Biomedical subjects

Maurizio Paci

Publications and source records attributed to Maurizio Paci.

24 records · Page 2Linked to original sources

PubMed2003

Two distinct calcium-calmodulin interactions with N-terminal regions of the olfactory and rod cyclic nucleotide-gated channels characterized by NMR spectroscopy.

The interactions of calcium-calmodulin with two fragments of the N-terminal domains of the olfactory alpha-subunit and rod beta-subunit cyclic nucleotide-gated channels have been investigated using nuclear magnetic resonance spectroscopy. The results indicate that in the two cases both the N-terminal and the C-terminal calmodulin lobes are involved in the interaction. The olfactory cyclic nucleotide-gated channel segment forms a 1:1 complex with calmodulin, whereas the rod fragment forms a 2:1 complex. The correlation times of the two complexes, as estimated by (15)N relaxation studies, are compatible with the observed stoichiometries. These results indicate differences in the mode of action by which calmodulin modulates the activity of both channels, and suggest either that the rod channel is modulated through a simultaneous interaction of two beta-subunits with calmodulin or that other regions of the N-terminus are necessarily implicated in the binding.

Amino Acid Sequence↗

PubMed2003

Surface changes and role of buried water molecules during the sulfane sulfur transfer in rhodanese from Azotobacter vinelandii: a fluorescence quenching and nuclear magnetic relaxation dispersion spectroscopic study.

The Azotobacter vinelandii rhodanese is a sulfurtransferase enzyme that catalyzes the transfer of the outer sulfur atom from thiosulfate to cyanide. Recently, investigations by NMR relaxation on the (15)N-enriched protein reported that interdomain contacts are rigidly maintained upon the sulfane sulfur transfer from the enzyme to the substrate. The modality of the enzymatic mechanism is then confined to a surface interaction, including dynamics of water molecules buried in the tertiary structure. Thus, investigations have been carried out by fluorescence, circular dichroism, and nuclear magnetic relaxation dispersion measurements. The comparison of circular dichroism spectra of the persulfurated enzyme and the sulfur-free form indicated that small changes occur. Fluorescence quenching studies have been performed to evaluate the conformational changes during catalysis using the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid, and acrylamide, iodide, and cesium ions as quenchers. Changes in exchange dynamics of water molecules buried in the structure with bulk water, observed by nuclear magnetic relaxation dispersion, are due to local conformational transitions, likely involving residues around the active site, and are consistent with the global correlation time found by (15)N relaxation. These results, taken together, provide important information for elucidating the conformational features of the mechanism of action of the enzyme either in the role of a selective donor of a sulfur atom to small-sized substrates (i.e., to cyanide, transforming it into thiocyanate) or in the role of sulfur insertase for the formation of the Fe(2)S(2) iron-sulfur cluster in sulfur-deprived ferredoxins.

Azotobacter vinelandii↗

PubMed2003

Contryphan-Vn: a modulator of Ca2+-dependent K+ channels.

Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.

Animals↗

PubMed2003

Structural rearrangements of the two domains of Azotobacter vinelandii rhodanese upon sulfane sulfur release: essential molecular dynamics, 15N NMR relaxation and deuterium exchange on the uniformly labeled protein.

The Azotobacter vinelandii rhodanese is a 31kDa sulfurtransferase protein that catalyzes the transfer of sulfur atom from thiosulfate to cyanide in the detoxification process from cyanide and is able to insert sulfur atom in the iron-sulfur cluster. A study of the uniformly 15N isotopic labeling by high resolution NMR, before obtaining the backbone sequential assignment, has been carried out. The sulfur loaded and the sulfur discharged forms of the enzyme show very similar HSQC spectra with a good spectral dispersion. Few resonances show changes in chemical shift between the two forms. Relaxation parameters T(1), T(2) and 1H-15N NOE of all amide nitrogen atoms, as well as isotope exchange kinetics, show that the two forms exhibit the same global correlation time and hydrodynamic properties. In parallel, essential dynamics studies show that formation and discharging of catalytic cysteine persulfide group has no significant impact on the overall conformation of the protein. These results, taken together, give a clearcut answer to the question if the catalytic mechanism of the enzyme involves a change in the conformation and/or in the mutual orientation of the two domains. On the contrary these results clearly indicate that upon the catalytic mechanism the two domains of the protein behave as a unique fold.

Azotobacter vinelandii↗

PubMed2003

Azotobacter vinelandii rhodanese: selenium loading and ion interaction studies.

Rhodanese is a sulfurtransferase which in vitro catalyzes the transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandii were studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using [15N]Cys, and changes in 1H and 15N NMR resonances on addition of different ions were monitored. The results clearly indicate that the sulfur transfer is due to a specific reaction of the persulfurated Cys residue with a sulfur acceptor such as cyanide and not to the presence of the anions. Moreover, the 1H-NMR spectrum of a defined spectral region is indicative of the status of the enzyme and can be used to directly monitor sulfur loading even at low concentrations. Selenium loading by the addition of selenodiglutathione was monitored by fluorescence and NMR spectroscopy. It was found to involve a specific interaction between the selenodiglutathione and the catalytic cysteine residue of the enzyme. These results indicate that rhodanese-like proteins may function in the delivery of reactive selenium in vivo.

Azotobacter vinelandii↗

PubMed2003

Interaction of DAPI with individual strands of trinucleotide repeats. Effects of replication in vitro of the AAT x ATT triplet.

The structural changes produced by the minor-groove binding ligand DAPI (4',6-diamidine-2-phenylindole) on individual strands of trinucleotide repeat sequences were detected by electrophoretic band-shift analysis and related to their effects on DNA replication in vitro. Among the 20 possible single-stranded trinucleotide repeats, only the T-rich strand of the AAT.ATT triplet exhibits an observable fluorescence band and a change in electrophoretic mobility due to the drug binding. This is attributable to the property of DAPI that favours folding of the random coil ATT strand into a fast-migrating hairpin structure by a minor-groove binding mechanism. Electrophoretic characteristics of AAT, ACT, AGT, ATG and ATC are unchanged by DAPI, suggesting the crucial role of T.T with respect to A.A, C.C and G.G mismatch, in favouring the binding properties and the structural features of the ATT-DAPI complexes. Primer extension experiments, using the Klenow fragment of DNA polymerase I, demonstrate that such a selective structural change at ATT targets presents a marked property to stall DNA replication in vitro in comparison with the complementary AAT and a random GC-rich sequence. The results suggest a novel molecular mechanism of action of the DNA minor-groove binding ligand DAPI.

Base Sequence↗