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J Parello

Publications and source records attributed to J Parello.

At least 19 recordsLinked to original sources
PubMed1992

Crystallization studies of the catalytic subunit of cAMP-dependent protein kinase: crystals of murine recombinant catalytic subunit and a mutant, Cys 343----Ser, diffract to 2.7 A resolution.

The recombinant mouse catalytic subunit of cAMP-dependent protein kinase, expressed and purified from E. coli, has been successfully cocrystallized as a binary complex with an inhibitor peptide and as a ternary complex with an inhibitor peptide and MgATP. In contrast to the catalytic subunit obtained from porcine heart, the recombinant catalytic subunit lacks a myristoyl group at the amino terminus and differs in sequence at nine positions out of 350 amino acids. The catalytic activities of the two enzymes, however, are nearly identical. Both enzymes cocrystallized with a 20-amino-acid inhibitor and MgATP; however, the porcine-heart enzyme crystallized in a hexagonal space group (P6(1)22) while the recombinant murine catalytic subunit crystallized in an orthorhombic space group (P2(1)2(1)2(1), a = 73.70, b = 76.26, c = 80.74 A). The orthorhombic crystals of the recombinant catalytic subunit exhibit the best diffraction characteristics of all catalytic subunit crystals obtained so far: 2.7 A resolution. Unlike the mammalian porcine-heart enzyme, no crystals of the recombinant apo-enzyme were obtained under the same crystallization conditions. These results are consistent with earlier conclusions that the catalytic subunit exists in at least two distinct conformational states and furthermore suggests that the inhibitor peptide alone is sufficient to induce the major conformational changes that distinguish the two states. A mutant form of the catalytic subunit where Cys343 was replaced with Ser was also cocrystallized with the 20-amino-acid peptide inhibitor and MgATP, and resulted in an orthorhombic crystal isomorphous to crystals of the unmutated enzyme with a similar diffraction of 2.7 A.

Amino Acid Sequence↗
PubMed1992

Crystal structure of the unique parvalbumin component from muscle of the leopard shark (Triakis semifasciata). The first X-ray study of an alpha-parvalbumin.

The three-dimensional structure of parvalbumin from leopard shark (Triakis semifasciata) with 109 amino acid residues (alpha-series) is described at 1.54 A resolution. Crystals were grown at 20 degrees C from 2.9 M-potassium/sodium phosphate solutions at pH 5.6. The space group is P3(1)21 and unit cell dimensions are a = b = 32.12 A and c = 149.0 A. The structure has been solved by the molecular replacement method using pike 4.10 parvalbumin as a model. The final structure refinement resulted in an R-factor of 17.3% for 11,363 independent reflections at 1.54 A resolution. The shark parvalbumin shows the main features of all parvalbumins: the folding of the chain including six alpha-helices, the salt bridge between Arg75 and Glu81, and the hydrophobic core. Compared to the structure of beta-parvalbumins from pike and carp, one main difference is observed: the chain is one residue longer and this additional residue, which extends the F helix, is involved through its C-terminal carboxylate group in a network of electrostatic contacts with two basic residues, His31 in the B helix and Lys36 in the BC segment. Furthermore, hydrogen bonds exist between the side-chains of Gln108 (F helix) and Tyr26 (B helix). There is therefore a "locking" of the tertiary structure through contacts between two sequentially distant regions in the protein and this is likely to contribute to making the stability of an alpha-parvalbumin higher in comparison to that of a beta-parvalbumin. The lengthening of the C-terminal F helix by one residue appears to be a major feature of alpha-parvalbumins in general, owing to the homologies of the amino acid sequences. Besides the lengthening of the C-terminal helix, the classification of the leopard shark parvalbumin in the alpha-series rests upon the observation of Lys13, Leu32, Glu61 and Val66. As this is the first crystal structure description of a parvalbumin from the alpha-phylogenetic lineage, it was hoped that it would clearly determine the presence or absence of a third cation binding site in parvalbumins belonging to the alpha-lineage. In beta-pike pI 4.10 parvalbumin, Asp61 participates as a direct ligand of a third site, the satellite of the CD site. In shark parvalbumin, as in nearly all alpha-parvalbumins, one finds Glu at position 61. Unfortunately, the conformation of the polar head of Glu61 cannot be inferred from the X-ray data.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Ionic interactions with parvalbumins. Crystal structure determination of pike 4.10 parvalbumin in four different ionic environments.

The crystal structure of the Ca-loaded form of pike 4.10 parvalbumin (minor component from pike muscle belonging to the beta phylogenetic series), with both its primary sites CD and EF occupied by Ca2+ ions and its third site occupied by an ammonium ion, as previously determined at 1.93 A resolution, has now been refined to a resolution of 1.65 A. The crystallization of this parvalbumin in different ionic environments has allowed three novel non-isomorphous crystalline forms to be obtained: (1) a first form, crystallized in the presence of a mixture of ammonium sulphate and manganese sulphate, for which all the cation binding sites in the protein are occupied by Mn2+; (2) a second form crystallized in the presence of MgSO4 as the precipitating agent, only differs from the Ca/NH4 form by the occupation of the third site by Mg2+, whereas the primary sites remain occupied by Ca2+; (3) a third form, also crystallized in the presence of MgSO4, corresponds to a well-defined molecular species with both the primary EF site and the third site occupied by Mg2+, whereas the primary CD site remains occupied by CA2+. The corresponding molecular structures reported here have been determined at resolutions between 1.8 and 2.4 A. The comparison of the different crystal structures allows the structural modifications accompanying the substitution of the primary sites by cations differing significantly in their ionic radii (Ca2+, Mn2+, Mg2+) to be investigated in detail, and it also leads to a precise description of the third site in a typical beta parvalbumin. The substitution Ca2+ by Mg2+ within the primary site EF is characterized by a "contraction" of the co-ordination sphere, with a decrease of the mean oxygen-metal distance by a value of 0.25 A and a decrease of the co-ordination number from 7 to 6, as a consequence of the loss of a bidentate ligand (Glu101), which becomes a monodentate one. Such an adaptation of the co-ordination sphere around a cation of smaller size involves, among others, the transformation of the Glu101 side-chain from the stable gauche(+) form to the less stable gauche(-) form. The third site is clearly described as a satellite of the CD primary site, since both sites possess common protein ligands, such as Asp53 and Glu59. Furthermore, Asp61 appears as a specific ligand of the third site in the different environments investigated in this work. We finally discuss the relevance of the third site to parvalbumin phylogeny.

Ammonia↗
PubMed1988

Two-dimensional 1H nuclear magnetic resonance study of pike pI 5.0 parvalbumin (Esox lucius). Sequential resonance assignments and folding of the polypeptide chain.

The structure of alpha pike 5.0 parvalbumin under its Ca-loaded form (or PaCa2) is studied in solution by two-dimensional 1H nuclear magnetic resonance (n.m.r.) at 360 MHz using a conventional strategy of sequential assignments, which involved correlated spectroscopy, relayed coherence transfer spectroscopy and nuclear Overhauser enhancement spectroscopy. In order to overcome the problem of spectral overlapping due to the presence of 108 residues in the protein, experiments were performed at different pH and temperature values, either in 1H2O or in 2H2O solutions. The amino acid sequence of pike 5.0 parvalbumin is thus fully characterized by nearly the totality of its NH, C alpha H and C beta H resonances originating from the different residues (421 protons assigned among 429 in total). When associated with the remaining side resonances, these sequence-specific assignments provide a basis for establishing the secondary organization and tertiary folding of the polypeptide chain. Pike 5.0 parvalbumin was selected as a characteristic representative of the alpha phylogenic series, for which no crystalline structure is presently available, in contrast with the beta series for which two crystalline structures have been determined. A parvalbumin molecule with a single polypeptide chain of 108 amino acids represents one of the highest molecular weights analyzed so far by two-dimensional n.m.r. spectroscopy. The use of a moderate magnetic field strength, with 1H nuclei resonating at 360 MHz, is justified by the fact that ring current effects are operating favorably in this globular protein with a high phenylalanine content. A three-dimensional structure has been generated by the "distance geometry" or DISGEO computational procedure on the basis of about 450 interproton nuclear Overhauser enhancement connectives (short, medium and long-range) in conjunction with a selection of phi and chi dihedral angle constraints. The coherence of the calculated structure, which displays all the features of the typical folding of a parvalbumin protein, provides a good test of reliability of the n.m.r. data collected so far. Although similar to a beta parvalbumin in the folding of its polypeptide chain, the alpha parvalbumin studied here differs markedly from a beta parvalbumin in the length of its C-terminal F-helix domain, which includes 11 residues instead of ten in the latter.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1988

Chromatin core particle obtained by selective cleavage of histones H3 and H4 by clostripain.

A new chromatin core particle characterized by a half-proteolyzed octamer is obtained by controlled digestion of the native core particle by clostripain. The proteolyzed histones correspond to four polypeptide fragments which are tentatively assigned to H2A[4-129], H2B[1-125], H3[27-135] and H4[18-102] on the basis of electrophoretic evidence and the known specificity of clostripain for arginyl residues. Despite the loss of the N-terminal regions of histones H3 and H4, the partially proteolyzed core particle retains the structural conformation of the native one as shown by circular dichroism. As expected, this half-proteolyzed core particle presents an intermediate accessibility to polycations, such as spermidine, in comparison with that observed with the native core particle and a fully proteolyzed core particle. The latter includes the polypeptide fragments H2A[12-129], H2B[21-125], H3[27-135] and H4[20-102].

Amino Acid Sequence↗
PubMed1988

Crystal structure determination and refinement of pike 4.10 parvalbumin (minor component from Esox lucius).

The crystal and molecular structure of the minor component of pike parvalbumins has been determined at 1.93 A resolution by molecular replacement (1 A = 0.1 nm). The crystals are orthorhombic, space group P2(1)2(1)2 with a = 59.62 A, b = 59.83 A and c = 26.35 A. A location of the secondary cation binding site is proposed for this parvalbumin of the beta phylogenetic series.

Amino Acid Sequence↗
PubMed1987

Synthesis of (pGlu-5, MePhe-8, Sar-9) substance P (5-11) (DiMe-C7) using a polyacrylamide resin and biological activity on guinea pig ileum and tracheal smooth muscle.

DiMe-C7 (pGlu-Gln-Phe-MePhe-MeGly-Leu-MetNH2), a metabolically stable analogue of Substance P, was prepared by solid-phase peptide synthesis using a polyacrylamide resin and a labile anchorage derived from glycolic acid. Myotropic activities in guinea pig ileum (ED50 = 4.0 +/- 1.5 10(-8) M) and guinea pig trachea (ED50 = 8.6 +/- 3.5 10(-8) M) are discussed in comparison with the corresponding activities of Substance P.

Animals↗
PubMed1986

A chromatin core particle obtained by selective cleavage of histones by clostripain.

Rat liver chromatin core particles digested with clostripain yield a structurally well-defined nucleoprotein particle with an octameric core made up of fragmented histone species (designated H'2A, H'2B, H'3 and H'4, respectively) after selective loss of a sequence segment located in the N-terminal region of each core histone. Sequential Edman degradation and carboxypeptidase digestion unambiguously establish that histones H2A, H2B, H3 and H4 are selectively cleaved at the carboxyl side of Arg 11, Lys 20, Arg 26 and Arg 19 respectively and that the C-terminal sequences remain unaffected. Despite the loss of the highly basic N-terminal regions, including approximately 17% of the total amino acids, the characteristic structural organization of the nucleosome core particle appears to be fully retained in the proteolyzed core particle, as judged by physicochemical and biochemical evidence. Binding of spermidine to native and proteolyzed core particles shows that DNA accessibility differs markedly in both structures. As expected the proteolyzed particle, which has lost all the in vivo acetylation sites, is not enzymatically acetylated, in contrast to the native particle. However, proteolyzed histones act as substrates of the acetyltransferase in the absence of DNA, as a consequence of the occurrence of potential acetylation sites in the core histones thus rendered accessible. The possible role of the histone N-terminal regions on chromatin structure and function is discussed in the light of the present observations with the new core particle obtained by clostripain proteolysis.

Amino Acid Sequence↗
PubMed1985

Metal-ion binding to parvalbumin. A 113Cd-n.m.r. study of the binding of different lanthanide ions.

113Cd-n.m.r. studies were used to investigate the binding of the lanthanide ions La3+, Gd3+, Tb3+, Yb3+ and Lu3+ to parvalbumins. It was shown that lanthanide ions with a smaller ionic radius bind sequentially to Cd2+-saturated parvalbumin, whereas those with a larger ionic radius bind with similar affinity to both the CD site and the EF site. The smallest ion, Lu3+, does in fact not compete significantly with Cd2+ for the CD site in carp parvalbumin, but appears to bind only to the EF site. This preference of the smaller lanthanide ions for the EF site was used to assign the n.m.r. signals for protein-bound 113Cd. By using Cd n.m.r. and Tb3+ fluorescence it was also shown for alpha-lineage parvalbumin from pike that these proteins possess a third site that can bind lanthanide ions. This site is, however, much weaker than in the beta-lineage parvalbumins. It was used to assign the 113Cd resonances from protein-bound Cd2+ ions in the spectrum of pike pI5.0 parvalbumin.

Animals↗
PubMed1984

[A new particle of chromatin obtained by proteolysis of histones with clostripain].

The proteolysis of the chromatin core particle by the arginine-specific endopeptidase clostripain yields a new nucleoprotein particle containing an unaltered DNA fragment of about 145 base pairs in length and a protein octameric core made up of twice the four histone fragments H2A, H2B, H3 and H4. This composition is suggested by the molecular weight of about 180 kd determined for the new particle by small angle neutron scattering. The histone fragments differ by about 2-3 kd each from the initial histones H2A, H2B, H3 and H4 and they correspond to the cleavage of the N-terminal part of the sequence (20-30 residues). A preliminary investigation by thermal denaturation, circular dichroism and small angle neutron scattering (measurement of a radius of gyration by the H2O-D2O contrast variation technique) indicates that the spatial organization of the new chromatin particle closely resembles that of the initial core particle.

Chemical Phenomena↗
PubMed1982

NMR studies on parvalbumin phylogeny and ionic interactions.

The inspection of several muscular parvalbumins from different species by two NMR methods (113Cd resonance and 1H relaxation measurements) allows two classes of parvalbumins to be distinguished according to their ion-binding properties. This result is in agreement with the phylogenetic classification of parvalbumins in two series, alpha and beta, which was established on the basis of the primary structures of these proteins. All parvalbumins are characterized by the presence of two primary cationic sites CD and EF, with structural features closely related to those already known on the basis of X-ray crystallographic studies of the beta parvalbumin pI 4.25 from carp muscle. However, parvalbumins of the beta series are characterized by a secondary cation (Ca2+, Mg2+ and other cations) binding site which is absent (or at least inaccessible) in parvalbumins of the alpha series. The major component from thornback ray (pI 4.45) behaves as an alpha parvalbumin as shown by the present NMR studies, although its primary structure suggests a closer similarity with the parvalbumins of the beta series.

Animals↗
PubMed1982

Effect of cations on the acetylation of chromatin in vitro.

The effect of the differently charged cations Na+, Mg2+, spermidine and spermine on the acetylation of histones in vitro in soluble chromatin and in core particles was investigated. Up to a given concentration, which depends on the charge of the cation, all four cations activate the acetylation of these fractions. Above this critical concentration a gradual inhibition of the acetyltransferase activity occurs. Spermine, spermidine and Mg2+, but not Na+, affect the relative accessibilities of the acetyltransferase to the individual histones. As the concentration of these three polyvalent cations increases there is a gradual increase in the relative acetyl incorporation in histones H3 accompanied by a corresponding decrease in H4. Kinetic aspects of the system were also studied. A minor fraction of the soluble chromatin (mononucleosomes and dinucleosomes) which is preferentially digested by micrococcal nuclease is also acetylated preferentially. The role chromatin structure plays in determining the specificity of histone acetylation is discussed.

Acetylation↗