[Show abstract][Hide abstract] ABSTRACT: Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues impairs the productive folding of the protein making the polypeptide chain highly prone to aggregate forming amyloid fibrils at physiological pH and room temperature. The role played by tryptophanyl residues in driving the productive folding process was investigated by providing structural details at low resolution of compact intermediate of three mutated apomyoglobins, i.e., W7F, W14F and the amyloid forming mutant W7FW14F. In particular, we followed the hydrogen/deuterium exchange rate of protein segments using proteolysis with pepsin followed by mass spectrometry analysis. The results revealed significant differences in the N-terminal region, consisting in an alteration of the physico-chemical properties of 7-11 segment for W7F and in an increase of local flexibility of 12-29 segment for W14F. In the double trypthophanyl substituted mutant, these effects are additive and impair the formation of native-like contacts and favours inter-chain interactions leading to protein aggregation and amyloid formation at physiological pH.
Protein and Peptide Letters 01/2013; · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W → F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.
Biophysics of Structure and Mechanism 06/2012; 41(7):615-27. · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has been recently hypothesized that BAG3 protein, a co-chaperone of Hsp70/Hsc70, is involved in the regulation of several cell processes, such as apoptosis, autophagy and cell motility. Following the identification of Hsc70/Hsp70, further BAG3 molecular partners such as PLC-gamma and HspB8 were likewise identified, thus contributing to the characterization of the mechanisms and the biological roles carried out by this versatile protein. By using a His-tagged BAG3 protein as bait, we fished out and identified the cytosolic chaperonin CCT, a new unreported BAG3 partner. The interaction between BAG3 and CCT was confirmed and characterized by co-immunoprecipitation experiments and surface plasmon resonance techniques. Furthermore, our analyses showed a slower CCT association and a faster dissociation with a truncated form of BAG3 containing the BAG domain, thus indicating that other protein regions are essential for a high-affinity interaction. ATP or ADP does not seem to significantly influence the chaperonin binding to BAG3 protein. On the other hand, our experiments showed that BAG3 silencing by small interfering RNA slowed down cell migration and influence the availability of correctly folded monomeric actin, analyzed by DNAse I binding assays and latrunculin A depolymerization studies. To our knowledge, this is the first report showing a biologically relevant interaction between the chaperonin CCT and BAG3 protein, thus suggesting interesting involvement in the folding processes regulated by CCT.
The international journal of biochemistry & cell biology 12/2009; 42(5):641-50. · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel diketopiperazine, named cyclo-(D-pipecolinyl-L-isoleucine) (DKP 1), and 7 known diketopiperazines were isolated from the cell-free culture supernatant of the Antarctic psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125. Two diketopiperazines containing pipecolinyl moiety were isolated for the first time from a natural source. Two new linear peptides, stable to bacterial proteolytic enzymes, were also characterized. The structures of the isolated compounds were elucidated by means of spectroscopic data (1D-, 2D-NMR, EIMS, FABMS, and ESIMS/MS) and chiral high-performance liquid chromatography. The potential antioxidant activity of the isolated compounds was evaluated by a DPPH free radical scavenging assay.
[Show abstract][Hide abstract] ABSTRACT: A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the beta-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of alpha-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.
Protein Science 06/2004; 13(5):1322-30. · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phytochelatin synthase (PCS) is a major determinant of heavy metal tolerance in plants and other organisms. No structural information on this enzyme is as yet available. It is generally believed, however, that the active site region is located in the more conserved N-terminal portion of PCS, whereas various, as yet unidentified (but supposedly less critical) roles have been proposed for the C-terminal region. To gain insight into the structural/functional organization of PCS, we have conducted a limited proteolysis analysis of the enzyme from Arabidopsis (AtPCS1), followed by functional characterization of the resulting polypeptide fragments. Two N-terminal fragments ending at positions 372 (PCS_Nt1) and 283 (PCS_Nt2) were produced sequentially upon V8 protease digestion, without any detectable accumulation of the corresponding C-terminal fragments. As revealed by the results of in vivo and in vitro functional assays, the core PCS_Nt2 fragment is biosynthetically active in the presence of cadmium ions and supports phytochelatin formation at a rate that is only approximately 5-fold lower than that of full-length AtPCS1. The loss of the C-terminal region, however, substantially decreases the thermal stability of the enzyme and impairs phytochelatin formation in the presence of certain heavy metals (e.g. mercury and zinc, but not cadmium or copper). The latter phenotype was shared by PCS_Nt2 and by its precursor fragment PCS_Nt1, which, on the other hand, was almost as stable and biosynthetically active (in the presence of cadmium) as the full-length enzyme. AtPCS1 thus appears to be composed of a protease-resistant (and hence presumably highly structured) N-terminal domain, flanked by an intrinsically unstable C-terminal region. The most upstream part of such a region (positions 284-372) is important for enzyme stabilization, whereas its most terminal part (positions 373-485) appears to be required to determine enzyme responsiveness to a broader range of heavy metals.
Journal of Biological Chemistry 05/2004; 279(15):14686-93. · 4.60 Impact Factor