Agitation and High Ionic Strength Induce Amyloidogenesis of a Folded PDZ Domain in Native Conditions |
| |
Authors: | Alessandro Sicorello Gemma Soldi Carlo Travaglini-Allocatelli Annalisa Relini Fabrizio Chiti |
| |
Institution: | † Dipartimento di Scienze Biochimiche, Università di Firenze, Florence, Italy ‡ Dipartimento di Fisica, Università di Genova, Genoa, Italy § Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza-Università di Roma, Rome, Italy ¶ Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia, Genoa, Italy ‖ Consorzio Interuniversitario “Istituto Nazionale Biostrutture e Biosistemi”, Rome, Italy |
| |
Abstract: | Amyloid fibril formation is a distinctive hallmark of a number of degenerative diseases. In this process, protein monomers self-assemble to form insoluble structures that are generally referred to as amyloid fibrils. We have induced in vitro amyloid fibril formation of a PDZ domain by combining mechanical agitation and high ionic strength under conditions otherwise close to physiological (pH 7.0, 37°C, no added denaturants). The resulting aggregates enhance the fluorescence of the thioflavin T dye via a sigmoidal kinetic profile. Both infrared spectroscopy and circular dichroism spectroscopy detect the formation of a largely intermolecular β-sheet structure. Atomic force microscopy shows straight, rod-like fibrils that are similar in appearance and height to mature amyloid-like fibrils. Under these conditions, before aggregation, the protein domain adopts an essentially native-like structure and an even higher conformational stability (ΔGU-FH2O). These results show a new method for converting initially folded proteins into amyloid-like aggregates. The methodological approach used here does not require denaturing conditions; rather, it couples agitation with a high ionic strength. Such an approach offers new opportunities to investigate protein aggregation under conditions in which a globular protein is initially folded, and to elucidate the physical forces that promote amyloid fibril formation. |
| |
Keywords: | |
本文献已被 ScienceDirect 等数据库收录! |
|