Residue-Specific, Real-Time Characterization of Lag-Phase Species and Fibril Growth During Amyloid Formation: A Combined Fluorescence and IR Study of p-Cyanophenylalanine Analogs of Islet Amyloid Polypeptide

TitleResidue-Specific, Real-Time Characterization of Lag-Phase Species and Fibril Growth During Amyloid Formation: A Combined Fluorescence and IR Study of p-Cyanophenylalanine Analogs of Islet Amyloid Polypeptide
Publication TypeJournal Article
Year of Publication2010
AuthorsMarek P, Mukherjee S, Zanni MT, Raleigh DP
Secondary TitleJournal of Molecular Biology
Volume400
Issue4
Pagination878 - 888
Date Published07/2010
Abstract

Amyloid formation normally exhibits a lag phase followed by a growth phase, which leads to amyloid fibrils. Characterization of the species populated during the lag phase is experimentally challenging, but is critical since the most toxic entities may be pre-fibrillar species. p-Cyanophenylalanine (FC≡N) fluorescence is used to probe the nature of lag-phase species populated during the formation of amyloid by human islet amyloid polypeptide. The polypeptide contains two phenylalanines at positions 15 and 23 and a single tyrosine located at the C-terminus. Each aromatic residue was separately replaced by FC≡N. The substitutions do not perturb amyloid formation relative to wild-type islet amyloid polypeptide as detected using thioflavin T fluorescence and electron microscopy. FC≡N fluorescence is high when the cyano group is hydrogen bonded and low when it is not. It can also be quenched via Förster resonance energy transfer to tyrosine. Fluorescence intensity was monitored in real time and revealed that all three positions remained exposed to solvent during the lag phase but less exposed than unstructured model peptides. The time course of amyloid formation as monitored by thioflavin T fluorescence and FC≡N fluorescence is virtually identical. Fluorescence quenching experiments confirmed that each residue remains exposed during the lag phase. These results place significant constraints on the nature of intermediates that are populated during the lag phase and indicate that significant sequestering of the aromatic side chains does not occur until β-structure sufficient to bind thioflavin T has developed. Seeding studies and analysis of maximum rates confirm that sequestering of the cyano groups occurs concomitantly with the development of thioflavin T binding capability. Overall, the process of amyloid formation and growth appears to be remarkably homogenous in terms of side-chain ordering. FC≡N also provides information about fibril structure. Fluorescence emission measurements, infrared measurements, and quenching studies indicate that the aromatic residues are differentially exposed in the fibril state with Phe15 being the most exposed. FC≡N is readily accommodated into proteins; thus, the approach should be broadly applicable.

URLhttp://www.ncbi.nlm.nih.gov/pubmed/20630475
DOI10.1016/j.jmb.2010.05.041.
Short TitleJournal of Molecular Biology