Amylin is an endocrine hormone that regulates metabolism. In patients afflicted with type 2 diabetes, amylin is found in fibrillar deposits in the pancreas. Membranes are thought to facilitate the aggregation of amylin, and membrane-bound oligomers may be responsible for the islet ␤-cell toxicity that develops during type 2 diabetes. To better understand the structural basis for the interactions between amylin and membranes, we determined the NMR structure of human amylin bound to SDS micelles. The first four residues in the structure are constrained to form a hairpin loop by the single disulfide bond in amylin. The last nine residues near the C terminus are unfolded. The core of the structure is an ␣-helix that runs from about residues 5-28. A distortion or kink near residues 18 -22 introduces pliancy in the angle between the N-and C-terminal segments of the ␣-helix. Mobility, as determined by 15 N relaxation experiments, increases from the N to the C terminus and is strongly correlated with the accessibility of the polypeptide to spin probes in the solution phase. The spin probe data suggest that the segment between residues 5 and 17 is positioned within the hydrophobic lipid environment, whereas the amyloidogenic segment between residues 20 and 29 is at the interface between the lipid and solvent. This orientation may direct the aggregation of amylin on membranes, whereas coupling between the two segments may mediate the transition to a toxic structure. Type 2 diabetes affects over 100 million people worldwide (1) and is thought to cost upward of $130 billion dollars a year to treat in the United States alone (2). The endocrine hormone amylin (also known as islet amyloid polypeptide) appears to have key roles in diabetes pathology (3-5). The normal functions of amylin include the inhibition of glucagon secretion, slowing down the emptying of the stomach, and inducing a feeling of satiety through the actions of the hormone on neurons of the hypothalamus in the brain (5). The effects of amylin are exerted in concert with those of insulin and reduce the level of glucose in the blood (3, 5). Circulating amylin levels increase in a number of pathological conditions, including obesity, syndrome X, pancreatic cancer, and renal failure (3). Amylin levels together with insulin are raised initially in type 2 diabetes but fall as the disease progresses to a stage where the pancreatic islets of Langerhans ␤-cells that synthesize amylin no longer function (3).One of the hallmarks of type 2 diabetes, found in 90% of patients, is the formation of extracellular amyloid aggregates composed of amylin (3-5). The amyloid deposits accumulate in the interstitial fluid between islet cells and are usually juxtaposed with the ␤-cell membranes (3). Aggregates of amylin are toxic when added to cultures of ␤-cells, so that the amyloid found in situ may be responsible for ␤-cell death as type 2 diabetes progresses (6, 7). Genetic evidence that amylin is directly involved in pathology includes a familial S20G mutation that leads to early ...

The intrinsically unfolded protein a-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pK a values for all 26 sites in the protein that ionize below pH 7 using 2D 1 H-15 N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pK a values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pK a values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For a-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pK a of His50, a residue of particular interest since it is flanked within one turn of the a-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pK a of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pK a shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pK a values of uncomplexed a-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the a-synuclein sequence may help nucleate the folding of the protein into an a-helical structure and confer protection from misfolding.

We characterized the interaction of amylin with heparin fragments of defined length, which model the glycosaminoglycan chains associated with amyloid deposits found in type 2 diabetes. Binding of heparin fragments to the positively charged N-terminal half of monomeric amylin depends on the concentration of negatively charged saccharides but is independent of oligosaccharide length. By contrast, amylin fibrillogenesis has a sigmoidal dependence on heparin fragment length, with an enhancement observed for oligosaccharides longer than four monomers and a leveling off of effects beyond 12 monomers. The length dependence suggests that the negatively charged helical structure of heparin electrostatically complements the positively charged surface of the fibrillar amylin cross-␤ structure. Fluorescence resonance energy transfer and total internal reflection fluorescence microscopy experiments indicate that heparin associates with amylin fibrils, rather than enhancing fibrillogenesis catalytically. Short heparin fragments containing two-or eight-saccharide monomers protect against amylin cytotoxicity toward a MIN6 mouse cell model of pancreatic ␤-cells.Type 2 diabetes accounts for ϳ90% of adult diabetes. The disease currently affects 200 million people worldwide, and its incidence is projected to rise to 300 million by 2025 (1). Like many complex diseases, type 2 diabetes has multifactorial origins (1-4). The disease is characterized by insulin resistance, which causes the pancreas to synthesize more of the hormones insulin and amylin (5). The late stages of the disease are associated with ␤-cell dysfunction and a loss of ␤-cell mass (3, 5).Amylin (also known as islet amyloid polypeptide) is a 37-residue (4-KDa) endocrine hormone that is synthesized by pancreatic ␤-cells and co-secreted with insulin. In its normal state, amylin works together with insulin to control blood sugar (5). Additional amylin functions include suppressing appetite, slowing down the emptying of the stomach, and inhibiting glucagon secretion (5, 6). Like some 30 other polypeptides associated with human amyloid pathologies (6), amylin can undergo aggregative misfolding into fibrils with a cross-␤ structure (5,7,8). Although it is still uncertain whether fibrils or soluble oligomeric precursors are responsible for adverse affects (8 -12), extracellular amylin aggregates have been implicated in the destruction of pancreatic ␤-cells during the progression of type 2 diabetes (5, 9, 13). Amylin is the main component of fibrillar amyloid deposits found in the interstitial fluid between pancreatic islet cells of type 2 diabetes patients tested post-mortem (3,5,14), and extracellular aggregates of amylin are toxic when added to cultures of ␤-cells (9, 13). Overexpression of human amylin has been found to correlate with ␤-cell apoptosis and diabetes-like symptoms in several transgenic mouse and rat models whose endogenous amylin does not fibrillize (15). Genetic evidence that amylin is involved in pathology comes from the familial S20G mutation, which leads...

Total internal reflection fluorescence microscopy has been used to visualize the fibrillization of amylin, a hormone which in aggregated forms plays a role in type 2 diabetes pathology. Data were obtained at acidic pH where fibrillization is hindered by the charging of histidine 18 and at slightly basic pH where the loss of charge on the histidine promotes aggregation. The experiments show three types of aggregate growth processes. In the earliest steps globular seeds are formed with some expanding radially during the course of the reaction. The dimensions of the globular seeds as well as their staining with the amyloid-specific dye thioflavin T indicate that they are plaques of short fibrils. The next species observed are fibrils that invariably grow from large globular seeds or smaller punctate granules. Fibril elongation appears to be unidirectional, although in some cases multiple fibrils radiate from a single seed or granule. After fibrils are formed, some show an increase in fluorescence intensity that we attribute to the growth of new fibrils alongside those previously formed. All three aggregation processes are suggestive of secondary (heterogeneous) nucleation mechanisms in which nucleation occurs on preformed fibrils. Consistently, electron micrographs show changes in fibril morphology well after fibrils are first formed, and the growth processes observed by fluorescence microscopy occur after the corresponding solution reactions have reached an initial apparent plateau. Taken together, the results highlight the importance of secondary nucleation in the fibrillization of amylin, as this could provide a pathway to continue fibril growth once an initial population of fibrils is established.

Particle size distribution, a measurable physicochemical quantity, is a critical quality attribute of drug products that needs to be controlled in drug manufacturing. The non-invasive methods of dynamic light scattering (DLS) and Diffusion Ordered SpectroscopY (DOSY) NMR can be used to measure diffusion coefficient and derive the corresponding hydrodynamic radius. However, little is known about their use and sensitivity as analytical tools for particle size measurement of formulated protein therapeutics. Here, DLS and DOSY-NMR methods are shown to be orthogonal and yield identical diffusion coefficient results for a homogenous monomeric protein standard, ribonuclease A. However, different diffusion coefficients were observed for five insulin drug products measured using the two methods. DOSY-NMR yielded an averaged diffusion coefficient among fast exchanging insulin oligomers, ranging between dimer and hexamer in size. By contrast, DLS showed several distinct species, including dimer, hexamer, dodecamer and other aggregates. The heterogeneity or polydisperse nature of insulin oligomers in formulation caused DOSY-NMR and DLS results to differ from each other. DLS measurements provided more quality attributes and higher sensitivity to larger aggregates than DOSY-NMR. Nevertheless, each method was sensitive to a different range of particle sizes and complemented each other. The application of both methods increases the assurance of complex drug quality in this similarity comparison.