| Name | Pietro |
| Surname | Pucci |
| Institution | Università degli Studi della Campania Luigi Vanvitelli |
| Telephone | +39 081 674 318 (UniNa) |
| Telephone 2 | +39 081 373 7896 (Ceinge) |
| pucci@unina.it | |
| Address | Department of Chemical Sciences, Federico II University, Via Cintia 6, 80126, Naples, Italy |
| Resume | Download |
The spectroscopic and reactivity properties of hemin complexes formed with cyanogen bromide fragments B (residues 1-123), C (124-298), A (299-585), and D (1-298) of human serum albumin (HSA) have been investigated. The complex hemin-D exhibits binding, spectral, circular dichroism, and reactivity characteristics very similar to those of hemin-HSA, indicating that fragment D contains the entire HSA domain involved in heme binding. The characteristics of the other hemin complexes are different, and a detailed investigation of the properties of hemin-C has been carried out because this fragment contains the HSA binding region of several important drugs. Hemin-C contains a low-spin Fe(III) center, with two imidazole ligands, but the complex undergoes a reversible structural transition at basic pH leading to a high-spin, five-coordinated Fe(III) species. This change determines a marked increase in the relaxation rate of water protons. Limited proteolysis experiments and mass spectral analysis carried out on fragment C and hemin-C show that the region encompassing residues Glu-208 to Trp-214 is protected from activity of proteases in the complex and, therefore, is involved in the interaction with hemin. A structural model of fragment C enables us to propose that His-242 and His-288 are the axial ligands for the Fe(III) center.
Amyloid fibrils of patients treated with regular hemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment has a more flexible three-dimensional structure and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology of synthetic fibrils obtained from intact beta2-m and truncated DeltaN6beta2-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of beta2-m within the fibrillar polymer. The distribution of prefential proteolytic sites observed in both fibrils revealed that the central region of the protein, which had been easily cleaved in the full-length globular beta2-m, was fully protected in the fibrillar form. In addition, the amino- and carboxy-terminal regions of beta2-m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that beta2-m molecules in the fibrils consist of an unaccessible core comprising residues 20-87 with the strands I and VIII being not constrained in the fibrillar polymer and exposed to the proteases. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to occur in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native beta2-m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given.
A combination of spectroscopic techniques, hydrogen/deuterium exchange, and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the topology of the monomeric M* partly folded intermediate of aspartate aminotransferase from Escherichia coli in wild type (WT) as well as in a mutant form in which the highly conserved cis-proline at position 138 was replaced by a trans-alanine (P138A). Fluorescence analysis indicates that, although M* is an off-pathway intermediate in the folding of WT aspartate aminotransferase from E. coli, it seems to coincide with an on-pathway folding intermediate for the P138A mutant. Spectroscopic data, hydrogen/deuterium exchange, and limited proteolysis experiments demonstrated the occurrence of conformational differences between the two M* intermediates, with P138A-M* being conceivably more compact than WT-M*. Limited proteolysis data suggested that these conformational differences might be related to a different relative orientation of the small and large domains of the protein induced by the presence of the cis-proline residue at position 138. These differences between the two M* species indicated that in WT-M* Pro138 is in the cis conformation at this stage of the folding process. Moreover, hydrogen/deuterium exchange results showed the occurrence of few differences in the native N(2) forms of WT and P138A, the spectroscopic features and crystallographic structures of which are almost superimposable.
The chemical assessment of the complete disulphide bridge pattern in the beta-chain of human recombinant follicotropin (betaFSH) was accomplished by integrating classical biochemical methodologies with mass spectrometric procedures. A proteolytic strategy consisting of a double digestion of native betaFSH using the broad-specificity protease subtilisin first, followed by trypsin, was employed. The resulting peptide mixture was directly analysed by FAB-MS, leading to the assignment of the first three disulphide bridges. The remaining S-S bridges were determined by HPLC fractionation of the proteolytic digest followed by ESMS analysis of the individual fractions. The pattern of cysteine couplings in betaFSH was determined as: Cys3-Cys5l, Cys17-Cys66, Cys20-Cys104, Cys28-Cys82, Cys32-Cys84 and Cys87-Cys94, confirming the arrangement inferred from the crystal structure of the homologous betaCG. A subset of the S-S bridge pattern comprising Cys3-Cys51, Cys28-Cys82 and Cys32-Cys84 constitutes a cysteine knot motif similar to that found in the growth factor superfamily.
Albumin Kenitra is a new type of genetic variant of human serum albumin that has been found in two members of a family of Sephardic Jews from Kenitra (Morocco). The slow-migrating variant and the normal protein were isolated by anion-exchange chromatography and, after treatment with CNBr, the digests were analyzed by two-dimensional electrophoresis in a polyacrylamide gel. The CNBr peptides of the variant were purified by reverse-phase high performance liquid chromatography and submitted to sequence analysis. Albumin Kenitra is peculiar because it has an elongated polypeptide chain, 601 residues instead of 585, and its sequence is modified beginning from residue 575. DNA structural studies showed that the variant is caused by a single-base insertion, an adenine at nucleotide position 15 970 in the genomic sequence, which leads to a frameshift with the subsequent translation to the first termination codon of exon 15. Mass spectrometric analyses revealed that the four additional cysteine residues of the variant form two new S-S bridges and showed that albumin Kenitra is partially O-glycosylated by a monosialylated HexHexNAc structure. This oligosaccharide chain has been located to Thr596 by amino-acid sequence analysis of the tryptic fragment 592-597.
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.
The structure of ecto-5'-nucleotidase from bull seminal plasma, containing a glycosyl-phosphatidylinositol anchor, was studied using mass spectrometry. MALDI-MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for approximately 6000 Da. MALDI-MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N-glycosylation. GC-MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high-mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high-mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl-phosphatidylinositol core contained EtN(P)Man3GlcNH2-myo-inositol(P)-glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylglycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S-S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51-Cys57, Cys353-Cys358, Cys365-Cys387 and Cys476-Cys479. This work resolves details of the structure of ecto-5'-nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl-phosphatidylinositol anchor.
Titin is an exceptionally large protein (M.Wt. approximately 3 MDa) that spans half the sarcomere in muscle, from the Z-disk to the M-line. In the Z-disk, it interacts with alpha-actinin homodimers that are a principal component of the Z-filaments linking actin filaments. The interaction between titin and alpha-actinin involves repeating approximately 45 amino acid sequences (Z-repeats) near the N-terminus of titin and the C-lobe of the C-terminal calmodulin-like domain of alpha-actinin. The conformation of Z-repeat 7 (ZR7) of titin when complexed with the 73-amino acid C-terminal portion of alpha-actinin (EF34) was studied by heteronuclear NMR spectroscopy using (15)N-labeling of ZR7 and found to be helical over a stretch of 18 residues. Complex formation resulted in the protection of one site of preferential cleavage of EF34 at Phe14-Leu17, as determined by limited proteolysis experiments coupled to mass spectrometry measurements. Intermolecular NOEs show Val16 of ZR7 to be positioned close in space to the backbone of EF34 around Phe14. These observations suggest that the mode of binding of ZR7 to EF34 is similar to that of troponin I to troponin C and of peptide C20W to calmodulin. These complexes would appear to represent a general alternative binding mode of calmodulin-like domains to target peptides.
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.
Human alpha1-microglobulin (alpha1-m; also called protein HC), a glycoprotein belonging to the lipocalin superfamily, was isolated by sequential anion-exchange chromatography and gel filtration from the urine of hemodialized patients and from amniotic fluid collected in the week 16-18 of pregnancy. The carbohydrate chains of the protein purified from the two sources, which are organized in two Asn-linked and one Thr-linked oligosaccharides, were structurally characterized using matrix-assisted laser desorption ionization and electrospray mass spectrometry. The glycans attached to Thr5 are differently truncated NeuHexHexNAc sequences, and O-glycosylation in the amniotic fluid protein is only partial. Asn96 has both diantennary and triantennary structures attached in the case of urinary alpha1-m and only diantennary glycans in the amniotic fluid protein. The main carbohydrate units attached to Asn17 are in both proteins monosialylated and disialylated diantennary glycans. The position of the oligosaccharide chains in a three-dimensional model of the protein, produced using the automated Swiss-Model service, is also discussed.