Biomedical subjects

A Tramontano

Publications and source records attributed to A Tramontano.

At least 73 records · Page 4Linked to original sources

PubMed1995

Redesigning the substrate specificity of the hepatitis C virus NS3 protease.

Backgound. Hepatitis C Virus (HCV) non-structural protein 3 (NS3) encodes a trypsin-like serine protease that catalyzes the cleavages at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions in the viral polyprotein and that shows a preference for a cysteine as the P1 residue. Results. We describe here a partial model of the HCV NS3 protease which allowed us to predict the position of the secondary structure elements of the enzyme and of the residues involved in its specificity. By replacing these with the corresponding residues of Streptomyces griseus protease B, we obtained a protease that, similar to the bacterial protein and unlike the wild-type enzyme, is able to cleave a substrate containing a phenylalanine in the P1 position. Conclusion. These results confirm the reliability of our model and represent one of the few examples of redesign of a serine protease substrate specificity directed by molecular modelling.

Journal Article↗

PubMed1994

Molecular cloning of a proteolytic antibody light chain.

The cDNA for an antibody light chain raised by immunization against vasoactive intestinal peptide (VIP) was cloned in a bacterial expression vector, and the recombinant light chain was purified to electrophoretic homogeneity. The light chain catalyzed the hydrolysis of VIP efficiently owing to its comparatively high affinity for the substrate. In control experiments, the catalytic activity was preserved at a constant level after further chromatography of the light chain on anion-exchange and gel-filtration fast protein liquid chromatography columns, and it was removed by immunoadsorption with immobilized anti-mouse light chain antibody. The amide bond linking methylcoumarinamide (MCA) and arginine in a tripeptide unrelated in sequence to VIP was cleaved by the light chain with lower affinity and kinetic efficiency (kcat/Km). Hydrolysis of the peptidyl-MCA conjugate was inhibited competitively by the alternate substrate, VIP. The Ki and Km values for VIP were in the same range, indicating that peptide-MCA and VIP hydrolysis occurs at a common catalytic site in the light chain. Molecular modeling suggested the presence of a serine protease-like site in the light chain. This was supported by inhibition of the hydrolytic activity by serine protease inhibitors, but not by inhibitors of other classes of proteases. These observations suggest a poorly discriminatory catalytic site, with specificity for VIP arising chiefly by means of the antigen recognition function of the light chain combining site.

Amino Acid Sequence↗

PubMed1994

High level expression and rational mutagenesis of a designed protein, the minibody. From an insoluble to a soluble molecule.

We recently described the design and chemical synthesis of the minibody, a 61-residue metal binding beta-protein with a novel fold. Characterization of the polypeptide by circular dichroism spectroscopy, size exclusion chromatography, and metal binding studies showed the molecule to be folded, monomeric, globular and able to bind metals. The main obstacle which prevented a more detailed characterization was the very low solubility of the protein in water (about 10 microM). To address this problem, we used two independent approaches: (1) mutagenesis of the beta-sheet framework residues and (2) addition of a solubilizing motif, made of three lysine residues, at the N or C termini. Engineering and production of mutants was facilitated by the achievement of high level expression of the protein in Escherichia coli. Both approaches led to minibody variants with a solubility ranging from tenfold higher up to millimolar levels. For the best-characterized variant obtained so far, the thermodynamic stability calculated from denaturant-induced transition is identical to that of the parent, poorly soluble, molecule.

Amino Acid Sequence↗

PubMed1994

Molecular model of the specificity pocket of the hepatitis C virus protease: implications for substrate recognition.

We have built a model of the specificity pocket of the protease of hepatitis C virus on the basis of the known structures of trypsin-like serine proteases and of the conservation pattern of the protease sequences among various hepatitis C strains. The model allowed us to predict that the substrate of this protease should have a cysteine residue in position P1. This hypothesis was subsequently proved by N-terminal sequencing of two products of the protease. The success of this "blind" test increases our confidence in the overall correctness of our proposed alignment of the enzyme sequence with those of other proteases of known structure and constitutes a first step in the construction of a complete model of the viral protease domain.

Amino Acid Sequence↗

PubMed1994

PUZZLE: a new method for automated protein docking based on surface shape complementarity.

We describe here a novel procedure for automated protein docking, based only on geometric criteria. In our algorithm we project protein surfaces into bi-dimensional matrices; the search for complementary regions is performed by detecting matching sub-matrices. An exhaustive sampling of the rotation space is made in order to analyse all the possible relative orientations of the two proteins, but nevertheless this procedure requires a relatively short processing time (3 h to 24 h cpu time on a SG4D320, depending on the complexity of the input information). When tested with co-crystallized, free components and models of components of known protein-protein complexes, the method gave very satisfactory results. The procedure selects no more than four relative orientations of the molecular components, but the correct orientation is always present among them, ranking either first or second. In more than half the cases the "wrong" solutions nevertheless correctly identify most of the residues involved in the interaction. This is remarkable also in view of the fact that the chosen test complexes (trypsin-trypsin inhibitor and antibody-lysozyme) have a very different geometry of surface complementarity: trypsin inhibitor inserts a long side-chain into the deep specificity pocket of the protease, while the interface between antibody and lysozyme is rather flat and contains buried water molecules (not included in the calculation). In order to simulate a more realistic protein docking problem, we also used a trypsin inhibitor and an anti-lysozyme antibody model in our simulations, again with satisfying results.

Algorithms↗

PubMed1994

The making of the minibody: an engineered beta-protein for the display of conformationally constrained peptides.

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost the drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue beta-protein aimed at retaining some desirable features of immunoglobulin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of the main chain conformation of the same regions, based on the 'canonical structures' model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical synthesis and expression in E. coli and characterized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low solubility. We were able to overcome this problem both by further mutagenesis of the framework and by addition of a solubilizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.

Amino Acid Sequence↗

PubMed1994

Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor.

In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.

Amino Acid Sequence↗

PubMed1994

Variability within the Candida rugosa lipases family.

Several fungi secrete lipase isozymes differing in biochemical properties and in some cases in substrate specificity. In the yeast Candida rugosa, a family of related genes encodes for multiple lipase proteins, highly homologous in sequence but partially different in the regions interacting with the substrate molecule. Analysis of these substitutions performed on the basis of multiple alignments and using a 3-D model of the enzyme, allows identification of a restricted number of amino acids possibly involved in substrate specificity of Candida lipases.

Amino Acid Sequence↗

PubMed1994

Spontaneous hydrolysis of vasoactive intestinal peptide in neutral aqueous solution.

Hydrolysis of radioiodinated vasoactive intestinal peptide (VIP) was observed in buffered aqueous solution at neutral pH and 38 degrees C. The reaction displayed apparent first-order kinetics at initial peptide concentrations below 3 nM (kobs = 1.5 x 10(-5) s-1), but the rate deviated below predicted values at higher peptide concentrations. The rate constant derived from the reaction progress curve over three half lives, starting at a concentration of 82 pM peptide, was also consistent with a first-order process. The reaction results in several products that were isolated and characterized as peptide fragments. Based on the identity of these fragments, we deduced hydrolysis at five different peptide bonds clustered between residues 17-25 of VIP. Control experiments were devised to eliminate trivial explanations for the peptide hydrolysis. Peptides representing the C-terminal segment 15-28 and the internal segment 14-22 assayed by analogous methods and under identical conditions were not degraded at a measurable rate. Sodium dodecyl sulfate and acetonitrile, agents known to influence the secondary structure of VIP, inhibited its spontaneous hydrolysis, as did chloride salts of sodium and calcium, albumin and a peptide unrelated to VIP. The rate and product distribution are inconsistent with known pathways of peptide degradation involving cyclic imide or anhydride formation at asparagine or aspartate residues. We suggest that the breakdown of VIP in dilute solutions represents an autolytic process.

Amino Acid Sequence↗

PubMed1993

Mimicking of discontinuous epitopes by phage-displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides.

We have constructed a random nonapeptide library in the N-terminal region of the major coat protein VIII of bacteriophage f1, with two cysteines flanking the insert, and preliminary data suggest that many of the clones display at least some of their peptides in cyclized form. This library was used to select oligopeptides binding to the monoclonal antibody (mAb) H107, recognising the assembled native conformation of recombinant human H-subunit ferritin (H Fer), whose three-dimensional structure is known. Comparison of the selected oligopeptides with one another allowed us to derive two consensus sequences characterized by conserved amino acid (aa) residues. Analysis of the distribution of the aa side chains exposed on the surface of H Fer reveals that most of the aa defining both consensus sequences are present either at the end of the big loop or at the end of the A helix. These two regions of the H Fer, though separated in the linear sequence, are very close in the folded molecule. Interestingly, each consensus sequence derived from the selected phage-displayed peptides is characterized by aa present both at the end of the big loop and at the end of the A helix. These two H Fer regions are good candidates for mimicry by the selected peptides and therefore for constituting part of the H107 epitope. To provide support to this hypothesis, we constructed several H Fer mutants carrying point mutations in different positions of these two regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

A database system for handling phage library-derived sequences.

We have implemented a system for creating and maintaining nucleotide and amino acid sequence databases especially suited for the handling of phage library-derived sequences. The system is currently used in our laboratory and has already proven to be useful for the comparison of sequences obtained by different investigators. We believe that the availability of this system will encourage and simplify the exchange of sequence data among different laboratories.

Amino Acid Sequence↗

PubMed1993

Saturation mutagenesis of the human interleukin 6 receptor-binding site: implications for its three-dimensional structure.

Interleukin 6 is a 184-aa polypeptide postulated to belong to the class of helical cytokines. We built a three-dimensional model of human interleukin 6 based on the similarity of its hydrophobicity pattern with that of other cytokines and on the x-ray structure of growth hormone, interleukin 2, interleukin 4, interferon beta, and granulocyte-macrophage colony-stimulating factor. The resulting model is a bundle of four alpha-helices and suggests possible alternative conformations for the 9 C-terminal amino acids; in this region, the importance of Arg-182 and Met-184 for biological activity has been demonstrated [Lutticken, C., Kruttgen, A., Moller, C., Heinrich, P.C. & Rose-John, S. (1991) FEBS Lett. 282, 265-267]. Therefore, we generated a large collection of single-amino acid variants in residues 175-181. Analysis of their biological activity in two systems and the receptor binding properties of a subset of the mutants indicates that the entire region is involved in forming the receptor binding surface and supports the hypothesis that this region does not assume an alpha-helical conformation. Remarkably, we also found a mutant with receptor affinity and biological activity much higher than wild type; the potential therapeutical value of this finding is discussed.

Amino Acid Sequence↗

PubMed1993

A designed metal-binding protein with a novel fold.

A major challenge in protein design is to create stable scaffolds into which tailored functions can be introduced. Here we present the design, synthesis and characterization of a 61-residue all-beta protein: the minibody. We used a portion of the heavy chain variable domain of an immunoglobulin as a template, obtaining a molecule with a novel beta-sheet scaffold and two regions corresponding to the hypervariable loops H1 and H2. To exploit the potential for creating functional centres in the minibody, we engineered a metal-binding site into it. This site is formed by one histidine in H1 and two in H2. The protein is folded, compact and able to bind metal, thus representing the first designed beta-protein with a novel fold and a tailored function. By randomizing the sequence of the hypervariable loops, we are using the minibody scaffold to construct a conformationally constrained peptide library displayed on phage.

Amino Acid Sequence↗

PubMed1993

Cloning and analysis of Candida cylindracea lipase sequences.

Lipases (Lip) hydrolyze triglycerides into fatty acids and glycerol. Lip produced by the yeast Candida cylindracea are encoded by multiple genomic sequences. We report the molecular cloning and characterization of three genes from this family. They encode putative mature 57-kDa proteins of 534 amino acids (aa). To date, five Lip-encoding genomic sequences from C. cylindracea have been characterized in our laboratory. The five deduced aa sequences share an overall homology of 80%. These sequences have been aligned with each other and with those of homologous enzymes, the Lip from the mould Geotrichum candidum and the acetylcholinesterase from Torpedo californica, whose three-dimensional structures have been solved by X-ray analysis. The C. cylindracea Lip appear to have a structural organization similar to that described for both enzymes.

Amino Acid Sequence↗

PubMed1993

Chemical synthesis of a designed beta-protein through the flow-polyamide method.

A designed, 61-residue long, metal-binding protein was synthesized through the flow-polyamide method. This protein, named Minibody (Mini-antibody), contains a beta-sheet scaffold and two regions corresponding to immunoglobulin hypervariable loops (H1 and H2), onto which a metal binding site was engineered. The protein is extremely hydrophobic, with a 70% beta structure. Accordingly, it was anticipated, and actually found, to be a "difficult sequence" for all its length. Comparison of the standard, "minimum redundancy" protocol (single coupling, low excess of activated species) with a different protocol (double and triple coupling plus capping) showed that the two produced approximately the same amount of full-length product (3.7% after extensive purification). Capping was effective in blocking the unreacted amino groups, converting most failure sequences into truncated ones, a possible aid to implement affinity-type chromatographic protocols. Purification was complicated by the very low solubility of the molecule, coupled to a high tendency to aggregate even in concentrated chaotropic media (8 M urea). Nevertheless, a multi-dimensional purification scheme produced a highly homogeneous product, with the expected Ion Spray mass spectrum. The Minibody produced by recDNA methods showed identical chromatographic, spectroscopic and biochemical properties to those of the synthetic product.

Amino Acid Sequence↗

PubMed1993

Affinity purification of a difficult-sequence protein. Implications for the inclusion of capping in synthetic protocols.

A biotinylated derivative of a designed, difficult-sequence protein (the Minibody, Bianchi, E., Tramontano, A., Sollazzo, M. & Pessi, A. (1993) Int. J. Peptide Protein Res. 41, 385-393) which represented only 3.7% of the crude, cleaved material was quantitatively recovered with about 70% purity, in a single step, by affinity chromatography on immobilised avidin. Purification to homogeneity was then easily achieved by preparative HPLC. This highly effective purification scheme must be contrasted with the previously shown multidimensional, low-yield chromatographic protocol. Since no facilitation of the purification had been obtained by capping alone, this result suggests that capping is useful only in conjunction with affinity chromatography.

Amino Acid Sequence↗

PubMed1992

Homology-derived three-dimensional structure prediction of Candida cylindracea lipase.

We propose a structural model of Candida cylindracea lipase (CCL) based on the reported X-ray structure of the highly homologous Geotrichum candidum lipase (GCL). The network of interactions around the active site, the salt and disulfide bridge pattern is conserved in the proposed structure. Functional, structural and evolutionary aspects of the peculiar usage of CTG codons by C. cylindracea ATCC 14830 are discussed.

Biological Evolution↗

PubMed1992

Common features of the conformations of antigen-binding loops in immunoglobulins and application to modeling loop conformations.

Using database screening techniques we have examined the relationship between antigen-binding loops in immunoglobulins, and regions of similar conformation in other protein families. The conformations of most antigen-binding loops are not unique to immunoglobulins. But in many cases, the geometrical relationship between the loop and the peptides flanking it differs between the immunoglobulins and other structures with the same loop. We assess model building by data base screening, compared with that based on canonical structures.

Amino Acid Sequence↗