Amyloid formation plays a role in a wide range of human diseases. The rate and extent of amyloid formation depends on solution conditions including pH and ionic strength. Amyloid fibrils often adopt structures with parallel, in-register β-sheets, which generate quasi-infinite arrays of aligned side chains. These arrangements can lead to significant electrostatic interactions between adjacent polypeptide chains. The effect of ionic strength and ion composition on the kinetics of amyloid formation by islet amyloid polypeptide (IAPP) is examined. IAPP is a basic 37-residue polypeptide responsible for islet amyloid formation in type 2 diabetes. Poisson–Boltzmann calculations revealed significant electrostatic repulsion in a model of the IAPP fibrillar state. The kinetics of IAPP amyloid formation are strongly dependent on ionic strength, varying by more than a factor of 10 over the range of 20 to 600 mM NaCl at pH 8.0, but the effect is not entirely due to Debye screening. At low ionic strength the rate depends strongly on the identity of the anion, nearly varying by a factor of four and scales with the electroselectivity series, implicating anion binding. At high ionic strength the rate varies by only 8% and scales with the Hofmeister series. At intermediate ionic strength no clear trend is detected, likely because of convolution of different effects. The effects of salts on the growth phase and lag phase of IAPP amyloid formation are strongly correlated. At pH 5.5, where the net charge on IAPP is larger, the effect of different anions scales with the electroselectivity series at all salt concentrations.

The use of noncoded amino acids as spectroscopic probes of protein folding and function is growing rapidly, in large part because of advances in the methodology for their incorporation. Recently p-cyanophenylalanine has been employed as a fluorescence and IR probe, as well as a FRET probe to study protein folding, protein-membrane interactions, protein-protein interactions and amyloid formation. The probe has been shown to be exquisitely sensitive to hydrogen bonding interactions involving the cyano group, and its fluorescence quantum yield increases dramatically when it is hydrogen bonded. However, a detailed understanding of the factors which influence its fluorescence is required to be able to use this popular probe accurately. Here we demonstrate the recombinant incorporation of p-cyanophenylalanine in the N-terminal domain of the ribosomal protein L9. Native state fluorescence is very low, which suggests that the group is sequestered from solvent; however, IR measurements and molecular dynamics simulations show that the cyano group is exposed to solvent and forms hydrogen bonds to water. Analysis of mutant proteins and model peptides demonstrates that the reduced native state fluorescence is caused by the effective quenching of p-cyanophenylalanine fluorescence via FRET to tyrosine side-chains. The implications for the interpretation of p-cyanophenylalanine fluorescence measurements and FRET studies are discussed.