Astrid Gräslund

EPR stopped-flow studies of the reaction of the tyrosyl radical of protein R2 from ribonucleotide reductase with hydroxyurea.

The reaction of the functional tyrosyl radical in protein R2 of ribonucleotide reductase from E. coli and mouse with the enzyme inhibitor hydroxyurea has been studied by EPR stopped-flow techniques at room temperature. The rate of disappearance of the tyrosyl radical in E. coli protein R2 is k2 = 0.43 M-1 s-1 at 25 degrees C. The reaction follows pseudo-first-order kinetics up to 450 mM hydroxyurea indicating that no saturation by hydroxyurea takes place even at this high concentration. Transient nitroxide-like radicals from hydroxyurea have been detected for the first time in the reaction of hydroxyurea with protein R2 from E. coli and mouse, indicating that 1-electron transfer from hydroxyurea to the tyrosyl radical is the dominating mechanism in the inhibitor reaction. The hydroxyurea radicals appear in low steady-state concentrations during 2-3 half-decay times of the tyrosyl radical and disappear thereafter.

The mechanism of action of the anti-herpes virus compound 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-(2,3-b)quinoxaline

The compound 2,3-dimethyl-6(2-dimethylaminoethyl)6H-indolo-(2,3-b)quinoxaline (B-220) has been shown to exhibit potent antiviral activity against herpes simplex virus type 1 (HSV-1), varicella-zoster virus (VZV) and cytomegalovirus (CMV). The mechanism of antiviral action of B-220 against HSV-1 has been studied; from the results it appears that B-220 binds by intercalation into the DNA helix and then disturbs steps that are vital for viral uncoating.

DNA-carcinogen interaction: covalent DNA-adducts of benzo(a)pyrene 7,8-dihydrodiol 9,10-epoxides studied by biochemical and biophysical techniques.

Exposure to various chemicals, either due to occupation or lifestyle, is considered to be a major contributing factor to tumour formation in man (Higginson, 1969; Doll and Peto, 1981). An important and prevalent class of potent carcinogenic compounds present in he environment is polycyclic aromatic hydrocarbons (PAHs), which are found in various petroleum and combustion products derived from heat and power generation and motor vehicle exhausts (Baum, 1978). Furthermore, since PAHs are generally formed by pyrolysis of organic matters such as tobacco smoking and certain procedures of food preparation, the PAH exposure to humans is extensive.

Induction of secondary structure in the peptide hormone motilin by interaction with phospholipid vesicles

Motilin is an intestinal peptide hormone that binds to a membrane bound receptor located in the gut tissue. Circular dichroism (CD) was used to study the interaction between either porcine or rabbit motilin or a 1-16 fragment of porcine motilin, with model systems of lipid membranes: sodium dodecyl sulphate (SDS), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The CD measurements show significant induction of secondary structure in both motilins and the fragment when negatively charged vesicles (DOPG) or negatively charged micelles (SDS) were present. In contrast, neutral DOPC vesicles did not induce any change in the secondary structure compared to water, in which a random-like secondary structure dominates. The induced secondary structure in the presence of DOPG vesicles is very close to that induced by a mixed aqueous solution containing 30% hexafluoroisopropanol, in which previous NMR-studies have resulted in a three-dimensional solution structure of porcine motilin. In both porcine and rabbit motilin the alpha-helix content is about 50%. This is in agreement with the presence of an amphipathic helix in the C-terminal half of motilin interacting with phospholipid membranes. The interaction appears to be mainly electrostatic in nature, and does not induce any significant alterations in the vesicle, as monitored by EPR studies of spin labels located at the fifth carbon atom of the backbone in a stearic acid molecule. In the 1-16 fragment the alpha-helical content induced by DOPG and SDS is only about 20%.

Thiols as peroxidase substrates

The abilities of haem peroxidases to catalyse the oxidation of various thiols were studied using the spin-trapping electron spin resonance (ESR) technique. Myeloperoxidase, a neutrophil and monocyte enzyme, catalysed the oxidation of cysteamine, cysteine methyl, and ethyl ester and to some extent 2-mercaptoethanol and thioglycollic acid. This peroxidase poorly catalysed the oxidation of cysteine, N-acetylcysteine, penicillamine, and glutathione under the same conditions. The dependence on pH of peroxidase-catalysed thiol oxidation may indicate that the thiolate anion form is the actual peroxidase substrate. Another leucocyte peroxidase, eosinophil peroxidase, had similar catalytic properties toward thiols as myeloperoxidase. Lactoperoxidase (found in milk, saliva, and tears) and the plant horseradish peroxidase were, however, different from the aforementioned leucocyte peroxidases in their abilities to catalyse the oxidation of thiols.

A refined three-dimensional solution structure of a carboxy terminal fragment of apolipoprotein CII.

The three-dimensional structure of a synthetic fragment of human apolipoprotein CII (apo-CII) in 35%, 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) has been determined on the basis of distance and intensity constraints derived from two-dimensional proton nuclear magnetic resonance measurements. The NOE crosspeak build-up rates were converted to distance constraints which were used in the distance geometry program DIANA. A set of one hundred structures were generated and of these ten structures were used in molecular dynamics simulations using the program XPLOR. This program enabled a direct minimization between the difference of the two-dimensional NOE intensities and those calculated from the full relaxation matrix. In this way spin diffusion is fully taken into account, which can be seen from the considerable improvement of the R-factor after the relaxation matrix refinement. These calculations show that this fragment, which corresponds to the carboxy terminal 30 amino acids of intact apo-CII and which retains its ability to activate lipoprotein lipase, is essentially flexible, but has three defined secondary structural elements. The most significant one is an α-helix between residues 67 and 74. The following three residues adopt a turn-like structure. Another turn of α-helix is seen between residues 56 and 59. The effect of the solvent system on the secondary structure was studied by circular dichroism spectroscopy. The results show that the mixed aqueous 35% HFP solvent induces secondary structure of a very similar nature to the one induced by sodium dodecyl sulphate.

Structure and dynamics of motilin. Time-resolved fluorescence of peptide hormone with single tyrosine residue

Time-resolved fluorescence and CD spectroscopy were used to characterize the structure and dynamics of the peptide hormone motilin with a single tyrosine residue among its 22 amino acids. CD spectroscopy showed that secondary structure is independent of concentration in the range 1 x 10(-5)-2.6 x 10(-4) M, and of the presence of DOPC lipid vesicles, but is strongly induced by addition of hexafluoroisopropanol. The fluorescence studies with tyrosine as the intrinsic fluorophore, performed at the MAX synchrotron laboratory at Lund, showed that three fluorescence lifetimes (0.4 ns, 1.7 ns and 3.6 ns at 20 degrees C) and two rotational correlation times (0.4 ns and 5 ns at 20 degrees C) were needed to account for the data. The different decay times are interpreted as representing ground-state rotamers interconverting slowly on the ns time scale. The rotational correlation times are ascribed to local angular motion of the tyrosyl ring, and global motion of the whole peptide, respectively.

Dynamics of benzo[a]pyrene diol epoxide adducts in poly(dG-dC · (dG-dC studied by synchrotron excited fluorescence polarization anisotropy decay

Time-resolved fluorescence studies have been performed on (+)-anti-7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene adducts in double-stranded poly(dG-dC).(dG-dC). Part of the adduct population gives rise to excimer fluorescence. The heterogeneous fluorescence emission decay curves at 22 degrees C could be resolved into three components with lifetimes: 0.4 ns, 3 ns and 24 ns for the total fluorescence (monomer and excimer emission), and 0.5 ns, 5 ns and 24 ns, respectively, for excimer emission alone. The relative amplitudes for the longer lifetimes were larger for the pure excimer population than for the mixed population. The fluorescence polarization anisotropy decay curves were resolved into two components of rotational correlation times: 0.4 ns and 25 ns for the total fluorescence and 0.3 ns and 33 ns for the excimer fluorescence. We interpret the two rotational correlation times to correspond to local motion of the adduct and segmental motion of the polynucleotide, respectively.

Interaction of the Deoxy-Oligonucleotide Duplex d(CGCGATCGCG)2 and Anti-Herpes Virus Active Indolo [2,3-b]-quinoxaline Derivatives

Abstract Ellipticine (1) and the derivatives 2,3-dimethyl-6(2-dimethyl-aminoethyl)6H-indolo-(2,3-b)quinoxaline (2) and 6-(2-dimethylaminoethyl)6H-indolo-(2,3-b)quinoxaline (3) intercalate in the duplex 4CGCGATCGCG)with slow exchange kinetics. (I) and (2) show a non-specific interaction. (3) shows an AT specific interaction.

Effect of length and netropsin binding on base-pair lifetimes of oligonucleotides with the center core GCGAATTCGC

Abstract The imino proton solvent exchange of three self-complementary oligonucleotides with different lengths 5′-d([G[C[GCGAATTCGCG]C]G])-3′ has been studied by 1H NMR selective saturation recovery. From measurements of the solvent exchange rates at different catalysing ammonia base concentrations it is possible to obtain the base pair lifetimes (1, 2). The results at 15°C shows that the base pair lifetime is an individual property dependent on the nature of the base pair and its neighbouring sequence and varies from about 10 to 80 ms for the different base pairs in the core of the oligonucleotides. End effects (“fraying at the ends”) with considerably shorter lifetimes are visible at least two or three base pairs from the ends of the oligonucleotide (see table below).