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L Paolillo

Publications and source records attributed to L Paolillo.

At least 37 records · Page 2Linked to original sources
PubMed1990

Evidence for beta-turn structure in model peptides reproducing pro-ocytocin/neurophysin proteolytic processing site.

The structural organization of small peptides reproducing the amino acid sequence of the common ocytocin/neurophysin precursor around the LysArg cleavage locus was investigated by a combination of spectroscopical techniques. In water both circular dichroism and [1H] NMR spectra indicated that these peptides adopted a random conformation. Evidence for folded structures was obtained when these compounds were placed in a membrane-like environment i.e. 40 mM SDS in phosphate buffer or trifluoroethanol. Whereas the CD spectra indicated the formation of various types of beta-turn in rapid equilibrium, measurements of NH temperature coefficients and Nuclear Overhauser Effects by 400 and 500 MHz NMR revealed the existence of contacts and of a folded conformation. These observations are discussed in relation with previous hypothesis made on the secondary structure organization of the proteolytic processing site of polypeptide hormone precursors.

Amino Acid Sequence↗
PubMed1990

S-deoxo-Abu1,Ile3-amaninamide, an inactive amatoxin analogue.

The title compound 3, an amatoxin analogue containing L-alpha-aminobutyric acid instead of L-asparagine in position 1, as in natural toad stool peptides, has been synthesized. It does not inhibit the eukaryotic DNA-dependent RNA polymerase form II (or B) in concentrations up to 10(-4)M, whereas 50% inhibition is exerted in 10(-6)M solution by the corresponding Asn-analogue S-deoxo-Ile3-amaninamide 2. The striking difference seems to be due to a relatively small variation of the conformation recognized by sensitive NMR spectroscopic methods.

Amanitins↗
PubMed1989

Conformation studies on bombesin receptor antagonists: 500 MHz NMR and CD characterization of synthetic (D-Phe12, Leu14)-bombesin.

The conformation flexibility of the tetradecapeptide hormone bombesin and its synthetic antagonist (DPhe12, Leu14)-bombesin has been studied using nuclear magnetic resonance and circular dichroism techniques. The spectral features observed indicate that the ordered structure present in the C-terminal pentapeptide moiety of native BBS is lost in the (DPhe12, Leu14) analog.

Bombesin↗
PubMed1989

Bicyclic peptides: solution conformation and Ca2+ binding of the heterodetic bicyclic decapeptide cyclo(Glu1-Leu2-Pro3-Gly4-Ser5-Ile6-Pro7-Ala8)- cyclo(1 gamma----5 beta)Phe9-Gly10.

The conformational behavior of a heterodetic bicyclic decapeptide (BCPLT) in the absence and in the presence of calcium ions has been studied by means of mono and two-dimensional nmr techniques. Free BCPLT possesses a quite compact structure stabilized by intramolecular bonds and turns. In the structure a cluster of carbonyls is located in a cavity that is supposed to be the cation binding site.

Amino Acid Sequence↗
PubMed1989

Mono and two-dimensional 500-MHz characterization of synthetic bombesin and related peptides in DMSO and DMSO-water.

The proton nmr characterization of bombesin (BBS) and of two peptide fragments corresponding to the (1-6) and (6-14) sequences has been carried out at 500 MHz in dimethyl sulfoxide (DMSO-d6) using two-dimensional (2D) homo and 1H-13C heterocorrelated techniques. All resonances in the nmr spectra have been assigned and several coupling constants have been measured. The backbone J alpha CH-NH coupling constants are quite similar and around 7.8-8.2 Hz, pointing to an unfolded structure in DMSO-d6. The possibility of secondary structures in highly viscous mixtures of DMSO-d6-water was investigated. The existence of sequential nuclear Overhauser enhancement (NOE) effects in the C-terminal nonapeptide section may indicate a preferential site for secondary structuring.

Bombesin↗
PubMed1989

1H-n.m.r. studies on the existence of substrate binding sites in bovine pancreatic ribonuclease A.

The reaction of ribonuclease A with either 6-chloropurine riboside 5'-monophosphate or the corresponding nucleoside yields one derivative, with the reagent covalently bound to the alpha-amino group of Lys-1, called derivative II and derivative E, respectively. We studied by means of 1H-n.m.r. at 270 MHz the interaction of these derivatives with different purine ligands. The pK values of His-12- and -119 were obtained and compared with those resulting from the interaction with ribonuclease A. The results showed that the interaction of derivative E with 3'AMP is similar to that described for RNase A as the pK2 of His-12 is increased while that of His-119 remains unaltered. However, derivative II presents some differences as it was found an enhancement of the pK2 values of both His-12 and His-119. Interaction of derivative II and derivative E with dApdA increases the pK2 of His-119, whereas a decrease is found when it interacts with ribonuclease A. These results suggest that the phosphate group and the nucleoside of both derivatives are located in regions of the enzyme where natural substrate analogues have secondary interactions and they can be interpreted as additional binding sites.

Adenosine Monophosphate↗
PubMed1988

500 MHz NMR characterization of synthetic bombesin and related peptides in DMSO-d6 by two-dimensional techniques.

The proton NMR characterization of bombesin has been carried out at 500 MHz in DMSO-d6 using two-dimensional homo- and 1H-13C hetero-correlated techniques. All resonances in the NMR spectra have been assigned and several coupling constants have been measured. The backbone J alpha CH-NH coupling constants have constant values that vary between 7.8 and 8.2 Hz and indicate an unfolded structure in DMSO-d6. Discrepancies with data recently obtained at 300 MHz [(1987) Eur. J. Biochem. 168, 193-199] are discussed.

Amino Acid Sequence↗
PubMed1988

H-n.m.r. studies on the specificity of the interaction between bovine pancreatic ribonuclease A and dideoxynucleoside monophosphates.

The titration curves of the C-2 histidine protons of bovine pancreatic ribonuclease A in the presence of several dideoxynucleoside monophosphates (dNpdN) were studied by means of proton nuclear magnetic resonance at 270 MHz in order to obtain information on the ligand--RNase A interaction. The changes in the chemical shift and pKs of the C-2 proton resonances of His-12, -48, -119 in the complexes RNase A--dNpdN were smaller than those previously found when the enzyme interacted with mononucleotides. The pK2 of His-12 was not affected by the interaction of the enzyme with these ligands, whereas, the perturbation of the pK2 of His-119 was clearly dependent on the nature of the ligand. If there is a pyrimidine nucleoside at the 3' side of the dideoxynucleoside monophosphates, as in TpdA and TpT, an enhancement due to the well known interaction of the phosphate in p1, the catalytic site, was found. However, when there is a purine nucleoside, as in dApT and dApdA, a decrease in the pK2 value was observed and we propose that in such cases the phosphate group interacts in a secondary phosphate binding site, p2. The results obtained suggest the existence of different specific interactions depending on the structure of the dideoxynucleoside monophosphate studied.

Deoxyribonucleotides↗
PubMed1988

Synthetic amatoxin analogues. II. A proton n.m.r. study of S-deoxo-Ile3-(L)Ala5 and S-deoxo-Ile3-(D)Ala5-amaninamide.

Amatoxin analogues with D and L-Ala substitutions in position 5 have been studied by means of 1- and 2-dimensional n.m.r. spectroscopy at 500 MHz. The assignment of all resonances for both analogues has been carried out mostly with the use of COSY and NOESY type experiments. Temperature coefficients for the amide NH protons have been measured and the data compared to known amatoxin structures. The results obtained demonstrate that the rigidity of the bicyclic amatoxin framework is preserved in the D and L-Ala5 analogues, although the temperature coefficients point to intramolecular hydrogen bonds stronger in the case of the L-Ala analogue. The 10-fold decrease of biological activity is discussed in terms of structural features involving also the Trp4 indole accessibility.

Amanitins↗
PubMed1986

Nuclear magnetic resonance of protamines. A 1H-NMR study of the interaction of clupeine fractions with mononucleotides.

The interaction of the three clupeine fractions, YI, YII, Z, and salmine fraction AI with mononucleotides has been examined by means of 1H nuclear magnetic resonance. The results obtained are interpreted in terms of electrostatic interactions between positive arginine guanidinyl groups and negative nucleotide phosphates. In addition, clupeine fraction YI and salmine fraction AI exhibit with guanine and adenine nucleotides a more specific interaction that leads to the formation of large aggregates in solution. The experimental data presented in this work demonstrate that the strength of interaction between clupeine YI and salmine AI with mononucleotides follows the order: 5'-dTMP approximately equal to 5'-dCMP much less than 5'-dAMP less than 5'-dGMP approximately equal to 5'-GMP.

Adamantane↗
PubMed1986

Synthetic amatoxin analogue. A two-dimensional proton NMR study of S-deoxy-(Ile3)-(D-Ala7)-amaninamide.

The effect of substitution of L and D amino acids in amatoxin analogues is discussed in this paper. The structure of the analog where D-alanine substitutes for glycine in position 7 has been worked out in solution by two-dimensional NMR methods using a 500 MHz instrument. The combined use of COSY and NOESY two-dimensional spectra allows a clear assignment of the resonances. The use of the coupling constants permits the calculation of the phi angles of the backbone. The NOE effects reveal the through-space contacts between protons of different peptide units, thus determining the rigidity of the amatoxin structure. On these grounds it has been possible to elucidate the conformation of the amatoxin analogue that resembles very closely that of beta-amanitin, thus explaining the high inhibitory activity toward RNA polymerase B.

Amanitins↗
PubMed1983

Comparative proton NMR studies of bovine semen and pancreas ribonucleases.

The fine structure of bovine semen RNAase was studied with proton NMR spectroscopy making use of the four-protein system constituted by dimeric bovine semen RNAase, its catalytically active monomeric bis-(S-carboxymethyl-31,32) derivative, the naturally monomeric RNAase A from the pancrease of the same species, and dimerized RNAase A. Only four histidine C-2 H resonances were observed in the aromatic spectrum of bovine semen RNAase, which belong to the four histidine residues present in the sequence of bovine semen RNAase subunits at positions identical with those of the histidines of RNAase A. This is indicative of identical environments for the individual histidine residues in both subunits. These resonances were assigned (i) by comparing their titration curves with the corresponding curves obtained with RNAase A and with monomeric bovine semen RNAase and (ii) by evaluating the effects on their titration curves of nucleotide binding. Very similar NMR parameters were measured for His-105 and also for His-119 of seminal and pancreatic RNAase, while His-12 was found to have different environments in the two proteins. The distinctive NMR features of His-48 in bovine semen RNAase confirmed the role of the hinge regions of the subunits in maintaining the dimeric structure of the protein. While monomerization of the seminal enzyme reduced the differences between the histidine C-2 H resonances of RNAase A and bovine semen RNAase, dimerization of RNAase A did not affect the NMR spectrum of this protein, thus indicating as unlikely the possibility that the quaternary structure of bovine semen RNAase resembles that of dimerized RNAase A.

Animals↗
PubMed1982

Evidence on the existence of a purine ligand induced conformational change in the active site of bovine pancreatic ribonuclease A studied by proton nuclear magnetic resonance spectroscopy.

The titration curves of the C-2 histidine protons of RNase A and of derivative II--a covalent derivative obtained by reaction of the enzyme with the halogenated nucleotide 9-beta-D-ribofuranosyl-6-chloropurine 5'-phosphate--in the presence of a number of purine nucleosides, nucleoside monophosphates, and nucleoside diphosphates were studied by means of proton nuclear magnetic resonance at 270 MHz. The examination of the perturbations found on the chemical shifts and pKs of the C-2 protons of His-12, -48, and -119 are consistent with the following conclusions: (1) The interaction of adenosine in the primary purine binding site of the enzyme (B2R2) induces a conformational change in the active center of the enzyme [for the nomenclature of the RNase A binding subsites, see Parés et al. [Parés, X., Llorens, R., Arús, C., & Cuchillo, C. M. (1980) Eur. J. Biochem. 105, 571-579]]. (2) The phosphate moiety of the ligands, independently of its position, probably acts as a general carrier of the nucleotide to the active center, while the substituents of the base are the generators of the specificity of the binding and control the binding equilibrium between subsites B2R2 and B1R1. (3) There is no overlapping between the binding sites occupied by the labeling nucleotide in derivative II (B3R3p2) and the primary binding site for purine mononucleotides (B2R2p1).

Adenosine↗
PubMed1982

Nuclear magnetic resonance of protamines. A 13C relaxation study of the three main fractions of clupeine.

The three main fractions of clupeine, the protamine extracted from herring sperm, have been investigated by 13C nuclear magnetic resonance techniques. The dynamic behaviour, examined through the evaluation of the spin lattice relaxation times (T1) of individual carbon resonances assigned to both backbone and side chains, reveals interesting features. The relaxation times of backbone alpha-carbons, interpreted on the basis of an axially symmetric ellipsoid, point to the clupeine fractions as being essentially extended in aqueous solution. These times remain constant along the polypeptide chain and are of the order of 0.16 +/- 0.02 s. Conversely, the side chains show different flexibilities in the presence of monophosphate counterions, thus demonstrating a diverging behaviour which may be biologically relevant. In particular, the side-chain flexibilities of fraction YI decrease, while those of fractions Z and YII are either constant or increase. Comparison of these data with the viscosity measurements helps in explaining the viscosity changes observed in the presence of phosphate.

Amino Acid Sequence↗
PubMed1981

1H-NMR studies on the binding subsites of bovine pancreatic ribonuclease A.

The titration curves of the C-2 histidine protons of an RNAase derivative (a covalent derivative obtained by reaction of bovine pancreatic RNAase A (EC 3.1.27.5) with 6-chloropurine 9-beta-D-ribofuranosyl 5'-monophosphate) were studied by means of 1H-NMR spectroscopy at 270 MHz. The interaction of natural (5'AMP, 5'GMP, 5'IMP) and halogenated purine mononucleotides (cl6RMP, br8AMP) with RNAase A was also monitored by using the same technique. The slight change observed in the pK values of the active centre histidine residues of the RNAase derivative, with respect to those in the native enzyme, can be considered as evidence that the phosphate of the label does not interact directly either with His-12 or 119 in the p1 site, but the p2 site as proposed previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571--579). Lys-7 and/or Arg-10 are proposed as part of the p2 phosphate-binding subsite. The pK values of His-12 and 119 and the shift of an aromatic resonance of the native enzyme found on interaction with some purine nucleotides, can be interpreted by postulating that the interaction of 5'AMP, 5'GMP and 5'IMP takes place not only in the so-called purine-binding site B2R2p1 but also in the primary pyrimidine-binding site B1R1 and p0 of RNAase A.

Animals↗
PubMed1979

13C Nuclear magnetic resonance of protamines. The three main components of clupeine.

The three main components (called YI, YII and Z) of clupeine a protamine from herring have been isolated and characterized by 13C nuclear magnetic resonance. Resonance belonging to the many different amino acid carbon atoms are observable and assigned. The effect of urea on the clupeine fractions points to the molecules being essentially in extended form in 2H2O solutions. Unusual properties of clupeine fractions can be observed in the presence of phosphate. Two of the fractions, i.e. YI and Z, show that a partial folding occurs in some well-defined whereas fraction YII remains essentially unaltered.

Animals↗
PubMed1976

A study of calf-thymus histone H2B using 13C magnetic resonance.

Calf thymus histone H2B has been studied by means of 13c magnetic resonance. Most sidechain resonances have been assigned. Analysis of 13c peak intensities by a simple computer-aided method proves that 13c magnetic resonance spectroscopy can be used to monitor aggregation phenomena more accurately than 1H magnetic resonance or other physico-chemical methods. Re-examination of 1H spectral simulations shows substantial agreement with 13C results. It is concluded that in 0.1M added NaCL the H2b aggregates include the chain segment from 50 (+/-5) to 108 (+/-5).

Animals↗