolated from sturgeon muscle. 2. Direct binding measurements at 25 degrees C by equilibrium gel filtration fit dissociation constants for the first two molecules that are too low to be measured by this technique and 0.9 micrometer for the third and fourth molecules. The dissociation constant of the fourth molecule is much lower than that previously reported for the rabbit-muscle enzyme. 3. In contrast to previous results with the rabbit-muscle enzyme, the increase in absorbance at 360 nm between three and four molecules of NAD+ bound to the enzyme was, within experimental error, the same as that with each of the first three molecules. 4. Data on the quenching of the protein fluorescence by NAD+ at 15 degrees C at different enzyme concentrations closely fit dissociation constants of 0.028 micrometer for the first two molecules and 0.27 micrometer for the third and fourth molecules.
Animals↗
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↗