NMR Studies of Polypeptide Structuring and Aggregation

Abstract

The importance of studying polypeptide structuring and folding mechanisms lies in the damage caused by misfolding proteins in the human body. The misfolding of proteins can lead to the off-pathway formation of aggreagates that can prove harmful to living cells. Several disease states result from this aggregation process, e.g. Parkinson's disease in which &alpha-synuclein is accumulated into Lewy bodies within the brain. In this work, the aggregation process of &alpha-synuclein is examined through the use of NMR, primarily <super>15</super>N-NMR. Aromatic-containing, well-folded &beta-hairpins have been shown to be effective at interfering with the fibril formation pathway. Through NMR, the binding of these hairpins to &alpha-synuclein and the divertion of the fibrils to a non-amyloid precipitate is explored. The design of stable protein-like structures allows for the study of specific interactions which contribute to folding. The trunctation and mutation of a poorly folded 39-residue peptide has produced 20-residue constructs that are > 95% folded in water. These constructs have been designated as the `Trp-cage' motif. This construct is characterised by an N-terminal &alpha-helix and hydrophobic collapse centred on a tryptophan indole ring. The majority of this work explores essential features of the Trp-cage that are responsible for its stability. The later part focuses on the circular permutation of the Trp-cage, followed by the cyclisation of the most stable motif. This results in a hyperstable Trp-cage (T_M >> 100&deg C at pH 7) which possesses the same diagnostic features as the standard Trp-cage structure.