Effects of succinylation on thermal induced amyloid formation in Concanavalin A

Dipartimento di Scienze Fisiche e Astronomiche, Università di Palermo, Via Archirafi 36, Palermo, Italy.
European Biophysics Journal (Impact Factor: 2.22). 10/2007; 36(7):733-41. DOI: 10.1007/s00249-007-0181-z
Source: PubMed


We have recently shown that upon slight thermal destabilization the legume lectin Concanavalin A may undergo two different aggregation processes, leading, respectively, to amyloid fibrils at high pH and amorphous aggregates at low pH. Here we present an experimental study on the amyloid aggregation of Succinyl Concanavalin A, which is a dimeric active variant of Concanavalin. The results show that, as for the native protein, the fibrillation process appears to be favoured by alkaline pH, far from the isoelectric point of the protein. Moreover, it strongly depends on temperature and requires large conformational changes both at secondary and tertiary structure level. With respect to the native protein, the succinyl derivative forms amyloid fibrils in considerably longer times and with a minor exposure of hydrophobic regions. At physiological conditions, Concanavalin A still displays a sizeable tendency to form amyloid fibril, while the succinyl variant does not. A close correlation was observed between the progress of amyloid formation and a narrowing of the tryptophans fluorescence emission band, indicating a reduction of protein conformational heterogeneity in amyloid fibrils.

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    • "Also, Con A has significant structural homology to the human serum amyloid protein (SAP) that is present in in vivo amyloid deposits [22]. Moreover, the absence of disulphide bonds in its structure enhances its flexibility, favoring conformational changes at the tertiary structure level [23]. Finally, the aggregation process of Con A is also associated with its antitumor activity [24], [25] and was found to be related to its property of inducing apoptosis on tumoral cells [26], [27]. "
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    ABSTRACT: Understanding the early events during amyloid aggregation processes is crucial to single out the involved molecular mechanisms and for designing ad hoc strategies to prevent and reverse amyloidogenic disorders. Here, we show that, in conditions in which the protein is positively charged and its conformational flexibility is enhanced, Concanavalin A leads to fibril formation via a non-conventional aggregation pathway. Using a combination of light scattering, circular dichroism, small angle X-ray scattering, intrinsic (Tryptophan) and extrinsic (ANS) fluorescence and confocal and 2-photon fluorescence microscopy we characterize the aggregation process as a function of the temperature. We highlight a multi-step pathway with the formation of an on-pathway long-lived intermediate and a subsequent coagulation of such "crinkled" precursors into amyloid-like fibrils. The process results in a temperature-dependent aggregation-coagulation pathway, with the late phase of coagulation determined by the interplay between hydrophobic and electrostatic forces. Our data provide evidence for the complex aggregation pathway for a protein with a highly flexible native conformation. We demonstrate the possibility to generate a long-lived intermediate whose proportion and occurrence are easily tunable by experimental parameters (i.e. temperature). As a consequence, in the case of aggregation processes developing through well-defined energy barriers, our results can open the way to new strategies to induce more stable in vitro on-pathway intermediate species through a minute change in the initial conformational flexibility of the protein. This will allow isolating and experimentally studying such transient species, often indicated as relevant in neurodegenerative diseases, both in terms of structural and cytotoxic properties.
    PLoS ONE 07/2013; 8(7):e68912. DOI:10.1371/journal.pone.0068912 · 3.23 Impact Factor
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    • "Finally, ordered protein aggregates can be of great interest in technological applications as a new potential nano-materials that can be exploited by research, industry and medicine [5]. As described in many studies, aggregation phenomena result from a partial unfolding of the tertiary structure of the protein and from the conformational changes of secondary structure as well [6] [7] [8]. Indeed, from recent studies on thermal aggregation process, we have concluded that the first step of aggregation consists on a partial opening of the protein native conformation. "
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    ABSTRACT: Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.
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