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The DNA microstates that regulate transcription include sequence-specific transcription factors (TFs), coregulatory complexes, nucleosomes, histone modifications, DNA methylation, and parts of the three-dimensional architecture of genomes, which could create an enormous combinatorial complexity across the genome. However, many proteins and epigenetic marks are known to colocalize, suggesting that the information content encoded in these marks can be compressed. It has so far proved difficult to understand this compression in a systematic and quantitative manner. Here, we show that simple linear models can reliably predict the data generated by the ENCODE and Roadmap Epigenomics consortia. Further, we demonstrate that a small number of marks can predict all other marks with high average correlation across the genome, systematically revealing the substantial information compression that is present in different cell lines. We find that the linear models for activating marks are typically cell line-independent, while those for silencing marks are predominantly cell line-specific. Of particular note, a nuclear receptor corepressor, transducin beta-like 1 X-linked receptor 1 (TBLR1), was highly predictive of other marks in two hematopoietic cell lines. The methodology presented here shows how the potentially vast complexity of TFs, coregulators, and epigenetic marks at eukaryotic genes is highly redundant and that the information present can be compressed onto a much smaller subset of marks. These findings could be used to efficiently characterize cell lines and tissues based on a small number of diagnostic marks and suggest how the DNA microstates, which regulate the expression of individual genes, can be specified. [ABSTRACT FROM AUTHOR]

31P-nuclear-magnetic-resonance spectroscopy has been employed to probe the structure of the detergent-solubilized form of liver microsomal NADPH—cytochrome-P-450 reductase. In addition to the resonances due to the FMN and FAD coenzymes, additional phosphorus resonances are observed and are assigned to the tightly bound adenosine 2′-phosphate (2′-AMP) and to phospholipids. The phospholipid content was found to vary with the preparation; however, the 2′-AMP resonance was observed in all preparations tested. In agreement with published results [Otvos et al. (1986) Biochemistry 25, 7220&nadsh;7228] for the protease-solubilized enzyme, the addition of Mn(II) to the oxidized enzyme did not result in any observable line-broadening of the FMN and FAD phosphorus resonances. The phospholipid resonances, however, were extensively broadened and the line width of the phosphorus resonance assigned to the bound 2′-AMP was broadened by &assymp;70 Hz. The data show that only the phosphorus moieties of the phospholipids and the 2′-AMP, but not the flavin coenzymes are exposed to the bulk solvent. Removal of the FMN moiety from the enzyme substantially alters the 31P-NMR spectrum as compared with the native enzyme. The 2′-AMP is removed from the enzyme during the FMN-depletion procedure and the pyrophosphate resonances of the bound FAD are significantly altered. Reconstitution of the FMN-depleted protein with FMN results in the restoration of the coenzyme spectral properties. Reduction of FMN to its air-stable paramagnetic semiquinone form results in broadening of the FMN and 2′-AMP resonances in the detergent-solubilized enzyme. In agreement with previous results, FMN semiquinone formation had little or no effect on the line width of the FMN phosphorus resonance for the proteolytically solubilized enzyme. 31P-NMR experiments with Azotobacter flavodoxin semiquinone, both in its free form and in a complex with spinach ferredoxin-NADP+ reductase, mimic the differential paramagnetic effects of the flavin semiquinone on the line width of the FMN phosphorus resonance, observed by comparison of the detergent-solubilized and protease-solubilized forms of the reductase. The data demonstrate that assignment of the site of flavin semiquinone formation to a particular flavin coenzyme may not always be possible by 31P-NMR experiments in multi-flavin containing enzymes. [ABSTRACT FROM AUTHOR]

The secondary structure of two-electron-reduced Megasphaera elsdenii flavodoxin has been determined by visual, qualitative inspection of the sequential connectivities involving CαH, CβH and NH protons observed in NOESY (two-dimensional nuclear Overhauser enhancement spectroscopy) spectra. Results from an amide proton exchange experiment were used to confirm the secondary structure assignment and to demonstrate the compactness and stability of the protein. After the secondary structure elements were established, the global fold of the protein and the flavin binding site have been determined using nonsequential interresidual NOE connectivities as primary source of information. The secondary structure and the global fold of M. elsdenii and Clostridium MP flavodoxin appeared to be very similar, differences are observed however, M. elsdenii flavodoxin consists of a central parallel β-sheet including five strands surrounded on both sides by a pair of α-helices. [ABSTRACT FROM AUTHOR]

Desulfovibrio vulgaris apolavodixin has been reconstituted with 15N and 13C-enriched riboflavin 5'-phosphate. For the first time all carbon atoms of the isoalloxazine ring of the protein-bound prosthetic group have been investigated. The reconstituted protein was studied in the oxidized and in the two-electron-reduced state. The results are interpreted in terms of specific interactions between the apoprotein and the prosthetic group, and the chemical structure of protein-bound FMN. In the oxidized state weak hydrogen bonds exist between the apoprotein and the N(5), N(3) and O(4α) atoms of FMN. The N(1) and O(2α) atoms of FMN form strong hydrogen bonds. The isoalloxazine ring of FMN is strongly polarized and the N(10) atom shows an increase sp² hybridisation compared to that of free FMN in aquenous solution. The N(3)-H group is not accessible to bulk solvent, as deduced from the coupling constant of the N(3)-H group. In the reduced state the hydrogen bond pattern is similar to that in the oxidized state and in addition a strong hydrogen bond is observed between the N(5)-H group of FMN and the apoprotein. The reduced prosthetic group possesses a coplanar structure and is ionized. The N(3)-H and N(5)-H groups are not accessible to solvent water. Two-electron reduction of the protein leads to a large electron density increase in the benzene subnucleus of bound FMN compared to that in free FMN. The results are discussed in relation to the published crystallographic data on the protein. [ABSTRACT FROM AUTHOR]

Selectively 13C-enriched free and protein-bound flavins in the oxidized and two-electron reduced state were investigated by the 13C nuclear magnetic resonance technique. FMN in aqueous solution and N(3)-methyl-2',3',4',5'-tetraacetylriboflavin in an apolar solvent were used as reference compounds for the protein-bound FMN. A comparison of the chemical shifts of FMN in aqueous solution with those of N(3)-methyltetraacetyl-riboflavin in CHCl3 reveals that FMN is strongly polarized yielding a pseudo-ionic molecule stabilized by hydrogen bonding of H2O with C-2α of the isoalloxazine molecule. The interpretation is fully supported by the one-bond (¹J) 13C coupling constants. The chemical shifts combined with the coupling constants indicate the change of charge distribution within the molecule when going from apolar to polar solutions. Binding of FMN to Megasphaera elsdenii and to Axotobacter vinelandii apoflavodoxins affects the resonances due to C-2 and C-10a as compared to free FMN. This shifts together with the coupling constants indicate the formation of a hydrogen bond between C-2α of FMN and an amino acid residue of the apoprotein. In M. elsdenii flavodoxin such a hydrogen bond also exists with C-4α which is not observed in A. vinelandii flavodoxin. The chemical shifts to the two-electron reduced derivatives of FMN and N(3)-methyl-2',3',4',5'-tetraacetylriboflavin clearly indicate that the FMN derivative possesses a more planar conformation than its analog; it show pH-dependent chemical shifts of the atoms C-10a, C-2 and c-4a. the chemical shift of C-4 is pH-independent. From the pH-dependence of the chemical shifts an ionization constant of 6,7 is calculated. The chemical shifts of protein-bound FMN in the reduced state are very similar to those of the anionic, reduced FMN. The data show that in both flavodoxin studied the prosthetic group when reduced is bound in the anionic form an possesses approximately a coplanar conformation. The results are discussed with respect to the possible biological implications. It is suggested that specific interaction of the prosthetic group with the apoprotein, the ionization state and the degree of planarity of the reduced prosthetic group are factors determining the biological functions of flavodoxins and possibly the functions of flavoproteins in general. [ABSTRACT FROM AUTHOR]

The thermodynamically stable and, therefore, analytically most important alloxazine and isoalloxazine radical cations have been studied in detail by electron paramagnetic resonance (EPR) spectroscopy. Isotopic substitutions have been made as in earlier studies with the less stable neural and anionic species. The experimental spectra have been calculated with the aid of a more sophisticated computer-simulation program than previously used. Excellent fits were obtained only when all of the following atoms were taken into account in the hypertime coupling scheme: N-5, H-10 or CH3, C-6 H, C-8 H or CH3 and C-9 H. An additional but small coupling constant was required for the fit. This latter coupling constant is assigned to the nitrogen atom(s) of the pyrimidine subnucleus of (iso)alloxazine radical cations. The EPR-active proton is attached to N-5 as we also found for the neutral flavosemiquinone. The alloxazine and is alloxazine radical cations exhibit an identical hyperfine coupling scheme but differ especially in th pyrazine nucleus with respect to the spin density distribution. This suggests that the geometrical structure of the two kinds of radicals is somewhat different. The highest spin density is, however, located at N-5 of (iso)alloxazine as has been found for the other flavorsemiquinone species. The hyperfine coupling constants are interpreted in terms of spin densities and comparison is made with the most recently available quantum chemical calculations. All monomeric flavosemiquinone species are compared with each other and their differences in the submolecular structure are discussed briefly. [ABSTRACT FROM AUTHOR]

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida have been reconstituted with 13C- and 15N-enriched FAD. The protein preparations were studied by 13C- NMR. 15N-NMR and 31P-NMR techniques in the oxidized and in the two-electron-reduced states. The chemical shift values are compared with those of free flavin in water or chloroform. It is shown that the n electron distribution in oxidized free p-hydroxybenzoate hydroxylase is comparable to free flavin in water, and it is therefore suggested that the flavin ring is solvent accessible. Addition of substrate has a strong effect on several resonances, e.g. C2 and N5, which indicates that the flavin ring becomes shielded from solvent and also that a conformational change occurs involving the positive pole of an α-helix microdipole. In the reduced state, the flavin in p-hydroxybenzoate hydroxylase is bound in the anionic form, i.e. carrying a negative charge at N1. The flavin is bound in a more planar configuration than when free in solution. Upon binding of substrate the resonances of N1, Cl0a and N10 shift upfield. It is suggested that these upfield shifts are the result of a conformational change similar, but not identical, to the one observed in the oxidized state. The 13C chemical shifts of FAD bound to apo(salicylate hydroxylase) indicate that in the oxidized state the flavin ring is also fairly solvent accessible in the free enzyme. Addition of substrate has a strong effect on the hydrogen bond formed with O4α. It is suggested that this is due to the exclusion of water from the active site by the binding of substrate. In the reduced state, the flavin is anionic. Addition of substrate forces the flavin ring to adopt a more planar configuration, i.e. a sp2-hybridized N5 atom and a slightly sp3-hybridizcd N10 atom. The NMR results are discussed in relation to the reaction catalyzed by the enzymes. [ABSTRACT FROM AUTHOR]

Assignments for the 137 amino acid residues of Megasphaera elsdenii flavodoxin in the reduced state have been made using the sequential resonance assignment procedure. Several hydroxyl and sulfhydryl protons were observed at 41 °C at pH 8.3. Spin systems were sequentially assigned using phase-sensitive two-dimensional-correlated spectroscopy and phase-sensitive nuclear Overhauser enhancement spectroscopy. Spectra of the protein in H2O and of protein preparations either completely or partly exchanged against ²H2O were obtained. Use of the fast electron shuttle between the paramagnetic semiquinone and the diamagnetic hydroquinone state greatly simplified the NMR spectra, making it possible to assign easily the ¹H resonances of amino acid residues located in the immediate neighbourhood of the isoalloxazine ring. The majority of the nuclear Overhauser effect contacts between the flavin and the apoprotein correspond to the crystal structure of the flavin domain of Clostridium MP flavodoxin, but differences are also observed. The assignments provide the basis for the structure determination of M. elsdenii flavodoxin in the reduced state as well as for assigning the resonances of the oxidized flavodoxin. [ABSTRACT FROM AUTHOR]

A new method is described for the large-scale reversible dissociation of flavoproteins into apoprotein and prosthetic group using hydrophobic-interaction chromatography. Lipoamide dehydrogenase from Azotobacter vinelandii and butyryl-CoA dehydrogenase from Megasphaera elsdenii are selected to demonstrate the usefulness of the method. In contrast to conventional methods, homogeneous preparations of apoproteins in high yields are obtained. The apoproteins show high reconstitutability. The holoenzymes are bound to phenyl-Sepharose CL-4B at neutral pH in the presence of ammonium sulfate. FAD is subsequently removed at pH 3.5-4.0 by addition of high concentrations of KBr. Large amounts of apoenzymes (200-500 mg), showing negligible residual activity, are eluted at neutral pH in the presence of 50% ethylene glycol. The holoenzyme of lipoamide dehydrogenase can be reconstituted while the apoprotein is still bound to the column or the apoenzyme can be isolated in the free state. In both cases the yield and degree of reconstitution of holoenzyme is more than 90% of starting material. Apo-lipoamide-dehydrogenase exists mainly as a monomer in solution and reassociates to the native dimeric structure in the presence of FAD. The apoenzyme is stable for a long period of time when kept in 50% ethylene glycol at -18°C. Steady-state fluorescence-polarization measurements of protein-bound FAD indicate that reconstituted lipoamide dehydrogenase possesses a high stability which is governed by the low dissociation rate constant of the apoenzyme-FAD complex. The holoenzyme of butyryl-CoA dehydrogenase cannot be reconstituted when the apoenzyme is bound to the column. However, stable apoprotein can be isolated in the free state yielding 50-80% of starting material, depending on the immobilization conditions. The coenzyme A ligand present in native holoenzyme is removed during apoprotein preparation. The apoenzyme is relatively stable when kept in 50% ethylene glycol at -18 °C. From kinetic and gel filtration experiments it is concluded that the reconstitution reaction of butyryl-CoA dehydrogenase is governed by both the pH-dependent hydrodynamic properties of apoenzyme and the pHdependent stability of reconstituted enzyme. At pH 7, the apoenzyme is in equilibrium between dimeric and tetrameric forms and reassociates to a native-like tetrameric structure in the presence of FAD. The stability of reconstituted enzyme is strongly influenced by the presence of CoA ligands as shown by fluorescence-polarization measurements. The degree of reconstitution of butyryl-CoA dehydrogenase is more than 80% of the original specific activity under certain conditions. Unliganded reconstituted enzyme is easily regreened in the presence of CoASH and Na2S. The large-scale preparation and reconstitution of the apoproteins of glutathione reductase from human erythrocytes and mercuric reductase from Pseudomonas aeruginosa is also described. [ABSTRACT FROM AUTHOR]