Structural Characterization of Fibrils from Recombinant Human Islet Amyloid Polypeptide by Solid-State NMR: The Central FGAILS Segment Is Part of the ß-Sheet Core.

Amyloid deposits formed from islet amyloid polypeptide (IAPP) are a hallmark of type 2 diabetes mellitus and are known to be cytotoxic to pancreatic ß-cells. The molecular structure of the fibrillar form of IAPP is subject of intense research, and to date, different models exist. We present results of solid-state NMR experiments on fibrils of recombinantly expressed and uniformly 13C, 15N-labeled human IAPP in the non-amidated, free acid form. Complete sequential resonance assignments and resulting constraints on secondary structure are shown. A single set of chemical shifts is found for most residues, which is indicative of a high degree of homogeneity. The core region comprises three to four ß-sheets. We find that the central 23-FGAILS-28 segment, which is of critical importance for amyloid formation, is part of the core region and forms a ß-strand in our sample preparation. The eight N-terminal amino acid residues of IAPP, forming a ring-like structure due to a disulfide bridge between residues C2 and C7, appear to be well defined but with an increased degree of flexibility. This study supports the elucidation of the structural basis of IAPP amyloid formation and highlights the extent of amyloid fibril polymorphism.

Single fibril growth kinetics of α-synuclein.

Neurodegenerative disorders associated with protein misfolding are fatal diseases that are caused by fibrillation of endogenous proteins such as α-synuclein (α-syn) in Parkinson's disease (PD) or amyloid-ß in Alzheimer's disease. Fibrils of α-syn are a major pathological hallmark of PD and certain aggregation intermediates are postulated to cause synaptic failure and cell death of dopaminergic neurons in the substantia nigra. For the development of therapeutic approaches, the mechanistic understanding of the fibrillation process is essential. Here we report real-time observation of α-syn fibril elongation on a glass surface, imaged by total internal reflection fluorescence microscopy using thioflavin T fluorescence. Fibrillation on the glass surface occurred in the same time frame and yielded fibrils of similar length as fibrillation in solution. Time-resolved imaging of fibrillation on a single fibril level indicated that α-syn fibril elongation follows a stop-and-go mechanism; that is, fibrils either extend at a homogenous growth rate or stop to grow for variable time intervals. The fibril growth kinetics were compatible with a model featuring two states, a growth state and a stop state, which were approximately isoenergetic and interconverted with rate constants of ~1.5×10(-4) s(-1). In the growth state, α-syn monomers were incorporated into the fibril with a rate constant of 8.6×10(3) M(-1) s(-1). Fibril elongation of α-syn is slow compared to other amyloidogenic proteins.

Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide.

Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a ß-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered ß-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting ß-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins.