Parkinson's disease is an age-related movement disorder characterized by the presence in the mid-brain of amyloid deposits of the 140-amino-acid protein AS (α-synuclein). AS fibrillation follows a nucleation polymerization pathway involving diverse transient prefibrillar species varying in size and morphology. Similar to other neurodegenerative diseases, cytotoxicity is currently attributed to these prefibrillar species rather than to the insoluble aggregates. Nevertheless, the underlying molecular mechanisms responsible for cytotoxicity remain elusive and structural studies may contribute to the understanding of both the amyloid aggregation mechanism and oligomer-induced toxicity. It is already recognized that soluble oligomeric AS species adopt β-sheet structures that differ from those characterizing the fibrillar structure. In the present study we used ATR (attenuated total reflection)-FTIR (Fourier-transform infrared) spectroscopy, a technique especially sensitive to β-sheet structure, to get a deeper insight into the β-sheet organization within oligomers and fibrils. Careful spectral analysis revealed that AS oligomers adopt an antiparallel β-sheet structure, whereas fibrils adopt a parallel arrangement. The results are discussed in terms of regions of the protein involved in the early β-sheet interactions and the implications of such conformational arrangement for the pathogenicity associated with AS oligomers.
The interaction between ligands and proteins usually induces changes in protein thermal stability with modifications in the midpoint denaturation temperature, enthalpy of unfolding, and heat capacity. These modifications are due to the coupling of unfolding with binding equilibrium. Furthermore, they can be attained by changes in protein structure and conformational flexibility induced by ligand interaction. To study these effects we have used bovine serum albumin (BSA) interacting with three different anilinonaphthalene sulfonate derivatives (ANS). These ligands have different effects on protein stability, conformation, and dynamics. Protein stability was studied by differential scanning calorimetry and fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism and infrared spectroscopy including kinetics of hydrogen/deuterium exchange. The order of calorimetric midpoint of denaturation was: 1,8-ANS-BSA > 2,6-ANS-BSA > free BSA >> (nondetected) bis-ANS-BSA. Both 1,8-ANS and 2,6-ANS did not substantially modify the secondary structure of BSA, whereas bis-ANS induced a distorted ␣-helix conformation with an increase of disordered structure. Protein flexibility followed the order: 1,8-ANS-BSA < 2,6-ANS-BSA < free BSA << bis-ANS-BSA, indicating a clear correlation between stability and conformational flexibility. The structure induced by an excess of bis-ANS to BSA is compatible with a molten globule-like state. Within the context of the binding landscape model, we have distinguished five conformers (identified by subscript): BSA 1,8-ANS , BSA 2,6-ANS , BSA free , BSA bis-ANS , and BSA unfolded among the large number of possible states of the conformational dynamic ensemble. The relative population of each distinguishable conformer depends on the type and concentration of ligand and the temperature of the system. Keywords: Protein stability; ligand binding; dynamic landscape; calorimetry; H/D exchangeThe interaction of proteins with small ligands often takes place with an increase in protein thermostability due to the coupling of binding with unfolding equilibrium (Fukada et al. 1983;Brandts and Lin 1990;Shrake and Ross 1990, 1992). Previous works from our laboratory have demonstrated that the tight binding of biotin to streptavidin or avidin increases the temperature of midpoint denaturation (T m ) from 75°and 85°C in the free form to 112°and 117°C, respectively, at full ligand saturation (González et al. 1997(González et al. , 1999. The increase in the thermal stability of streptavidin goes together with an increase in the unfolding cooperativity and a substantial increase in the structural order as measured by infrared spectroscopy (González et al. 1997).It has been observed that mutations that change the packing of the inner core modify protein stability (Munson et al. 1996;Richards 1997). In this connection, it could be expected that modifications in protein stability correlate with changes in the protein packing induced by ligand binding. Article and publication are...
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