Biomedical subjects

P Pucci

Publications and source records attributed to P Pucci.

At least 37 records · Page 2Linked to original sources

PubMed2000

Characterization of five new low-molecular-mass trypsin inhibitors from white mustard (Sinapis alba L.) seed.

Five new low-molecular-mass trypsin inhibitors belonging to the RTI/MTI-2 family were identified from white mustard (Sinapis alba L. ; MTI-2) seed. Purified MTI-2 consisted of a peptide mixture, displaying Ile or Arg at position 43, Trp or kynurenine (Kyn) at position 44, and C-terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI-2 inhibitors originated from different genes. The presence of 5-oxo-proline (pyroglutamic acid; 5-oxoPro1) and Kyn44 reflected post-translational processing of the serine proteinase inhibitor. MTI-2 showed approximately 70% amino-acid identity with low-molecular-mass trypsin inhibitors isolated from oil rape (Brassica napus var. oleifera; RTI-III) seed and with serine proteinase inhibitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MTI-2 was homologous to brazzein, the sweet-tasting protein from Pentadiplandra brazzeana Baillon fruit ( approximately 30% amino-acid identity). Although snake-venom toxins showed a low amino-acid identity (< 20%) with MTI-2, RTI-III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI-2 (Cys5-Cys27, Cys18-Cys31, Cys42-Cys52, and Cys54-Cys57) corresponded to that of RTI-III and of snake-venom toxins, being different from that of brazzein. Therefore, protein similarity might be attributable to the three-dimensional arrangement rather than to the amino-acid sequence. Values of Ka for MTI-2 binding to bovine beta-trypsin (trypsin) and bovine alpha-chymotrypsin were 6.3 x 109 M-1 and 2.0 x 106 M-1, respectively, at pH 8.0 and 21.0 degrees C. Moreover, values of kon for MTI-2 binding to trypsin and of koff for the dissociation of the serine proteinase:inhibitor complex were 5.6 x 105 M-1.s-1 and 8.9 x 10-5 M-1.s-1, respectively, at pH 8.0 and 21.0 degrees C. Despite the heterogeneity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI-2 inhibitors were indistinguishable.

Amino Acid Sequence↗

PubMed2000

Early intermediates in the PDI-assisted folding of ribonuclease A.

The oxidative refolding of ribonuclease A has been investigated in several experimental conditions using a variety of redox systems. All these studies agree that the formation of disulfide bonds during the process occurs through a nonrandom mechanism with a preferential coupling of certain cysteine residues. We have previously demonstrated that in the presence of glutathione the refolding process occurs through the reiteration of two sequential reactions: a mixed disulfide with glutathione is produced first which evolves to form an intramolecular S-S bond. In the same experimental conditions, protein disulfide isomerase (PDI) was shown to catalyze formation and reduction of mixed disulfides with glutathione as well as formation of intramolecular S-S bonds. This paper reports the structural characterization of the one-disulfide intermediate population during the oxidative refolding of Ribonuclease A under the presence of PDI and glutathione with the aim of defining the role of the enzyme at the early stages of the reaction. The one-disulfide intermediate population occurring at the early stages of both the uncatalyzed and the PDI-catalyzed refolding was purified and structurally characterized by proteolytic digestion followed by MALDI-MS and LC/ESIMS analyses. In the uncatalyzed refolding, a total of 12 disulfide bonds out of the 28 theoretical possible cysteine couplings was observed, confirming a nonrandom distribution of native and nonnative disulfide bonds. Under the presence of PDI, only two additional nonnative disulfides were detected. Semiquantitative LC/ESIMS analysis of the distribution of the S-S bridged peptides showed that the most abundant species were equally populated in both the uncatalyzed and the catalyzed process. This paper shows the first structural characterization of the one-disulfide intermediate population formed transiently during the refolding of ribonuclease A in quasi-physiological conditions that mimic those present in the ER lumen. At the early stages of the process, three of the four native disulfides are detected, whereas the Cys26-Cys84 pairing is absent. Most of the nonnative disulfide bonds identified are formed by nearest-neighboring cysteines. The presence of PDI does not significantly alter the distribution of S-S bonds, suggesting that the ensemble of single-disulfide species is formed under thermodynamic control.

Catalysis↗

PubMed2000

Removal of the N-terminal hexapeptide from human beta2-microglobulin facilitates protein aggregation and fibril formation.

The solution structure and stability of N-terminally truncated beta2-microglobulin (deltaN6beta2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that deltaN6beta2-m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at microM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that deltaN6beta2-m is significantly less protected than its wild-type counterpart. Analysis of deltaN6beta2-m by NMR shows that this loss of protection occurs in beta strands I, III, and part of II. At mM concentration gel filtration analysis shows that deltaN6beta2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of beta2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that deltaN6beta2-m could be a key intermediate of a proteolytic pathway of beta2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of beta2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between beta-strands far removed from this constrain is greatly perturbed.

Amino Acid Sequence↗

PubMed2000

Structural and functional features of modified heat-stable toxins produced by enteropathogenic Klebsiella cells.

Heat-stable enterotoxins (STs) are 18- or 19-amino acid peptides (STa or ST1) produced by enteropathogenic bacteria with small differences in their amino acid sequence and a highly conserved carboxy terminus. All STs contain a core of three disulfide bridges whose integrity is believed to be necessary for full biologic activity. We previously reported that strains of Klebsiella pneumoniae transformed by the plasmid pSLM004 produce a modified toxin not recognized by MAb raised against genuine Escherichia coli ST. Investigation of the chemical structure of the modified toxins revealed that three new toxins were present. These were purified to homogeneity by a series of sequential chromatography on reverse-phase columns using guanylate cyclase to monitor the enterotoxic activity during purification procedures. The sequence of the modified toxins was obtained by a combination of Edman degradation and mass spectrometry, showing that they are proteolytically processed forms of E. coli ST1b. In particular, toxin A-2 lacks the cysteine at position 18 and then is not able to form the disulfide bridge cysteine-10-cysteine-18. All three toxins showed the ability to stimulate guanylate cyclase and to elicit chloride secretion in Caco-2 cell monolayers mounted in Ussing chambers. Toxin A-1 and toxin B demonstrated greatly reduced immunoreactivity whereas toxin A-2 was not recognized at all in the ELISA. It is likely that the three modified toxins were generated by Klebsiella specific proteolytic processing of the original pretoxin. These results have important implications for the diagnosis and prevention of heat-stable toxin-induced diarrhea.

Antibodies, Bacterial↗

PubMed2000

Structural analysis of modified forms of recombinant IFN-beta produced under stress-simulating conditions.

The present study focused on the investigation of the chemical stability of recombinant human interferon-beta (rhIFN-beta) tested in vitro by chemical treatments that simulate stress-induced conditions that may occur during handling, storage or ageing of protein samples. Mild oxidation and/or alkylation of the recombinant protein showed that the four methionines occurring in the interferon displayed different chemical susceptibility in that Met36 and Met117 were fully modified, whereas Met1 showed only little modification and Met62 was completely resistant. Moreover, incubation of rhIFN-beta under alkaline conditions resulted in the formation of a covalent dimeric species stabilised by an intermolecular disulphide bridge involving the free SH group of Cys17 from each polypeptide chain. Analysis of biological activity of the different IFN-beta derivatives showed that rhIFN-beta fully retains its specific activity following mild oxidation treatments whereas reaction with a high concentration of alkylating agents or incubation under alkaline conditions strongly reduce its specific antiviral activity.

Antiviral Agents↗

PubMed2000

The mutation associated with Hb Peterborough [beta111(G13)Val-->Phe] originated from Southern Italy.

Hb Peterborough [beta111(G13)Val-->Phe], an unstable hemoglobin variant with low oxygen affinity, was first described in two patients of Italian origin. This paper reports the first observation of this variant in Campania, Southern Italy, in two unrelated patients suffering from mild anemia. The variant was separated from Hb A by cation exchange chromatography on a high performance liquid chromatographic system with an automated procedure that might be useful for diagnostic purposes. The amino acid replacement, Val-Phe at [beta111, was assessed by tandem electrospray mass spectrometry analysis, and the corresponding DNA mutation was established as G-->T at the first position of codon 111 (GTC-TTC) by polymerase chain reaction amplification techniques. A family study showed that the two original carriers of Hb Peterborough were members of the same family as the proband examined in this study. This finding, and the presence of a second unrelated family carrying Hb Peterborough in Campania, strongly suggests that the DNA mutation associated with this variant originated in Southern Italy.

Adult↗

PubMed1999

Conformational changes in human hepatitis C virus NS3 protease upon binding of product-based inhibitors.

One of the most promising approaches to anti-hepatitis C virus drug discovery is the development of inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is essential for the maturation of the viral polyprotein, and processing requires complex formation between NS3 and its cofactor NS4A. Recently, we reported on the discovery of potent cleavage product-derived inhibitors [Ingallinella et al. (1998) Biochemistry 37, 8906-8914]. Here we study the interaction of these inhibitors with NS3 and the NS3/cofactor complex. Inhibitors bind NS3 according to an induced-fit mechanism. In the absence of cofactor different binding modes are apparent, while in the presence of cofactor all inhibitors show the same binding mode with a small rearrangement in the NS3 structure, as suggested by circular dichroism spectroscopy. These data are consistent with the hypothesis that NS4A complexation induces an NS3 structure that is already (but not entirely) preorganized for substrate binding not only for what concerns the S' site, as already suggested, but also for the S site. Inhibitor binding to the NS3/cofactor complex induces the stabilization of the enzyme structure as highlighted by limited proteolysis experiments. We envisage that this may occur through stabilization of the individual N-terminal and C-terminal domains where the cofactor and inhibitor, respectively, bind and subsequent tightening of the interdomain interaction in the ternary complex.

Amino Acid Sequence↗

PubMed1999

Stem cell factor is localized in, released from, and cleaved by human mast cells.

Stem cell factor (SCF) is the most important cytokine regulating human mast cell growth and functions. The immunogold technique showed SCF in the secretory granules of skin mast cells and in lung parenchymal mast cells (HLMC). Immunoreactive SCF (iSCF) was detected in cell lysates of HLMC, but not in basophils; iSCF and histamine were detected in supernatants of HLMC 3 min after challenge with anti-FcepsilonRI or anti-IgE, and iSCF in supernatants rapidly declined after 30 min, whereas histamine remained unchanged for 120 min. HPLC and electrospray mass spectrometry (ES/MS) analysis of recombinant human SCF1-166 (18,656. 9 +/- 0.9 Da) treated with chymase showed a polypeptide of 17,977.1 +/- 0.6 Da and a minor component of 697.4 +/- 0.1 Da generated by specific cleavage at Phe159. SCF1-166 and SCF1-159 similarly activated HLMC, potentiated anti-IgE-induced activation of these cells, and stimulated HLMC chemotaxis. SCF159-166 had no effect on mast cells. Western blot analysis of supernatants of anti-IgE-activated HLMC incubated with recombinant human SCF1-166 showed that SCF1-166 was rapidly cleaved to SCF1-159 and SCF1-144. Experiments with supernatants of anti-IgE-activated HLMC incubated with SCF1-166 yielded similar results. In conclusion, SCF is stored in mast cell secretory granules and is immunologically released by human mast cells. SCF1-166 is rapidly and specifically cleaved to SCF1-159 by chymase, which retains its biological effect on mast cells. SCF is also cleaved by other proteases to several SCF species whose possible biological activities remain to be established.

Adolescent↗

PubMed1999

Multiple determinants influence complex formation of the hepatitis C virus NS3 protease domain with its NS4A cofactor peptide.

The interaction of the hepatitis C virus (HCV) NS3 protease domain with its NS4A cofactor peptide (Pep4AK) was investigated at equilibrium and at pre-steady state under different physicochemical conditions. Equilibrium dissociation constants of the NS3-Pep4AK complex varied by several orders of magnitude depending on buffer additives. Glycerol, NaCl, detergents, and peptide substrates were found to stabilize this interaction. The extent of glycerol-induced stabilization varied in an HCV strain-dependent way with at least one determinant mapping to an NS3-NS4A interaction site. Conformational transitions affecting at least the first 18 amino acids of NS3 were the main energy barriers for both the association and the dissociation reactions of the complex. However, deletion of this N-terminal portion of the protease molecule only slightly influenced equilibrium dissociation constants determined under different physicochemical conditions. Limited proteolysis experiments coupled with mass spectrometric identification of cleavage fragments suggested a high degree of conformational flexibility affecting at least the first 21 residues of NS3. The accessibility of this region of the protease to limited chymotryptic digestion did not significantly change in any condition tested, whereas a significant reduction of chymotryptic cleavages within the NS3 core was detected under conditions of high NS3-Pep4AK complex affinity. We conclude the following: (1) The N-terminus of the NS3 protease that, according to the X-ray crystal structure, makes extensive contacts with the cofactor peptide is highly flexible in solution and contributes only marginally to the thermodynamic stability of the complex. (2) Affinity enhancement is accomplished by several factors through a general stabilization of the fold of the NS3 molecule.

Amino Acid Sequence↗

PubMed1999

Topology of the thyroid transcription factor 1 homeodomain-DNA complex.

The topology of the thyroid transcription factor 1 homeodomain (TTF-1HD)-DNA complex was investigated by a strategy which combines limited proteolysis and selective chemical modification experiments with mass spectrometry methodologies. When limited proteolysis digestions were carried out with the protein in the absence or presence of its target oligonucleotide, differential peptide maps were obtained from which the amino acid residues involved in the interaction could be inferred. Similarly, selective acetylation of lysine residues in both the isolated and the complexed homeodomain allowed us to identify the amino acids protected by the interaction with DNA. Surface topology analysis of isolated TTF-1HD performed at neutral pH was in good agreement with the three-dimensional structure of the molecule as determined by NMR studies under acidic conditions. Minor differences were detected in the C-terminal region of the protein which, contrary to NMR data, showed no accessibility to proteases. Analysis of the complex provided an experimental validation of the model proposed on the basis of the homology with the homeodomain structures described so far. An increased accessibility of the C-terminal region was observed following the interaction, suggesting its displacement from the protein core by the oligonucleotide molecule. Comparative experiments with DNA fragments differing in sequence and binding capabilities highlighted structural differences among the complexes, mainly located in the N-terminal region of the homeodomain, thus accounting for their different dissociation constants.

Acetylation↗

PubMed1999

Characterization of low-molecular-mass trypsin isoinhibitors from oil-rape (Brassica napus var. oleifera) seed.

A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P1-P1' reactive site bond (where residues forming the reactive site have been identified as PnellipsisP1 and P1'ellipsisPn', where P1-P1' is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of alphaalphaNOEs and 3JalphaNH coupling constants, typical of the beta-structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine beta-trypsin and bovine alpha-chymotrypsin are 3.3 x 109 m-1 and 2.4 x 106 m-1, respectively, at pH 8.0 and 21.0 degrees C. The serine proteinase : inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.

Amino Acid Sequence↗

PubMed1999

Structural and membrane-binding properties of saposin D.

Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor, called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has been reported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yet been clearly established. In the present study we examined structural and membrane-binding properties of saposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionic phospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization of the lipid surface and, conversely, the association with the membrane markedly affected the fluorescence properties of saposin D. The presence of phosphatidylserine-containing vesicles greatly enhanced the intrinsic tyrosine fluorescence of saposin D, which contains tyrosines but not tryptophan residues. The structural properties of saposin D were investigated in detail using advanced MS analysis. It was found that the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked in the following order: Cys5-Cys78, Cys8-Cys72 and Cys36-Cys47. The disulfide pattern of saposin D is identical with that previously established for two other saposins, B and C, which also exhibit a strong affinity for lipids. The common disulfide structure probably has an important role in the interaction of these proteins with membranes. The analysis of the sugar moiety of saposin D revealed that the single N-glycosylation site present in the molecule is mainly modified by high-mannose-type structures varying from two to six hexose residues. Deglycosylation had no effect on the interaction of saposin D with phospholipid membranes, indicating that the glycosylation site is not related to the lipid-binding site. The association of saposin D with membranes was highly dependent on the composition of the bilayer. Neither ceramide nor sphingomyelin, sphingolipids whose hydrolysis is favoured by saposin D, promoted its binding, while the presence of an acidic phospholipid such as phosphatidylserine or phosphatidic acid greatly favoured the interaction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles where saposins are localized, anionic phospholipids may be determinants of the saposin D topology and, conversely, saposin D may affect the lipid organization of anionic phospholipid-containing membranes.

Amino Acid Sequence↗

PubMed1999

Conformational changes in the NS3 protease from hepatitis C virus strain Bk monitored by limited proteolysis and mass spectrometry.

Conformational changes occurring within the NS3 protease domain from the hepatitis C virus Bk strain (NS3(1-180)) under different physico-chemical conditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface accessibility of the protein is affected by conformational changes, when comparative experiments were carried out on NS3(1-180) either at different glycerol concentrations or in the presence of Pep4A, differential peptide maps were obtained from which protein regions involved in the structural changes could be inferred. The surface topology of isolated NS3(1-180) in solution was essentially consistent with the crystal structure of the protein with the N-terminal segment showing a high conformational flexibility. At higher glycerol concentration, the protease assumed a more compact structure showing a decrease in the accessibility of the N-terminal segment that either was forced to interact with the protein or originate intermolecular interactions with neighboring molecules. Binding of the cofactor Pep4A caused the displacement of the N-terminal arm from the protein moiety, leading this segment to again adopt an open and flexible conformation, thus suggesting that the N-terminus of the protease contributes only marginally to the stability of the complex. The observed conformational changes might be directly correlated with the activation mechanism of the protease by either the cosolvent or the cofactor peptide because they lead to tighter packing of the substrate binding site.

Amino Acid Sequence↗

PubMed1999

Hb Rainier [beta145(HC2)Tyr-->Cys] in Italy. Characterization of the amino acid substitution and the DNA mutation.

A high oxygen affinity hemoglobin variant was identified in a 53-year-old male patient from Napoli (Italy), suffering from pulmonary thromboembolism and polycythemia. A detailed structural characterization of the variant hemoglobin was carried out, both at the protein and DNA levels, by a combination of DNA sequencing and allele-specific amplification techniques with mass spectrometric procedures. The amino acid substitution was found to be Tyr-->Cys, and the corresponding DNA mutation was identified as A-->G at the second position of codon 145 of the beta chain. These variations indicated the presence of Hb Rainier. Haplotype analysis of DNA polymorphisms showed that the beta-globin gene from Hb Rainier was associated with haplotype II. Moreover, structural analyses provided direct identification of an intramolecular disulphide bridge joining the newly inserted beta145Cys with beta93Cys. This is the first report of the occurrence of Hb Rainier in Italy.

Adolescent↗

PubMed1999

[Systematic treatment of acute myocardial infarction with primary angioplasty. Early clinical and angiographic results and after 6 months].

UNLABELLED: Starting in 1995, at our institution all patients with acute myocardial infarction (AMI) who gave informed consent were treated by primary percutaneous transluminal coronary angioplasty (PTCA) without limitations in entry criteria. This report presents early and six-month clinical and angiographic results of the 720 patients (77% male, median age 64 years) treated by direct PTCA between January 1, 1995 and July 31, 1998. On admission, 33% of patients were in Killip class > 1, and 101 patients (14%) were in early cardiogenic shock. Optimal acute angiographic success (TIMI grade 3 flow with residual stenosis < 30%) was achieved in 683 patients (95%). Primary or unplanned stenting of infarct related artery (IRA) for a suboptimal or poor angiographic result after primary PTCA was performed in 454 patients (63%). The mean time from hospital arrival to recanalization was 62 +/- 28 min. At 30 days, the mortality rate was 4.9% (1.8% in Killip class < 4 patients and 24% in patients with cardiogenic shock). The reinfarction rate was 1.2%. At 30 days, coronary angiography showed restenosis or reocclusion of the IRA in 55 patients (8.9%). During the six-month follow-up (30-180 days), there were 11 deaths (1.5%) and 2 non-fatal reinfarctions (0.3%). At six months, the IRA patency rate was 95%, while the mean ejection fraction improvement in 422 patients with paired ventriculograms was 7%. Recurrent ischemia occurred in 144 patients (20%) and resulted in 7 deaths, 11 non-fatal reinfarctions and 126 repeat targeted vessel revascularization. CONCLUSIONS: The major finding of our experience is that direct coronary angioplasty may result in excellent early and late outcome in a population without limitations in entry criteria. The low mortality and the few recurrent myocardial ischemic events are connected with the high patency rate at 6 months. The extensive use of stents improves the angiographic results and the clinical outcome.

Adult↗

PubMed1998

The length of a single turn controls the overall folding rate of "three-fingered" snake toxins.

Snake curaremimetic toxins are short all-beta proteins, containing several disulfide bonds which largely contribute to their stability. The four disulfides present in snake toxins make a "disulfide beta-cross"-fold that was suggested to be a good protein folding template. Previous studies on the refolding of snake toxins (Ménez, A. et al. (1980) Biochemistry 19, 4166-4172) showed that this set of natural homologous proteins displays different rates of refolding. These studies suggested that the observed different rates could be correlated to the length of turn 2, one out of five turns present in the toxins structure and close to the "disulfide beta-cross". To demonstrate this hypothesis, we studied the refolding pathways and kinetics of two natural isotoxins, toxin alpha (Naja nigricollis) and erabutoxin b (Laticauda semifasciata), and two synthetic homologues, the alpha mutants, alpha60 and alpha62. These mutants were designed to probe the peculiar role of the turn 2 on the refolding process by deletion or insertion of one residue in the turn length that reproduced the natural heterogeneity at that locus. The refolding was studied by electrospray mass spectrometry (ESMS) time-course analysis. This analysis permitted both the identification and quantitation of the population of intermediates present during the process. All toxins were shown to share the same sequential scheme for disulfide bond formation despite large differences in their refolding rates. The results presented here demonstrate definitely that no residues except those forming turn 2 accounted for the observed differences in the refolding rate of toxins.

Alkylation↗

PubMed1998

Corpeptins, new bioactive lipodepsipeptides from cultures of Pseudomonas corrugata.

The structure of the corpeptins, bioactive lipodepsipeptides produced in culture by Pseudomonas corrugata, the causal agent of tomato pith necrosis, has been determined. The combined use of FAB-mass spectrometry, NMR spectroscopy and chemical procedures has allowed us to assign the following primary structure to the peptide moiety: Dhb-Pro-Ala-Ala-Ala-Val-Val-Dhb-Hse-Val-alle-Dhp-Ala-Ala-Ala-Val-D hb-aThr-Ala-Dab-Ser-Ile with the terminal carboxy group closing a macrocyclic ring on the hydroxy group of the allo-threonine residue. The N-terminus is in turn acylated by 3-hydroxydecanoate in corpeptin A and by cis-3-hydroxy-5-dodecenoate in corpeptin B. Some preliminary data on the biological activity of corpeptins are included.

Acylation↗

PubMed1998

Analysis of human serum albumin variants by mass spectrometric procedures.

A new strategy for the structural characterisation of human albumin variants has been developed which makes extensive use of mass spectrometric methodologies. The rationale behind the method is to provide a rapid and effective screening of the entire albumin structure. The first step in this strategy consists in the attempt to determine the accurate molecular mass of the intact variant by electrospray mass spectrometry often providing a first indication on the presence of the variant. An HPLC procedure has been developed io isolate all the seven fragments generated by CNBr hydrolysis of HSA in a single chromatographic step. A rapid screening of the entire albumin structure is achieved by the ESMS analysis of the peptide fragments and the protein region(s) carrying the structural abnormality is identified by its anomalous mass value(s). Mass mapping of the corresponding CNBr peptide, either by Fast Atom Bombardment Mass Spectrometry (FABMS) or by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDIMS), leads to the definition of the site and the nature of the variation. This combined strategy was applied to the structural characterisation of three HSA genetic variants and provided to be an effective procedure for the rapid assessment of their structural modifications showing considerable advantages over the classical approach.

Humans↗