Amyloid fibril formation by a partially structured intermediate state of alpha-chymotrypsin.
ABSTRACT Here we investigated the effects of 2,2,2-trifluoroethanol (TFE) on the structure of alpha-chymotrypsin. The protein aggregates maximally in 35% (v/v) TFE. Congo red and thioflavin-T binding experiments suggest that the aggregates induced by TFE have amyloid-like properties, and transmission electron microscopy data show that these aggregates have a fibrilar morphology. Fluorescence, circular dichroism, anilino-8-napthalene sulfonate binding, and Fourier-transformed infrared spectroscopy data suggest that formation of a partially structured intermediate state precedes the onset of the aggregation process. The native beta-barrel structure of alpha-chymotrypsin appears to be disrupted in the partially structured intermediate state in favour of a non-native extended beta-sheet conformation with exposed hydrophobic surfaces. The protein becomes "sticky" under these conditions and aggregates into amyloid-like structures. The data support the hypothesis that amyloid formation involves the ordered self-assembly of partially folded species that are critical soluble precursors of fibrilar aggregates.
Article: Amyloidogenic regions and interaction surfaces overlap in globular proteins related to conformational diseases.[show abstract] [hide abstract]
ABSTRACT: Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins.PLoS Computational Biology 09/2009; 5(8):e1000476. · 5.22 Impact Factor
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ABSTRACT: Neuroserpin, a member of the Serine Proteinase Inhibitor (Serpin) superfamily, is known to be a neuroprotective factor in the focal ischemic stroke followed by reducing the microglial activation. Neuroserpin is a protein rich of methionine residues that can scavenge the free radical species which may increase its neuroprotective effect. On the other hand, the oxidative modifications of the amino acid residues in neuroserpin may lead to changes in its conformation and function. In this study, it was investigated the changes in the conformation and the function of the oxidized neuroserpin. Neuroserpin expressed in E. coli, BL21 or M15 harboring plasmid pQE81L containing neuroserpin cDNA. Expressed neuroserpin was purified by resin sulfopropyl A50 precharged with 0.1 M NiSO4 under denaturing condition. Neuroserpin was oxidized under oxidative stress condition in the presence of different concentration of hydrogen peroxide. The oxidation of neuroserpin was conveniently detected by a carbonyl content assay using 2, 4 dinitrophenylhydrazine. Changes in tertiary structure of neuroserpin were monitored by spectrofluorimeter to study the alteration of intrinsic fluorescence and also fluorescence of 8-anilinonaphthalin-1 sulfonic acid (ANS) in native and oxidized form of neuroserpin. Total expressed neuroserpin was estimated 4-5 mg/lit in 2XYT culture media. SDS-PAGE analysis of purified neuroserpin showed a single band which reflects the efficiency of the resin SP A50 for purification of the proteins containing 6xHis tag. Carbonyl content of oxidized and native neuroserpin was estimated 12.3 +/- 0.3 and 0.45 +/- 0.05, respectively. The inhibitory activity of oxidized neuroserpin decreased up to 40-60% as compared with native form of neuroserpin. Intrinsic fluorescence and also the emission of ANS bind to the hydrophobic region of the protein altered from 380 to 85 and in the case of ANS from 105 to 150 in oxidized and native form of neuroserpin, respectively. The decreased intrinsic fluorescence intensity, an enhancement in the fluorescence of ANS, and loss of the inhibitory activity up to 40-60% in neuroserpin, all suggested a conformational modification in the protein under the oxidative stress condition. Remaining the inhibitory activity of neuroserpin reflects that the protein tolerates the oxidative stress condition effectively.Iranian biomedical journal 02/2007; 11(1):41-6.