Amylin, also known as islet amyloid polypeptide (IAPP), is the major protein component of the fibril deposits found in the pancreas of individuals with type II diabetes. The central region of amylin, residues 20-29, has been implicated as a key determinate of amyloid formation. To establish which positions are most important for amyloid formation, the wild-type sequence of the 20-29 fragment and a set of 10 variants have been synthesized in which a proline was placed at each position. Proline is energetically unfavorable in the extended cross-beta structure found in amyloid. If a particular position is critical for amyloid formation, then substitution with a proline should inhibit amyloid formation. A proline substitution at any position inhibited aggregation and amyloid formation. Substitution of Asn22, Gly24, and residues 26-28 had the largest effect. Fourier transform infrared (FTIR) spectroscopy showed little secondary structure in these peptides, and transmission electron microscopy (TEM) showed mostly amorphous material. The peptides were much more soluble than the wild-type sequence, and no birefringence was observed with Congo Red staining. Proline substitutions at the N (residues 20 and 21) and C termini showed the least effect. These peptides showed the classic fibril morphology, a significant amount of beta-sheet structure, and exhibited green birefringence when stained with Congo Red. The results indicate that residues 22, 24, and 26-28 play a key role in formation of amyloid by amylin. Positions 23 and 25 also appear to be important, but may be less critical than positions 22, 24, and 26-28.

No abstract

The Factor for Inversion Stimulation (FIS) is a dimeric DNA binding protein found in enteric bacteria that is involved in various cellular processes, including stimulation of certain specialized DNA recombination events and transcription regulation of a large number of genes. The intracellular FIS concentration, when cells are grown in rich media, varies dramatically during the early logarithmic growth phase. Its broad range of concentrations could potentially affect the nature of its quaternary structure, which in turn, could affect its ability to function in vivo. Thus, we examined the stability of FIS homodimers under a wide range of concentrations relevant to in vivo expression levels. Its urea-induced equilibrium denaturation was monitored by far-and near-UV circular dichroism (CD), tyrosine fluorescence, and tyrosine fluorescence anisotropy. The denaturation transitions obtained were concentration-dependent and showed similar midpoints (C m ) and m values, suggesting a two-state denaturation process involving the native dimer and unfolded monomers (N 2 ↔ 2U). The ⌬G H 2 O for the unfolding of FIS determined from global and individual curve fitting was 14.2 kcal/mole. At concentrations <9 M, the FIS dimer began to dissociate, as noted by the change in CD signal and size-exclusion high-pressure liquid chromatography retention times and peak width. The estimated dimer dissociation constant based on the CD and size-exclusion chromatography data is in the micromolar range, resulting in a ⌬G H 2 O of at least 5 kcal/mole less than that calculated from the urea denaturation data. This discrepancy suggests a deviation from a two-state denaturation model, perhaps due to a marginally stable monomeric intermediate. These observations have implications for the stability and function of FIS in vivo.

No abstract

No abstract

No abstract