Wednesday, November 10, 2010: 2:10 PM
Canyon A (Hilton)
The objective of this study is to determine the effect of the presence of an interface on the ability of peptides to form amyloid deposits. The following questions will be addressed: 1) Is the formation of amyloid deposits a truly universal phenomenon? 2) Does the surface function as a nucleation site? And 3) What features of the surface are more likely to induce the formation of aggregates and secondary structure within those aggregates? Should the surface be hydrophobic or hydrophilic? Do specific sites on the surface play a dominant role in the formation of the aggregates? To identify and then synthesize a series of peptides able to form aggregates rich in beta-sheet structure will be like finding a needle in a haystack. We have decided to take a pragmatic approach by using diblock and multiblock polypeptides. We argue that either diblock or multiblock polypeptides resemble protein surface patches and thus they are better protein mimics than homopeptides. We decided to focus our experimental work on a family of peptides containing one “beta-sheet forming amino acid” (Val, Ile, and Tyr) and one ‘alpha-helix forming amino acid' (Glu, Lys, Ala). This selection covers a wide range of hydrophillicites and charges and, by avoiding highly hydrophobic amino acids (tryptophan or phenylalanine) we can assure that the diblock peptides are soluble in a wide range of concentrations and solvent conditions. Peptides were synthesized in two different lengths (20 and 40 amino acids). Three different diblocks (20:80; 50:50 and 80:20) and two different multiblock (alpha2beta2)n and (alpha5beta5)n. All peptides were synthesized by solid state synthesis using Fmoc chemistry and Wang resins. The peptides were purified by reverse phase chromatography and characterized by HPLC-MS and NMR. The aggregation studies were done in the absence and in the presence of solid liquid interfaces. Two types of surfaces were used: polystyrene latexes with different surface chemistry and negatively and positively charged liposomes. The rate and extent of aggregation was followed by dynamic light scattering, which was used to monitor the size of the aggregates as a function of time. Congo Red assays were performed to confirm if the aggregates were amyloidic and selected samples were inspected by atomic force microscopy. Under some conditions and depending on the chemistry of the peptide and of the surface the aggregation process was reversible and no evidence was found on the formation of intermolecular beta-sheet formation (based on circular dichroism experiments). Under some other conditions and depending on the chemistry of the peptides aggregation was irreversible and occurred in the presence of interfaces at peptide concentrations order of magnitude lower than in bulk. Some deposits irreversibly formed were amorphous whereas some others show the formation of fibrils mixed with amorphous deposits.