Browsing by Author "Carver, JA"
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- ItemThe chaperone activity of a-synuclein: Utilizing deletion mutants to map its interaction with target proteins(Wiley-Blackwell, 2012-03-01) Rekas, A; Ahn, KJ; Kim, J; Carver, JAa-Synuclein is the principal component of the Lewy body deposits that are characteristic of Parkinson's disease. In vivo, and under physiological conditions in vitro, a-synuclein aggregates to form amyloid fibrils, a process that is likely to be associated with the development of Parkinson's disease. a-Synuclein also possesses chaperone activity to prevent the precipitation of amorphously aggregating target proteins, as demonstrated in vitro. a-Synuclein is an intrinsically disordered (i.e., unstructured) protein of 140 amino acids in length, and therefore studies on its fragments can be correlated directly to the functional role of these regions in the intact protein. In this study, the fragment containing residues 61140 [a-syn(61140)] was observed to be highly amyloidogenic and was as effective a chaperone in vitro as the full-length protein, while the N- and C-terminal fragments a-syn(160) and a-syn(96140) had no intrinsic chaperone activity. Interestingly, full-length fibrillar a-synuclein had greater chaperone activity than nonfibrillar a-synuclein. It is concluded that the amyloidogenic NAC region (residues 6195) contains the chaperone-binding site which is optimized for target protein binding as a result of its beta-sheet formation and/or ordered aggregation by a-synuclein. On the other hand, the first 60 residues of a-synuclein modulate the protein's chaperone-active site, while at the same time protecting a-synuclein from fibrillation. On its own, however, this fragment [a-syn(160)] had a tendency to aggregate amorphously. As a result of this study, the functional roles of the various regions of a-synuclein in its chaperone activity have been delineated. Proteins 2012; (c) 2011 Wiley Periodicals, Inc.
- ItemMonitoring the interaction between beta(2)-microglobulin and the molecular chaperone alpha B-crystallin by NMR and mass spectrometry alpha b-crystallin dissociates beta(2)-microglobulin oligomers(American Society of Biochemistry and Molecular Biology, 2013-06-14) Esposito, G; Garvey, M; Alverdi, V; Pettirossi, F; Corazza, A; Fogolari, F; Polano, M; Mangione, PP; Giorgetti, S; Stoppini, M; Rekas, A; Bellotti, V; Heck, AJR; Carver, JAThe interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β2-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation. © 2013, The American Society for Biochemistry and Molecular Biology, Inc.
- ItemNative disulphide-linked dimers facilitate amyloid fibril formation by bovine milk αS2-casein(Elsevier, 2021-03) Thorn, DC; Bahraminejad, E; Grosas, AB; Kouldelka, T; Hoffmann, P; Mata, JP; Devlin, GL; Sunde, M; Ecroyd, H; Holt, C; Carver, JABovine milk αS2-casein, an intrinsically disordered protein, readily forms amyloid fibrils in vitro and is implicated in the formation of amyloid fibril deposits in mammary tissue. Its two cysteine residues participate in the formation of either intra- or intermolecular disulphide bonds, generating monomer and dimer species. X-ray solution scattering measurements indicated that both forms of the protein adopt large, spherical oligomers at 20 °C. Upon incubation at 37 °C, the disulphide-linked dimer showed a significantly greater propensity to form amyloid fibrils than its monomeric counterpart. Thioflavin T fluorescence, circular dichroism and infrared spectra were consistent with one or both of the dimer isomers (in a parallel or antiparallel arrangement) being predisposed toward an ordered, amyloid-like structure. Limited proteolysis experiments indicated that the region from Ala81 to Lys113 is incorporated into the fibril core, implying that this region, which is predicted by several algorithms to be amyloidogenic, initiates fibril formation of αS2-casein. The partial conservation of the cysteine motif and the frequent occurrence of disulphide-linked dimers in mammalian milks despite the associated risk of mammary amyloidosis, suggest that the dimeric conformation of αS2-casein is a functional, yet amyloidogenic, structure. © 2020 Elsevier B.V
- ItemA quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, alpha A- and alpha B-crystallin(Elsevier, 2010-11-01) Treweek, TM; Rekas, A; Walker, MJ; Carver, JAThe principal lens proteins alpha A- and alpha B-crystallin are members of the small heat-shock protein (sHsp) family of molecular chaperone proteins. Via their chaperone action, alpha A- and alpha B-crystallin play an important role in maintaining lens transparency by preventing crystallin protein aggregation and precipitation. alpha B-crystallin is found extensively extralenticularly where it is stress inducible and acts as a chaperone to facilitate general protein stabilization. The structure of either alpha A- or alpha B-crystallin is not known nor is the mechanism of their chaperone action. Our earlier H-1 NMR spectroscopic studies determined that mammalian sHsps have a highly dynamic, polar and unstructured region at their extreme C-terminus (summarized in Carver (1999) Prog. Ret. Eye Res. 18, 431). This C-terminal extension acts as a solubilizing agent for the relatively hydrophobic protein and the complex it makes with its target proteins during chaperone action. In this study, alpha A- and alpha B-crystallin were N-15-labelled and their H-1-N-15 through-bond correlation, heteronuclear single-quantum coherence (HSQC) NMR spectra were assigned via standard methods. H-1-N-15 spin-lattice (T-1) and spin spin (T-2) relaxation times were measured for alpha A- and alpha B-crystallin in the absence and presence of a bound target protein, reduced alpha-lactalbumin. H-1-N-15 Nuclear Overhauser Effect (NOE) values provide an accurate measure, on a residue-by-residue basis, of the backbone flexibility of polypeptides. From measurement of these NOE values, it was determined that the flexibility of the extension in alpha A- and alpha B-crystallin increased markedly at the extreme C-terminus. By contrast, upon chaperone interaction of alpha A-crystallin with reduced alpha-lactalbumin, flexibility was maintained in the extension but was distributed evenly across all residues in the extension. Two mutants of alpha B-crystallin in its C-terminal region: (i) 1159A and 1161A and (ii) K175L, have altered chaperone ability (Treweek et al. (2007) PLoS One 2, e1046). Comparison of H-1-N-15 NOE values for these mutants with wild type alpha B-crystallin revealed alteration in flexibility of the extension, particularly at the extremity of K175L alpha B-crystallin, which may affect chaperone ability. (C) 2010 Elsevier Ltd. All rights reserved.
- ItemQuaternary organization and dynamics of the molecular chaperone HSP26 are thermally regulated(Elsevier, 2010-09-24) Benesch, JLP; Aquilina, JA; Baldwin, AJ; Rekas, A; Stengel, F; Lindner, RA; Basha, E; Devlin, GL; Horwitz, J; Vierling, E; Carver, JA; Robinson, CVThe function of ScHSP26 is thermally controlled: the heat shock that causes the destabilization of target proteins leads to its activation as a molecular chaperone. We investigate the structural and dynamical properties of ScHSP26 oligomers through a combination of multiangle light scattering, fluorescence spectroscopy, NMR spectroscopy, and mass spectrometry. We show that ScHSP26 exists as a heterogeneous oligomeric ensemble at room temperature. At heat-shock temperatures, two shifts in equilibria are observed: toward dissociation and to larger oligomers. We examine the quaternary dynamics of these oligomers by investigating the rate of exchange of subunits between them and find that this not only increases with temperature but proceeds via two separate processes. This is consistent with a conformational change of the oligomers at elevated temperatures which regulates the disassembly rates of this thermally activated protein. © 2010, Elsevier Ltd.
- ItemStructure/function studies of dogfish α-crystallin, comparison with bovine α-crystallin(National Center for Biotechnology Information, 2009-11-20) Ghahghaei, A; Rekas, A; Carver, JA; Augusteyn, RCPurpose: α-Crystallin is the major protein of the mammalian lens where it contributes to the refractive properties needed for vision and possibly to the stability of the tissue. The aim of this study was to determine whether the properties of α-crystallin have changed during the course of evolution. Methods: Dogfish α-crystallin, which appeared over 420 million years ago, has been contrasted with bovine α-crystallin, which emerged around 160 million years later, by comparing their sizes, the microenvironments of their cysteine and tryptophan residues, their chaperone-like activities and the flexibility of their COOH-terminal extensions. Results: Dogfish α-crystallin consists of α A- and α B-polypeptides, in a 1: 5 ratio, and has a molecular mass of around 400 kDa. By contrast, the bovine protein is around 600-800 kDa in mass and has a 3: 1 subunit ratio. Cysteine residues in the proteins were equally accessible to reaction with 5,5'-dithiobis-(2-nitrobenzoic acid). Quenching of fluorescence with acrylamide indicated tryptophan residues in the two proteins were in similar environments. The chaperone activity of dogfish α-crystallin was comparable to that of bovine α-crystallin in preventing the heat-induced precipitation of β(L)-crystallin but the dogfish protein was three times more effective at preventing insulin precipitation after reduction at 37 degrees C. H-1 nuclear magnetic resonance spectroscopic studies showed that the last 17 amino acids of the dogfish α B polypeptide (V162-K178) have great conformational flexibility, are highly exposed to solvent and adopt little ordered conformation. This is comparable to, but slightly longer in length, than the COOH-terminal extension observed in mammalian alpha-crystallins. Conclusions: The structure and properties of α-crystallin have changed relatively little during the evolutionary period from the emergence of sharks and mammals. © US National Library of Medicine National Institutes of Health