Biomedical subjects

Alessandro Pintar

Publications and source records attributed to Alessandro Pintar.

12 recordsLinked to original sources

PubMed2005

Gene synthesis, expression, purification, and characterization of human Jagged-1 intracellular region.

Notch signaling plays a key role in cell differentiation and is very well conserved from Drosophila to humans. Ligands of Notch receptors are type I, membrane spanning proteins composed of a large extracellular region and a 100-150 residue cytoplasmic tail. We report here, for the first time, the expression, purification, and characterization of the intracellular region of a Notch ligand. Starting from a set of synthetic oligonucleotides, we assembled a synthetic gene optimized for Escherichia coli codon usage and encoding the cytoplasmic region of human Jagged-1 (residues 1094-1218). The protein containing a N-terminal His(6)-tag was over-expressed in E. coli, and purified by affinity and reversed phase chromatography. After cleavage of the His(6)-tag by a dipeptidyl aminopeptidase, the protein was purified to homogeneity and characterized by spectroscopic techniques. Far-UV circular dichroism, fluorescence emission spectra, fluorescence anisotropy measurements, and (1)H nuclear magnetic resonance spectra, taken together, suggest that the cytoplasmic tail of human Jagged-1 behaves as an intrinsically unstructured domain in solution. This result was confirmed by the high susceptibility of the recombinant protein to proteolytic cleavage. The significance of this finding is discussed in relation to the recently proposed role of the intracellular region of Notch ligands in bi-directional signaling.

Calcium-Binding Proteins↗

PubMed2005

The "first in-last out" hypothesis on protein folding revisited.

We calculated profiles for mean residue depth, contact order, and number of contacts in the native structure of a series of proteins for which folding has been studied extensively, the chymotrypsin inhibitor 2, the SH3 module from the src tyrosine kinase, the small ribonuclease barnase, the bacterial immunity protein Im7, and apomyoglobin. We compared these profiles with experimental data from equilibrium or pulse labeling hydrogen-deuterium exchange obtained from NMR and phi values obtained from the protein engineering approach. We find a good qualitative agreement between the hierarchy of formation of topological elements during the folding process and the ranking of secondary structure elements in terms of residue depth. Residues that are most deeply buried in the core of the native protein usually belong to stretches of secondary structure elements that are formed early in the folding pathway. Residue depth can thus provide a useful and simple tool for the design of folding experiments.

Algorithms↗

PubMed2005

CX, DPX and PRIDE: WWW servers for the analysis and comparison of protein 3D structures.

The WWW servers at http://www.icgeb.org/protein/ are dedicated to the analysis of protein 3D structures submitted by the users as the Protein Data Bank (PDB) files. CX computes an atomic protrusion index that makes it possible to highlight the protruding atoms within a protein 3D structure. DPX calculates a depth index for the buried atoms and makes it possible to analyze the distribution of buried residues. CX and DPX return PDB files containing the calculated indices that can then be visualized using standard programs, such as Swiss-PDBviewer and Rasmol. PRIDE compares 3D structures using a fast algorithm based on the distribution of inter-atomic distances. The options include pairwise as well as multiple comparisons, and fold recognition based on searching the CATH fold database.

Algorithms↗

PubMed2005

Efficient synthesis and comparative studies of the arginine and Nomega,Nomega-dimethylarginine forms of the human nucleolin glycine/arginine rich domain.

The Gly- and Arg-rich C-terminal region of human nucleolin is a 61-residue long domain involved in a number of protein-protein and protein-nucleic acid interactions. This domain contains 10 aDma residues in the form of aDma-GG repeats interspersed with Phe residues. The exact role of Arg dimethylation is not known, partly because of the lack of efficient synthetic methods. This work describes an effective synthetic strategy, generally applicable to long RGG peptides, based on side-chain protected aDma and backbone protected dipeptide Fmoc-Gly-(Dmob)Gly-OH. This strategy allowed us to synthesize both the unmodified (N61Arg) and the dimethylated (N61aDma) peptides with high yield ( approximately 26%) and purity. As detected by NMR spectroscopy, N61Arg does not possess any stable secondary or tertiary structure in solution and N(omega),N(omega)-dimethylation of the guanidino group does not alter the overall conformational propensity of this peptide. While both peptides bind single-stranded nucleic acids with similar affinities (K(d) = 1.5 x 10(-7) M), they exhibit a different behaviour in ssDNA affinity chromatography consistent with the difference in pK(a) values. It has been previously shown that N61Arg inhibits HIV infection at the stage of HIV attachment to cells. This study demonstrates that Arg-dimethylated C-terminal domain lacks any inhibition activity, raising the question of whether nucleolin expressed on the cell-surface is indeed dimethylated.

Adenosine Triphosphatases↗

PubMed2004

Exon 6 of human Jagged-1 encodes an autonomously folding unit.

Human Jagged-1 is predicted to contain 16 epidermal growth factor-like (EGF) repeats. The oxidative folding of EGF-2, despite the several conditions tested, systematically led to complex mixtures. A longer peptide spanning the C-terminal part of EGF-1 and the complete EGF-2 repeat, on the contrary, could be readily refolded. This peptide, which corresponds to the entire exon 6 of the Jagged-1 gene, thus represents an autonomously folding unit. We show that it is structured in solution, as suggested by circular dichroism and NMR spectroscopy, and displays an EGF-like disulfide bond topology, as determined by disulfide mapping.

Amino Acid Sequence↗

PubMed2004

Folding of epidermal growth factor-like repeats from human tenascin studied through a sequence frame-shift approach.

In order to investigate the factors that determine the correct folding of epidermal growth factor-like (EGF) repeats within a multidomain protein, we prepared a series of six peptides that, taken together, span the sequence of two EGF repeats of human tenascin, a large protein from the extracellular matrix. The peptides were selected by sliding a window of the average length of tenascin EGF repeats over the sequence of EGF repeats 13 and 14. We thus obtained six peptides, EGF-f1 to EGF-f6, that are 33 residues long, contain six cysteines each, and bear a partial overlap in the sequence. While EGF-f1 corresponds to the native EGF-14 repeat, the others are frame-shifted EGF repeats. We carried out the oxidative folding of these peptides in vitro, analyzed the reaction mixtures by acid trapping followed by LC-MS, and isolated some of the resulting products. The oxidative folding of the native EGF-14 peptide is fast, produces a single three-disulfide species with an EGF-like disulfide topology and a marked difference in the RP-HPLC retention time compared with the starting product. On the contrary, frame-shifted peptides fold more slowly and give mixtures of three-disulfide species displaying RP-HPLC retention times that are closer to those of the reduced peptides. In contrast to the native EGF-14, the three-disulfide products that could be isolated are mainly unstructured, as determined by CD and NMR spectroscopy. We conclude that both kinetics and thermodynamics drive the correct pairing of cysteines, and speculate about how cysteine mispairing could trigger disulfide reshuffling in vivo.

Amino Acid Sequence↗

PubMed2003

DPX: for the analysis of the protein core.

SUMMARY: In order to obtain an accurate description of the protein interior, we describe a simple and fast algorithm that measures the depth of each atom in a protein (dpx), defined as its distance (A) from the closest solvent accessible atom. The program reads a PDB file containing the atomic solvent accessibility in the B-factor field, and writes a file in the same format, where the B-factor field now contains the dpx value. Output structure files can be thus directly displayed with molecular graphics programs like RASMOL, MOLMOL, Swiss-PDB View and colored according to dpx values. AVAILABILITY: The algorithm is implemented in a standalone program written in C and its source is freely available at ftp.icgeb.trieste.it/pub/DPX or on request from the authors.

Algorithms↗

PubMed2003

Atom depth in protein structure and function.

Atom depth, originally defined as the distance between a protein atom and the nearest water molecule surrounding a protein, is a simple but valuable geometrical descriptor of the protein interior. It can be easily computed from the 3D structure of a protein, thus complementing the information provided by the calculation of the solvent accessible surface area and buried surface area. Depth has been found to be correlated with several molecular, residue and atomic properties, such as average protein domain size, protein stability, free energy of formation of protein complexes, amino acid type hydrophobicity, residue conservation and hydrogen/deuterium amide proton exchange rates.

Algorithms↗

PubMed2003

Atom depth as a descriptor of the protein interior.

Atom depth, defined as the distance (dpx, A) of a nonhydrogen atom from its closest solvent-accessible protein neighbor, provides a simple but precise description of the protein interior. Mean residue depths can be easily computed and are very sensitive to structural features. From the analysis of the average and maximum atom depths of a set of 136 protein structures, we derive a limit of approximately 200 residues for protein and protein domain size. The average and maximum atom depths in a protein are related to its size but not to the fold type. From the same set of structures, we calculated the mean residue depths for the 20 amino acid types, and show that they correlate well with hydrophobicity scales. We show that dpx values can be used to partition atoms in discrete layers according to their depth and to identify atoms that, although buried, are potential targets for posttranslational modifications like phosphorylation. Finally, we find a correlation between highly conserved residues in structural neighbors of the same fold type, and their mean residue depth in the reference structure.

Algorithms↗

PubMed2002

Repeats with variations: accelerated evolution of the Pin2 family of proteinase inhibitors.

The Pin2 genes encode potato type II proteinase inhibitors that act against pathogenic attack. The first examples were found only in the Solanaceae family, but, using new EST and genomic data, we have found 11 homologous genes dispersed through almost the whole range of mono- and di-cotyledonous plants. In contrast to the repetitive precursor sequences of the Solanaceae Pin2 genes, the new homologs have only a single repeat unit. The gene family appears to have evolved from a single-domain ancestral gene through a series of gene-duplication and domain-duplication steps. A number of unequal cross-over and gene conversion events could explain the current gene and domain pattern of the Solanaceae Pin2 subfamily.

Amino Acid Sequence↗

PubMed2002

Structure and dynamics of the anticodon arm binding domain of Bacillus stearothermophilus Tyrosyl-tRNA synthetase.

The structure of a recombinant protein, TyrRS(delta4), corresponding to the anticodon arm binding domain of Bacillus stearothermophilus tyrosyl-tRNA synthetase, has been solved, and its dynamics have been studied by nuclear magnetic resonance (NMR). It is the first structure described for such a domain of a tyrosyl-tRNA synthetase. It consists of a five-stranded beta sheet, packed against two alpha helices on one side and one alpha helix on the other side. A large part of the domain is structurally similar to other functionally unrelated RNA binding proteins. The basic residues known to be essential for tRNA binding and charging are exposed to the solvent on the same face of the molecule. The structure of TyrRS(delta4), together with previous mutagenesis data, allows one to delineate the region of interaction with tRNATyr.

Amino Acid Sequence↗

PubMed2002

CX, an algorithm that identifies protruding atoms in proteins.

MOTIVATION: A simple and fast algorithm is described that calculates a measure of protrusion (cx) for atoms in protein structures, directly useable with the common molecular graphics programs. RESULTS: A sphere of predetermined radius is centered around each non-hydrogen atom, and the volume occupied by the protein and the free volume within the sphere (internal and external volumes, respectively) are calculated. Atoms in protruding regions have a high ratio (cx) between the external and the internal volume. The program reads a PDB file, and writes the output in the same format, with cx values in the B factor field. Output structure files can be directly displayed with standard molecular graphics programs like RASMOL, MOLMOL, Swiss-PDB Viewer and colored according to cx values. We show the potential use of this program in the analysis of two protein-protein complexes and in the prediction of limited proteolysis sites in native proteins. AVAILABILITY: The algorithm is implemented in a standalone program written in C and its source is freely available at ftp.icgeb.trieste.it/pub/CX or on request from the authors.

Algorithms↗