Some consequences of the covalent and non-covalent binding modes of plasmin with alpha 2-macroglobulin

Analysis of plasmin-alpha 2-macroglobulin interactions by polyacrylamide gel electrophoresis showed that both the light and heavy chains of the proteinase have covalent links with the inhibitor. This covalent binding occurs with a 95 +/- 5% yield and can be abolished in the presence of hydroxylamine without modification of the plasmin-alpha 2-macroglobulin stoichiometry, the extent of the 180-kDa peptide chain cleavage and the generation of the -SH groups. However, these two different binding modes greatly influence the enzymatic properties of the proteinase as well as the occupancy by an other proteinase molecule of the free binding site of the (1:1) plasmin-alpha 2-macroglobulin complex. Non-covalently bound plasmin is more active on synthetic substrates and interacts more tightly with the basic pancreatic trypsin inhibitor than the covalently bound enzyme. Furthermore, the former complex incorporates significantly more chymotrypsin than the latter. The incorporation of chymotrypsin influences the catalytic properties of plasmin within the ternary complex.     

The microsomal metabolism of pentachlorophenol and its covalent binding to protein and DNA

The microsomal metabolism of pentachlorophenol (PCP) was investigated, with special attention to the conversion dependent covalent binding to protein and DNA. The two metabolites detected were tetrachloro-1,2- and tetrachloro-1,4-hydroquinone. Microsomes from isosafrole (ISF)-induced rats were by far the most effective in catalyzing the reaction: the rate of conversion was increased 7-fold over control microsomes. All other inducers tested (hexachlorobenzene (HCB), phenobarbital (PB) and 3-methylcholanthrene (3MC) gave 2--3-fold increases over control. There are indications that the 1,2- and 1,4-isomers are produced in different ratio's by various cytochrome P-450 isoenzymes: Microsomes from PB- and HCB-treated rats produced the tetrachloro-1,4- and tetrachloro-1,2-hydroquinone in a ratio of about 2, while microsomes from rats induced with 3 MC and ISF showed a ratio of about 1.3. When PCP was incubated with microsomes from rats treated with HCB, a mixed type inducer of P-450, the ratio between formation of the 1,4- and 1,2-isomers decreased with increasing concentration of PCP, suggesting the involvement of at least two P-450 isoenzymes with different Km-values. The overall apparent Km-value for HCB-microsomes was 13 microM both for the formation of the soluble metabolites and the covalent binding to microsomal protein, suggesting both stem from the same reaction. The covalent binding could be inhibited by ascorbic acid and this inhibition was accompanied by an increase in formation of tetrachlorohydroquinones (TCHQ). Although a large variation was observed in rates of conversion between microsomes treated with different (or no) inducers, the rate of covalent binding to microsomal protein was remarkably constant. A conversion-dependent covalent binding to DNA was observed in incubations with added DNA which was 0.2 times the amount of binding to protein (37 pmol/mg DNA).     

Changing the determinants of protein stability from covalent to non-covalent interactions by in vitro evolution: a structural and energetic analysis

The three disulfide bonds of the gene-3-protein of the phage fd are essential for the conformational stability of this protein, and it unfolds when they are removed by reduction or mutation. Previously, we used an iterative in vitro selection strategy to generate a stable and functional form of the gene-3-protein without these disulfides. It yielded optimal replacements for the disulfide bonds as well as several stabilizing second-site mutations. The best selected variant showed a higher thermal stability compared with the disulfide-bonded wild-type protein. Here, we investigated the molecular basis of this strong stabilization by solving the crystal structure of this variant and by analyzing the contributions to the conformational stability of the selected mutations individually. They could mostly be explained by improved side-chain packing. The R29W substitution alone increased the midpoint of the thermal unfolding transition by 14 deg and the conformational stability by about 25 kJ mol^− 1. This key mutation (i) removed a charged side chain that forms a buried salt bridge in the disulfide-containing wild-type protein, (ii) optimized the local packing with the residues that replace the C46-C53 disulfide and (iii) improved the domain interactions. Apparently, certain residues in proteins indeed play key roles for stability.     

Consequences of quercetin methylation for its covalent glutathione and DNA adduct formation

This study investigates the pro-oxidant activity of 3′- and 4′-O-methylquercetin, two relevant phase II metabolites of quercetin without a functional catechol moiety, which is generally thought to be important for the pro-oxidant activity of quercetin. Oxidation of 3′- and 4′-O-methylquercetin with horseradish peroxidase in the presence of glutathione yielded two major metabolites for each compound, identified as the 6- and 8-glutathionyl conjugates of 3′- and 4′-O-methylquercetin. Thus, catechol-O-methylation of quercetin does not eliminate its pro-oxidant chemistry. Furthermore, the formation of these A-ring glutathione conjugates of 3′- and 4′-O-methylquercetin indicates that quercetin o-quinone may not be an intermediate in the formation of covalent quercetin adducts with glutathione, protein and/or DNA. In additional studies, it was demonstrated that covalent DNA adduct formation by a mixture of [4-¹⁴C]-3′- and 4′-O-methylquercetin in HepG2 cells amounted to only 42% of the level of covalent adducts formed by a similar amount of [4-¹⁴C]-quercetin. Altogether, these results reveal the effect of methylation of the catechol moiety of quercetin on its pro-oxidant behavior. Methylation of quercetin does not eliminate but considerably attenuates the cellular implications of the pro-oxidant activity of quercetin, which might add to the mechanisms underlying the apparent lack of in vivo carcinogenicity of this genotoxic compound. The paper also presents a new mechanism for the pro-oxidant chemistry of quercetin, eliminating the requirement for formation of an o-quinone, and explaining why methylation of the catechol moiety does not fully abolish formation of reactive DNA binding metabolites.     

HU binding to DNA: evidence for multiple complex formation and DNA bending

HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities.     

High-performance liquid chromatography separation of some NAD+ derivatives suitable for covalent binding

Separation of NAD⁺, N¹-carboxymethyl-NAD⁺, N⁶-carboxymethyl-NAD⁺, and N⁶-[N-(6-aminohexyl)carbamoylmethyl]-NAD⁺ by high performance liquid chromatography is described. Reversed-phase chromatography with the acidic mobile phase (phosphate buffer pH 2.0–3.6) proved to be the most suitable method, particularly for the separation of impurities. The proposed method can be used for monitoring the course of the synthesis of N⁶-[N-(6-aminohexyl)carbamoylmethyl]-NAD⁺ and for the separation of the intermediates. Identification of the peaks was performed by means of spectroscopic measurement as well as a specific coenzyme activity test. Performance of the described method is greater in comparison with thin-layer chromatography.     

Radiation chemical mechanisms of single- and double-strand break formation in irradiated SV40 DNA

Using an electrophoresis assay system developed in our laboratory, we have simultaneously measured single- and double-strand DNA breaks (SSBs and DSBs) induced by gamma radiation in small SV40 viral DNA molecules, under conditions of greatly varying radical scavenger concentration and DNA configuration. In our experiments with aqueous solutions of SV40 DNA, we observe that SSB induction is linear with dose (one-hit response), over the entire hydroxyl scavenger efficiency range examined, from approximately 0 to 5 x 10(9) s-1, while DSB induction shifts from having a major quadratic component (two-hit response) at very low scavenger efficiencies to nearly pure linear for efficiencies greater than 10(7) s-1. The mean ratio of SSBs to one-hit DSBs remains relatively constant with increasing scavenger efficiency, decreasing from about 100:1 to 40:1 as the scavenger efficiency increases from 2 x 10(5) s-1 to 5 x 10(9) s-1, and the absolute induction efficiencies for breaks decrease by three orders of magnitude. This decrease takes place primarily at scavenger efficiencies above 1 x 10(8) s-1. Irradiation of intranuclear SV40 minichromosomes induces SSBs and DSBs at nearly the same efficiencies as does irradiation of free DNA at the highest scavenger concentrations examined, and at only about twice the efficiencies observed at -75 degrees C, where direct effects are believed to predominate. Our observations that the linear-quadratic mix of the dose-response curve for DSBs depends critically on scavenger efficiency may help to clarify the considerable confusion in the literature on the shape of such curves. Our observations of a relatively constant ratio between one-hit SSBs and DSBs at low and moderate scavenger efficiencies are in agreement with the recent hypothesis of Siddiqi and Bothe (Radiat. Res. 112, 449-463 (1987)) that, contrary to widely and long-held beliefs, the formation by indirect effects of a one-hit DSB in DNA occurs under these conditions predominantly by a mechanism involving a single OH radical, with a presumed radical transfer between complementary DNA strands. In contrast, our results for strongly protective conditions are not consistent with this hypothesis, but are consistent with the predictions of Ward's hypothesis (Radiat. Res. 86, 185-195, (1981)) that one-hit DSBs from indirect effects are produced predominantly by local clusters of OH radicals from single energy deposition events (locally multiply damaged sites) rather than by single OH radicals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Strategies for covalent attachment of DNA to beads

Several covalent attachment chemistries were tested for the immobilization of DNA onto glass beads. The comparison was based on the ability of these chemistries to produce derivatized beads that give good hybridization signals. Cyanuric chloride, isothiocyanate, nitrophenyl chloroformate, and hydrazone chemistries gave us the best (yet comparable) hybridization signals. We further characterized the cyanuric chloride method for the number of attachment sites, number of hybridizable sites, hybridization kinetics, effect of linker length on hybridization intensity and stability of the derivatized beads.     

Covalent and non-covalent chemical engineering of actin for biotechnological applications

The cytoskeletal filaments are self-assembled protein polymers with 8–25 nm diameters and up to several tens of micrometres length. They have a range of pivotal roles in eukaryotic cells, including transportation of intracellular cargoes (primarily microtubules with dynein and kinesin motors) and cell motility (primarily actin and myosin) where muscle contraction is one example. For two decades, the cytoskeletal filaments and their associated motor systems have been explored for nanotechnological applications including miniaturized sensor systems and lab-on-a-chip devices. Several developments have also revolved around possible exploitation of the filaments alone without their motor partners. Efforts to use the cytoskeletal filaments for applications often require chemical or genetic engineering of the filaments such as specific conjugation with fluorophores, antibodies, oligonucleotides or various macromolecular complexes e.g. nanoparticles. Similar conjugation methods are also instrumental for a range of fundamental biophysical studies. Here we review methods for non-covalent and covalent chemical modifications of actin filaments with focus on critical advantages and challenges of different methods as well as critical steps in the conjugation procedures. We also review potential uses of the engineered actin filaments in nanotechnological applications and in some key fundamental studies of actin and myosin function. Finally, we consider possible future lines of investigation that may be addressed by applying chemical conjugation of actin in new ways.     

A new approach to possible substrate binding mechanisms for nitrile hydratase

We combined normal mode analysis (NMA) with cavity calculations as a method to get more insight into static crystal structures. We used nitrile hydratase (NHase) as a case study, and the crystal structure of a complex of Pseudonocardia thermophila NHase (1UGP) with n-butyric acid was chosen as a reference structure. The reference structure was compared with the other available NHase crystal structures. Cavity calculations of the static structures showed the entrances to the active site and also a possible function of the N-terminal in the substrate selection of the Co-type NHase. When NMA was combined with cavity calculations, a closing-opening passage was observed. Analysis of low frequency modes combined with cavity calculations led us to propose "breathing" and "flip-flop" mechanisms which might be a key part of the substrate binding mechanism.     

An empirical study of the universal chemical key algorithm for assigning unique keys to chemical compounds

In this paper, we introduce an algorithm that assigns an essentially unique key called the Universal Chemical Key (UCK) to molecular structures. The molecular structures are represented as labeled graphs whose nodes abstract atoms and whose edges abstract bonds. The algorithm was tested on 236,917 compounds obtained from the National Cancer Institute (NCI) database of chemical compounds. On this database, the UCK algorithm assigned unique keys for chemicals with distinct molecular structures.