Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.99.3 (PRE)
1,923 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mode of interaction between human epidermal growth factor (hEGF) and its receptor has been investigated by immunochemical studies and a synthetic peptide approach. Two types of monoclonal and five different monospecific polyclonal antibodies against hEGF have been prepared, whose epitopes are regions 1-13, 13-32, 33-53, 33-43, 22-32, and discontinuous sequences of hEGF. Antibody against 22-32 (Type I) and antibody against 33-53 (PRE 4) inhibited the binding of 125I-hEGF to membrane receptor on A 431 cells more markedly than the other antibodies. When hEGF was bound to the receptor, only antibody against 13-32 (PRE 2) could bind to hEGF-receptor complex whereas antibody against 22-32 (Type I) could not. These data suggest that region 13-20 is exposed outside during receptor-binding and both region 22-32 and region 33-53 contact the hEGF receptor. The activity of synthetic peptides corresponding to the amino acid residues 1-13, 13-32, 33-53, 13-20, 22-32, and 33-43 of hEGF was also examined. Out of the six peptides, only 13-32 stimulated DNA synthesis of BALB 3T3 cells. The activity was approximately 1/10(6) of that of intact hEGF. All of these data suggest that region 22-32 is responsible for binding to the receptor for signal transduction and region 33-53 binds to the receptor to stabilize the ligand-receptor interaction. This dual binding model fits in well with the three-dimensional hEGF structure deduced from NMR spectra.
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PMID:Topographic analysis of human epidermal growth factor by monospecific antibodies and synthetic peptides. 247 32

Irradiation of the double-stranded octamer d(GCGTTGCG).d(CGCAACGC) with UV light causes dimerization of the two central thymine residues. Proton NMR data reveal that this photodimer has the same chemical structure as the photodimer, which is formed upon UV irradiation of the single strand d(GCGTTGCG), a cis-syn-cyclobutane-type thymine dimer. Irradiation of the purified thymine dimer double-stranded octamer d(GCGTTGCG).d(CGCAACGC) with visible light in the presence of photoreactivating enzyme isolated from Anacystis nidulans leads to an increase in absorbance at 260 nm, which is characteristic for the repair of thymine dimers. The NMR spectrum recorded after the photoreactivating treatment indeed shows that a complete conversion to the starting octamer has occurred.
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PMID:Photoreactivation of the thymine dimer containing DNA octamer d(GCGTTGCG).d(CGCAACGC) by the photoreactivating enzyme from Anacystis nidulans. 314 67

A strategy was developed to assemble nucleosomes specifically damaged at only one site and one structural orientation. The most prevalent UV photoproduct, a cis-syn cyclobutane thymine dimer (cs CTD), was chemically synthesized and incorporated into a 30 base oligonucleotide harboring the glucocorticoid hormone response element. This oligonucleotide was assembled into a 165 base pair double stranded DNA molecule with nucleosome positioning elements on each side of the cs CTD-containing insert. Proton NMR verified that the synthetic photoproduct is the cis-syn stereoisomer of the CTD. Moreover, two different pyrimidine dimer-specific endonucleases cut approximately 90% of the dsDNA molecules. This cleavage is completely reversed by photoreactivation with E. coli UV photolyase, further demonstrating the correct stereochemistry of the photoproduct. Nucleosomes were reconstituted by histone octamer exchange from chicken erythocyte core particles, and contained a unique translational and rotational setting of the insert on the histone surface. Hydroxyl radical footprinting demonstrates that the minor groove at the cs CTD is positioned away from the histone surface about 5 bases from the nucleosome dyad. Competitive gel-shift analysis indicates there is a small increase in histone binding energy required for the damaged fragment (DeltaDeltaG approximately 0.15 kcal/mol), which does not prevent complete nucleosome loading under our conditions. Finally, folding of the synthetic DNA into nucleosomes dramatically inhibits cleavage at the cs CTD by T4 endonuclease V and photoreversal by UV photolyase. Thus, specifically damaged nucleosomes can be experimentally designed for in vitro DNA repair studies.
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PMID:Synthesis and nucleosome structure of DNA containing a UV photoproduct at a specific site. 1041 26

Prior theoretical work has predicted that the NMR paramagnetic relaxation enhancement (NMR-PRE) produced by electron spin S = 1 ions is highly sensitive to orthorhombic terms in the static zero field splitting (zfs) tensor. Zfs orthorhombicity (which implies chemical inequivalence of the three principal directions of the zfs-principal axis system and is described by the zfs E-parameter) is predicted to suppress the NMR-PRE profoundly relative to the reference cylindrical zfs-limit situation. This expectation was tested experimentally by a comparison of the zfs-limit NMR-PRE produced by [Ni(II)(en)(3)](2+) (en = ethylenediamine), a trigonal complex which lacks zfs-rhombicity, with the zfs-limit NMR-PRE produced by two orthorhombic complexes, [Ni(II)(en)(2)(H(2)O)(2)](2+) and [Ni(II)(en)(H(2)O)(4)](2+). As predicted, the zfs-limit NMR-PRE produced by the orthorhombic complexes in the proton resonance of a dioxane probe species in the solvent was strongly suppressed (by factors of approximately 5 and 7, respectively) relative to the comparable measurement on the trigonal complex. The suppression of the NMR-PRE due to the orthorhombic zfs terms is counteracted by an applied Zeeman field, leading to a predicted rise in the NMR-PRE with increasing Zeeman field strength; this rise occurs when the Zeeman energy is comparable to the orthorhombic zfs splitting, 2E. This second prediction of theory was likewise confirmed: the expected rhombicity-induced magnetic field dependence in the NMR-PRE was observed for the orthorhombic complexes but not for the trigonal complex.
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PMID:NMR paramagnetic relaxation enhancement: test of the controlling influence of zfs rhombicity for S = 1. 1123 32

Paramagnetic enhancement of NMR relaxation (NMR-PRE) depends on thermal relaxation of the electron spin system. Most previous analyses of experimental NMR-PRE data have relied on Bloembergen--Morgan (B--M) theory to describe the magnetic field dependence of electron spin relaxation in liquid samples. However, B--M theory assumes a Zeeman-limit situation and is not physically appropriate to the common case of S > or = 1 transition metal ions which possess a permanent zero-field splitting (zfs) that is comparable to or larger than the Zeeman splitting. Theory has been needed which (1) includes the effects of the zfs interaction, thus providing a realistic description of the magnetic field dependence of the NMR-PRE outside the Zeeman limit, and (2) describes electron spin relaxation phenomena at a comparable level of complexity to that of B--M theory, i.e., with two magnetic field-dependent electron spin relaxation times, tau(S1) and tau(S2), defined in the laboratory coordinate frame. Theory of this kind is developed. Expressions derived in a previous study (R. R. Sharp and L. L. Lohr, J. Chem. Phys. 115, 5005 (2001).) for level-specific relaxation rates have been averaged over spin eigenstates to give level-averaged quantities, tau(S1,2). This kind of averaging leads to a great simplification in the mathematical form of the results. Simple zfs-limit molecular-frame and laboratory-frame expressions are given for electron spin S=1, 3/2, 2, and 5/2. General expressions, valid for S > or = 1 and for arbitrary magnitudes of the Zeeman and zfs energies, are derived for level-averaged electron spin relaxation times defined in both the laboratory- and the molecule-fixed coordinate frames. The new theory coincides with B--M theory in the Zeeman limit.
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PMID:Closed-form expressions for level-averaged electron spin relaxation times outside the Zeeman limit: application to paramagnetic NMR relaxation. 1184 84

The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from (31)P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.
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PMID:Investigation of the cyclobutane pyrimidine dimer (CPD) photolyase DNA recognition mechanism by NMR analyses. 1516 80

Cyclobutane pyrimidine dimer (CPD) photolyases, which contain FAD as a cofactor, use light to repair CPDs. We performed structural analyses of the catalytic site of the Thermus thermophilus CPD photolyase-DNA complex, using FAD-induced paramagnetic relaxation enhancement (PRE). The distances between the tryptophan residues and the FAD calculated from the PRE agree well with those observed in the x-ray structure (with an error of <3 A). Subsequently, a single-stranded DNA containing 13C-labeled CPD was prepared, and the FAD-induced PRE of the NMR resonances from the CPD lesion in complex with the CPD photolyase was investigated. The distance between the FAD and the CPD calculated from the PRE is 16 +/- 3 A. The FAD-induced PRE was also observed in the CPD photolyase-double-stranded DNA complex. Based on these results, a model of the CPD photolyase-DNA complex was constructed, and the roles of Arg-201, Lys-240, Trp-247, and Trp-353 in the CPD-repair reaction are discussed.
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PMID:NMR study of repair mechanism of DNA photolyase by FAD-induced paramagnetic relaxation enhancement. 1546 18

Two new artificial photolyase models that recognize pyrimidine dimers in protic and aprotic organic solvents as well as in water through a combination of charge and hydrogen-bonding interactions and use a mimic of the flavine to achieve repair through reductive photoinduced electron transfer are presented. Fluorescence and NMR titration studies show that it forms a 1:1 complex with pyrimidine dimers with binding constants of approximately 10(3) M(-1) in acetonitrile or methanol, while binding constants in water at pH 7.2 are slightly lower. Excitation of the complex with visible light leads to clean and rapid cycloreversion of the pyrimidine dimer through photoinduced electron transfer catalysis. The reaction in water is significantly faster than in organic solvents. The reaction slows down at higher conversions due to product inhibition.
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PMID:Design, synthesis, and evaluation of a biomimetic artificial photolyase model. 1554 85

The metalloporphyrins, Me-TSPP [Me=Cr(III), Mn(III), Mn(II), Fe(III), and TSPP=meso-(tetra-p-sulfonatophenyl)porphyrin], which possess electron spins S=3/2, 2, 5/2, and 5/2, respectively, comprise an important series of model systems for mechanistic studies of NMR paramagnetic relaxation enhancement (NMR-PRE). For these S>1/2 spin systems, the NMR-PRE depends critically on the detailed form of the zero-field splitting (zfs) tensor. We report the results of experimental and theoretical studies of the NMR relaxation mechanism associated with Fe(III)-TSPP, a spin 5/2 complex for which the overall zfs is relatively large (D approximately = 10 cm(-1)). A comparison of experimental data with spin dynamics simulations shows that the primary determinant of the shape of the magnetic relaxation dispersion profile of the water proton R1 is the tetragonal fourth-order component of the zfs tensor. The relaxation mechanism, which has not previously been described, is a consequence of zfs-induced mixing of the spin eigenfunctions of adjacent Kramers doublets. We have also investigated the magnetic-field dependence of electron-spin relaxation for S=5/2 in the presence of a large zfs, such as occurs in Fe(III)-TSPP. Calculations show that field dependence of this kind is suppressed in the vicinity of the zfs limit, in agreement with observation.
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PMID:NMR paramagnetic relaxation due to the S=5/2 complex, Fe(III)-(tetra-p-sulfonatophenyl)porphyrin: central role of the tetragonal fourth-order zero-field splitting interaction. 1591 23

Four theoretical and computational approaches used at the University of Michigan to analyze NMR paramagnetic relaxation enhancement (NMR-PRE) are described. The primary objective of the theory is to describe the relationship of the NMR-PRE phenomenon to the electron spin hamiltonian and the spin energy level structure when zero field splitting interactions are significant. Four formulations of theory are discussed: (1) spin dynamics simulation; (2) the laboratory frame "constant H(S)" formulation; (3) the Molecular Frame "constant H(S)" formulation; and (4) the zfs-limit "constant H(S)" formulation. No single theoretical approach describes all important aspects of the relaxation mechanism in a fully satisfactory way. We use the four formulations in a complementary manner to provide as complete a picture of the relaxation mechanism as possible. We also discuss the integration of NMR-PRE theory and recently developed theory of electron spin relaxation which accounts for effects of the permanent zfs hamiltonian.
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PMID:Four complementary theoretical approaches for the analysis of NMR paramagnetic relaxation. 1600 86


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