Biomedical subjects

J Parello

Publications and source records attributed to J Parello.

At least 19 recordsLinked to original sources

PubMed2001

Photochemical rearrangement of oxaziridines and nitrones in the hexahydroindole series: a convenient synthetic route to 1-azabicyclo[5.2.0]nonan-2-ones as novel RGD mimetics.

[reaction: see text] Photolysis of oxaziridines a or nitrones b provides a convenient synthetic route to fused bicyclic lactams c adequately substituted on both cycles A and B as scaffolds for mimicking conformationally constrained beta-turn peptides as in the tripeptide RGD signaling motif of fibronectin.

Bridged Bicyclo Compounds↗

PubMed2000

Leukotriene B4 photoaffinity probes: design, synthesis and evaluation of new arylazide-1,3-disubstituted cyclohexanes.

The synthesis and the binding affinities of new leukotriene B4 receptor photoaffinity probes, where a 1,3-disubstituted cyclohexane ring replaces the conjugated delta6,7 and delta8,9 double bonds of the natural eicosanoid, are described. One enantiomeric compound, 4b alpha, is specifically cross-linked upon photolysis to the recombinant leukotriene B4 receptor from human origin (h-BLTR) solubilized in a micellar medium. This probe appears as a good candidate for identifying the ligand binding site of this receptor.

Cyclohexanes↗

PubMed2000

A minimized human integrin alpha(5)beta(1) that retains ligand recognition.

Two isolated recombinant fragments from human integrin alpha(5)beta(1) encompassing the FG-GAP repeats III to VII of alpha(5) and the insertion-type domain from beta(1), respectively, are structurally well defined in solution, based on CD evidence. Divalent cation binding induces a conformational adaptation that is achieved by Ca(2+) or Mg(2+) (or Mn(2+)) with alpha(5) and only by Mg(2+) (or Mn(2+)) with beta(1). Mn(2+) bound to beta(1) is highly hydrated ( approximately 3 water molecules), based on water NMR relaxation, in agreement with a metal ion-dependent adhesion site-type metal coordination. Each fragment saturated with Mg(2+) (or Mn(2+)) binds a recombinant fibronectin ligand in an RGD-dependent manner. A conformational rearrangement is induced on the fibronectin ligand upon binding to the alpha(5), but not to the beta(1) fragment, based on CD. Ligand binding results in metal ion displacement from beta(1). Both alpha(5) and beta(1) fragments form a stable heterodimer (alpha(5)beta(1) mini-integrin) that retains ligand recognition to form a 1:1:1 ternary complex, in the presence of Mg(2+), and induces a specific conformational adaptation of the fibronectin ligand. A two-site model for RGD binding to both alpha and beta integrin components is inferred from our data using low molecular weight RGD mimetics.

Calcium↗

PubMed1999

Ca2+/Mg2+ exchange in parvalbumin and other EF-hand proteins. A theoretical study.

A remarkable conformational rearrangement occurs upon Ca2+/Mg2+ exchange in the C-terminal EF-hand site (labelled site EF or EF-4) of parvalbumin, as initially established by X-ray crystallography. Such a conformational rearrangement is characterised as follows: (i) the co-ordination number decreases from seven oxygen atoms in the Ca-loaded form to six oxygen atoms in the Mg-loaded form, the heptaco-ordination of Ca2+ corresponding with a skewed pentagonal bipyramid configuration of the seven oxygen atoms, whereas the hexaco-ordination of Mg2+ corresponds with a regular octahedral configuration of the six oxygen atoms; and (ii) Glu101, at the relative position 12 in the EF-hand loop sequence (labelled "Glu12"), acts as a bidentate ligand in the Ca-loaded form and as a monodentate ligand in the Mg-loaded form. As part of the conformational rearrangement, the chi1 dihedral angle undergoes a gauche(+) to gauche(-) transition upon substitution of Ca2+ by Mg2+, whereas the chi2 angle remains practically unchanged and the chi3 angles in both forms adopt a nearly mirror image relationship. In order to understand the molecular mechanisms underlying such a conformational rearrangement, we undertook a theoretical study using the free energy perturbation (FEP) method, starting from high-resolution crystal structures of the same parvalbumin (pike 4. 10 isoform) differing by the substitution of their two cationic sites EF-3 (or CD) and EF-4 (or EF), i.e. the 1pal structure with EF-3(Ca2+) and EF-4(Ca2+), the 4pal structure with EF-3(Ca2+) and EF-4(Mg2+). When Mg2+ is "alchemically" transformed into Ca2+ within the EF-4 site of 4pal, the conformational rearrangement of Glu12 is correctly predicted by the FEP calculation. When Ca2+ is transformed into Mg2+ within the EF-3 site of 4pal, the FEP calculation predicts the topology of the fully Mg-loaded form for which no crystallographic data is presently available. As expected, Glu62 (at the relative position 12 in EF-3 loop) is predicted to be a monodentate residue within a regular octahedral arrangement of six oxygen atoms around Mg2+. We also investigated the behaviour during Ca2+/Mg2+ exchange of two other typical EF-hand proteins, troponin C (TnC) and calmodulin (CaM), for which no three-dimensional structure of their Mg-loaded forms is available so far. It is also predicted that the EF-3 site of TnC and the EF-1 site of CaM have their invariant Glu12 residues switching from the bidentate to the monodentate configuration when Ca2+ is substituted by Mg2+, with six oxygen atoms being observed in the co-ordination sphere of the alchemically generated Mg2+ cation.

Binding Sites↗

PubMed1999

Hydration-coupled dynamics in proteins studied by neutron scattering and NMR: the case of the typical EF-hand calcium-binding parvalbumin.

The influence of hydration on the internal dynamics of a typical EF-hand calciprotein, parvalbumin, was investigated by incoherent quasi-elastic neutron scattering (IQNS) and solid-state 13C-NMR spectroscopy using the powdered protein at different hydration levels. Both approaches establish an increase in protein dynamics upon progressive hydration above a threshold that only corresponds to partial coverage of the protein surface by the water molecules. Selective motions are apparent by NMR in the 10-ns time scale at the level of the polar lysyl side chains (externally located), as well as of more internally located side chains (from Ala and Ile), whereas IQNS monitors diffusive motions of hydrogen atoms in the protein at time scales up to 20 ps. Hydration-induced dynamics at the level of the abundant lysyl residues mainly involve the ammonium extremity of the side chain, as shown by NMR. The combined results suggest that peripheral water-protein interactions influence the protein dynamics in a global manner. There is a progressive induction of mobility at increasing hydration from the periphery toward the protein interior. This study gives a microscopic view of the structural and dynamic events following the hydration of a globular protein.

Alanine↗

PubMed1999

Crystal structure of the EF-hand parvalbumin at atomic resolution (0.91 A) and at low temperature (100 K). Evidence for conformational multistates within the hydrophobic core.

Several crystal structures of parvalbumin (Parv), a typical EF-hand protein, have been reported so far for different species with the best resolution achieving 1.5 A. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+-loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 A). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein three-dimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well-defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 A3. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy-barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a time-dependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed.

Binding Sites↗

PubMed1999

Two well-defined motifs in the cAMP-dependent protein kinase inhibitor (PKIalpha) correlate with inhibitory and nuclear export function.

The heat stable inhibitor of cAMP-dependent protein kinase (PKIalpha) contains both a nuclear export signal (NES) and a high affinity inhibitory region that is essential for inhibition of the catalytic subunit of the kinase. These functions are sequentially independent. Two-dimensional NMR spectroscopy was performed on uniformly [15N]-labeled PKIalpha to examine its structure free in solution. Seventy out of 75 residues were identified, and examination of the CaH chemical shifts revealed two regions of upfield chemical shifts characteristic of alpha-helices. When PKIalpha was fragmented into two functionally distinct peptides for study at higher concentrations, no significant alterations in chemical shifts or secondary structure were observed. The first ordered region, identified in PKIalpha (1-25), contains an alpha-helix from residues 1-13. This helix extends by one turn the helix observed in the crystal structure of a PKIalpha (5-24) peptide bound to the catalytic subunit. The second region of well-defined secondary structure, residues 35-47, overlaps with the nuclear export signal in the PKIalpha (26-75) fragment. This secondary structure consists of a helix with a hydrophobic face comprised of Leu37, Leu41, and Leu44, followed by a flexible turn containing Ile46. These four residues are critical for nuclear export function. The remainder of the protein in solution appears relatively unstructured, and this lack of structure surrounding a few essential and well-defined signaling elements may be characteristic of a growing family of small regulatory proteins that interact with protein kinases.

Amino Acid Sequence↗

PubMed1998

The cation-binding domain from the alpha subunit of integrin alpha5 beta1 is a minimal domain for fibronectin recognition.

The cation-binding domain from the alpha subunit of human integrin alpha5beta1 was produced as a recombinant protein, alpha5-(229-448). This protein displays a well defined fold with a content of 30-35% alpha-helix and 20-25% beta-strand, based on circular dichroism. The binding of Ca2+ or Mg2+ to alpha5-(229-448) results in a biphasic conformational rearrangement consistent with the occurrence of two classes of cation-binding sites differing by their affinities. The two classes of sites are located in two conformationally independent lobes, as established by a parallel study of two recombinant half-domains (N- and C-terminal) that also adopt stable folds. Upon saturation with divalent cations, alpha5-(229-448) binds an Arg-Gly-Asp (RGD)-containing fibronectin ligand to form a 1:1 complex. Complex formation is associated with a specific conformational adaptation of the ligand, suggesting an induced fit mechanism. In contrast, neither of the half-domains is competent for ligand binding. The alpha5-(229-448)-fibronectin complex is dissociated in the presence of an RGD peptide, as well as of a simple carboxylic acid, suggesting that the RGD aspartyl carboxylate is an essential element that directly interacts with the alpha5 cation-binding domain.

Amino Acid Sequence↗

PubMed1997

The N tails of histones H3 and H4 adopt a highly structured conformation in the nucleosome.

The histone N tails correspond to conserved amino acid sequences that are peripherally located in the nucleosome and undergo a variety of post-synthetic modifications during cell cycle. These N tails have been recently recognized as directly interacting with transcription-related proteins. We show here, based on circular dichroic evidence, that the N tails of both tetrameric histones H3 and H4 are highly organized as DNA-bound polypeptide segments in the nucleosome core particle, with about half of their residues, taken together, being alpha-helical. In contrast, the N tails of both dimeric histones H2A and H2B are found essentially in a random-coil conformation. The implications of these findings on nucleosome structure and recognition are discussed.

Animals↗

PubMed1997

Solution structure of synthetic peptide inhibitor and substrate of cAMP-dependent protein kinase. A study by 2D H NMR and molecular dynamics.

Peptides derived from the inhibitor of cAMP-dependent protein kinase. PKI, have been studied by 2D 1H NMR techniques. These include the inhibitor PKI(6-22), the substrate [Ala20-Ser21]PKI(5-24), and a phosphorylated form of the latter [Ala20-Ser21P]PKI(5-24). A homologous fold was found in the three peptides which consisted of an N-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI(5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI(5-24) complex, was conserved in the MD simulations with the enzyme-free inhibitor. Similarly the Gly14-Gly17 turn was apparent in all MD structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the N-terminal helix in the crystal structure. This is primarily due to ionic interaction between Asp9 and Arg15. Indeed, a detailed analysis of the NOE contacts by NOESY at low temperature (2 degrees C) shows the occurrence of pH-dependent contacts with Phe10. We conclude that the binding of short inhibitors, such as PKI(5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate [Ala20-Ser21]PKI(5-24) and the product [Ala20-Ser21P]PKI(5-24), give very similar structures with local rearrangements involving some of the side chains.

Amino Acid Sequence↗

PubMed1996

X-ray structure of a new crystal form of pike 4.10 beta parvalbumin.

A new crystal form of pike (pI 4.10) parvalbumin has been crystallized in presence of EDTA at pH 8.0. The crystals are orthorhombic, space group P2(1)2(1)2(1), with a = 51.84, b = 49.95, c = 34.96 A. Diffractometer data were collected to 1.75 A. The structure was solved by molecular replacement and refined to R = 0.168 for 7774 observed reflections [I>/= 2sigma(I)] in the range 8.0-1.75 A. In spite of the presence of EDTA, calcium ions are present in both primary binding sites. As compared to the previously reported structures, the main differences concern the conformation of the N-terminal residues and the packing in the unit cell.

Journal Article↗

PubMed1994

Evidence indicating proximity in the nucleosome between the histone H4 N termini and the globular domain of histone H1.

Proteolysis of rat liver chromatin by the Arg-C peptidase, clostripain, is characterized by a progressive fragmentation of the N-terminal segments of the four core histones H2A, H2B, H3 and H4, until a well-defined limit digest is reached. This work addresses the case of histone H4. Two intermediate proteolytic sites are identified for this histone, i.e. Arg3 and Arg17, before the limit digest is achieved through cleavage of the polypeptide chain after Arg19. The accessibility of these intermediate sites depends strongly on the presence or absence of histone H1. When H1 is absent, both intermediate sites of histone H4 are similarly accessible, whereas one of them, Arg3, becomes totally inaccessible in the presence of histone H1. Di- and trinucleosomes were used with the aim of avoiding any interference with superstructural effects which can occur with longer polynucleosomes in the presence of H1. We also investigated the accessibility of the Arg sites of H1 that are located primarily in the central globular domain of this histone. In free histone H1, all the centrally located Arg sites are accessible to clostripain. In contrast, in the chromatin-bound state none of these sites is accessible. Besides the arginyl sites in the central globular domain of H1, two Arg residues are observed with the most abundant H1d variant in rat chromatin, one in the N-terminal region and the other in the C-terminal region. The restricted number of proteolytic fragments observed with chromatin-bound H1 is accounted for by the cleavage of H1 after these Arg residues located on the outside of the globular domain. Our results suggest that mutual steric effects are at play between histones H1 and H4 and indicate that the N termini of both histones H4 in the nucleosome lie in close proximity to the globular domain of H1. Based on these observations and taking into account the known structural features of the nucleosome, we propose a model for positioning the N-terminal segments of both histones H4 at the periphery of the nucleoprotein structure. In this model both H4 segments are located within the expanded DNA minor grooves, at periods +/- 1, symmetrically disposed relatively to the nucleosome dyad axis. This arrangement brings the amino ends of both H4 molecules in close contact with the H1 globular domain thus accounting for the observed inaccessibility of the Arg3 site of H4 in the presence of H1.

Amino Acid Sequence↗

PubMed1994

Proton and nitrogen NMR sequence-specific assignments and secondary structure determination of the Bacillus subtilis SPO1-encoded transcription factor 1.

Sequence-specific 1H and 15N NMR1 assignments are reported for the transcription factor 1 (TF1), a 22-kDa type II DNA-binding protein (DBPII) that consists of two 99-residue monomers. An assignment strategy is employed that uses six complementary selectively deuterium-labeled TF1 variants and an uniformly 15N-labeled TF1 variant. Two-dimensional and three-dimensional homonuclear and heteronuclear NMR correlated spectra are analyzed and yield nearly complete assignments for the 1H and 15N resonances. Discrete protein secondary structure domains are also defined; in each monomer, three alpha-helices, an antiparallel beta-sheet, and an antiparallel beta-ribbon are identified. Analyses of two dimers formed from two distinct selectively deuteriated monomers serve to identify a number of interproton contacts as either intermonomeric or intramonomeric. An analysis of amide proton exchange reveals that the carboxy-terminal alpha-helix is less stable than the other two alpha-helices in each monomer. A previously proposed working structural model of the TF1 dimer [Geiduschek et al. (1990) J. Struct. Biol. 104, 84-90], based on the crystal structure of a highly homologous DBPII, the Bacillus stearothermophilus-encoded HU protein, is generally supported by our results. Several departures from this model, however, are noted. Most notably, the carboxy-terminal tail of TF1 adopts an alpha-helical conformation with a backbone distortion at Lys93.

Amino Acid Sequence↗

PubMed1994

Crystals of the cell-binding module of fibronectin obtained from a series of recombinant fragments differing in length.

A recombinant fragment corresponding to the cell adhesion module (FNIII10) of human fibronectin has been crystallized at pH 8.6 from solutions containing polyethylene glycol as precipitant. The crystals formed in the space group P2(1) with a = 30.76 A, b = 35.07 A, c = 37.66 A, beta = 106.9 degrees. There is one molecule per asymmetric unit and the crystals diffract beyond 1.75 A resolution. To improve the prospects for successful crystallization of the FNIII10 module, a series of recombinant fragments was produced with minor differences in the length of N or C-terminal segments. Only one of these variants crystallized. Interestingly, the C-terminal residue of this variant formed stable intermolecular contacts with a symmetry-related molecule in the crystal lattice.

Amino Acid Sequence↗

PubMed1993

A 1H-NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective 2H-labeling. Complete assignment and structural analysis of the aromatic resonances for a 22-kDa homodimer.

1H-NMR experiments have been performed on transcription factor 1 (TF1) encoded by Bacillus subtilis phage SPO1. To study this 22-kDa homodimeric DNA-binding protein, a selective 2H-labeling strategy has been employed. Complete sequence-specific assignments of all the resonances from the five aromatic residues were determined by a modified standard sequential-assignment procedure. The reduced contribution of spin diffusion upon the long-mixing-time nuclear-Overhauser-enhancement spectroscopy for the selectively 2H-labeled variants, as opposed to the fully 1H-containing protein, has allowed for the identification of the spin systems and of the long-range dipolar contacts between Phe28 and Phe47 protons in the protein core and between Phe61 and Phe97 protons. The latter suggests an interaction between the proposed beta-ribbon DNA-binding arm and the carboxy terminus of the paired monomer. A previously proposed TF1 structural model [Geiduschek, E. P., Schneider, G. J. & Sayre, M. H. (1990) J. Struct. Biol. 104, 84-90)] has been modified using constrained-energy-minimization calculations incorporating the experimentally determined set of aromatic-to-aromatic contacts. This new model has been analyzed with regard to the relative mobility and the relative solvent accessibility of the aromatic residues which have been measured by the nonselective T1 relaxation times of the aromatic resonances for the fully 1H-containing protein and the relaxation time enhancements upon selective 2H-labeling, respectively.

Amino Acid Sequence↗

PubMed1993

Symmetrical rearrangement of the cation-binding sites of parvalbumin upon Ca2+/Mg2+ exchange. A study by 1H 2D NMR.

Two forms of parvalbumin, i.e., the fully Ca-loaded form PaCa2 and the fully Mg-loaded form PaMg2, are investigated by 2D 1H NMR in solution. A detailed analysis of the resonances, which belong to residues involved in direct coordination of Ca2+ and Mg2+, establishes that the sixth ligand, a highly conserved Glu residue at the relative position 12 in both cation-binding sites CD and EF, undergoes a conformational rearrangement through a 120 degrees rotation of its side chain about the C alpha-C beta bond with PaMg2 adopting the less energetically favored g- conformation, as inferred from scalar coupling constants and dipole-dipole contacts measured on the COSY and NOESY spectra, respectively. Similarly, chemical shift effects, which selectively involve NH and C alpha H resonances (as well as side-chain resonances) in both CD and EF sites, point to a symmetrical behavior of both cation-binding sites upon Ca2+/Mg2+ exchange.

Animals↗

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↗