Contribution of simple saccharides to the stabilization of amyloid structure

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Abstract

The use of osmolytes or chaperones to stabilize proteins/peptides that misfold in neurodegenerative diseases is an attractive concept for drug development. We have investigated the role of a series of small carbohydrates for protection of the natively structured Alzheimer’s amyloid-β peptides (Aβ). Using circular dichroism spectroscopy to follow the β-structural transitions and electron microscopy to examine tertiary structural characteristics, we demonstrate that the hydrogen bonding capacity of the carbohydrate determines the inhibition or promotion of fibrillogenesis. Three sugar molecules that vary only in their distribution of potential H-bonding partners promote various structural changes in Aβ. Two of these sugar molecules are excluded from Aβ during aggregation and promote mature fibre growth, while the other binds Aβ promoting nucleation and the accumulation of protofibrils. Our studies suggest that utilization of a combinatorial strategy to alter H-bonding capacity across a simple carbohydrate molecule may represent a novel drug design strategy.

Section snippets

Materials and methods

Peptides. Aβ40/42 were synthesized by solid phase Fmoc-chemistry by the Hospital for Sick Children’s Biotechnology Centre (Toronto, Ont., Canada). They were purified by reverse phase HPLC on a C18 μbondapak column. Aβ peptides were initially dissolved in 0.5 ml of 100% trifluoroacetic acid (Aldrich Chemicals, Milwaukee, WI) to ensure that the peptide remained monomeric and free of fibril seeds, diluted in distilled H2O, and immediately lyophilized [14]. Aβ peptides were then dissolved at 1 mg/ml

Cosolvent-induced secondary structural transitions of Aβ40/42

In order to address the role of osmolyte–peptide interactions from macromolecular crowding, we chose to examine a series of monosaccharides with identical molecular weights, similar hydrodynamic volumes, and hard sphere radii (Table 1). This series of monosaccharides allowed us to dissect properties important for the stability and kinetics of peptide folding. We have also included the disaccharide, sucrose, which is comprised of two of the monosaccharides examined and has been previously shown

Conclusions

Many studies have shown that the distribution of H-bonding partners across a compound will determine the interaction with amyloid forming proteins/peptides [22], [23]. Sugar cosolvents replace hydrogen-bonded water molecules, which are important for protein stability, therefore structural stability is determined by the hydrogen bond potential of the sugar [24]. The less mobile sugars would be expected to form more stable, stronger hydrogen bonds, and therefore increase native protein stability

Acknowledgments

The authors acknowledge support from the Ontario Alzheimer’s Society (J.M.), Canadian Institutes of Health Research (J.M.), Natural Science and Engineering Research Council of Canada (J.M.), and Scottish and Cryptic Rite Charitable Foundations (J.M.).

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      Additionally, the positive values of enthalpy change and entropy change suggest that the binding is predominantly driven by the hydrophobic interactions [43]. The results from previous studies suggested that the binding between sugars and polypeptides may involve two processes: (1) the interaction between the hydrophobic rings of sugars and hydrophobic area of proteins and (2) the stabilization by hydrogen bonding. [44] In addition, evidence indicated that the microenvironment of tryptophan residues of proteins tended to be more hydrophobic than the solvent environment [33].

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