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Early Events in Insulin Fibrillization Studied by Time-Lapse Atomic Force Microscopy

Alessandro Podestà ^*, Guido Tiana , Paolo Milani ^* and Mauro Manno 

^* Istituto Nazionale per la Fisica della Materia, Dipartimento di Fisica, and Cimaina, Università di Milano, Milan, Italy; Dipartimento di Fisica, Università di Milano and Istituto Nazionale di Fisica Nucleare, Milan, Italy; and Italian National Research Council, Institute of Biophysics at Palermo, Palermo, Italy

Correspondence: Address reprint requests to Mauro Manno, Italian National Research Council, Institute of Biophysics at Palermo, via U. La Malfa 153, 90146 Palermo, Italy. Tel: 39-091-680-9305; Fax: 39-091-680-9349; E-mail: mauro.manno{at}pa.ibf.cnr.it.

The importance of understanding the mechanism of protein aggregation into insoluble amyloid fibrils lies not only in its medical consequences, but also in its more basic properties of self-organization. The discovery that a large number of uncorrelated proteins can form, under proper conditions, structurally similar fibrils has suggested that the underlying mechanism is a general feature of polypeptide chains. In this work, we address the early events preceding amyloid fibril formation in solutions of zinc-free human insulin incubated at low pH and high temperature. Here, we show by time-lapse atomic force microscopy that a steady-state distribution of protein oligomers with a quasiexponential tail is reached within a few minutes after heating. This metastable phase lasts for a few hours, until fibrillar aggregates are observable. Although for such complex systems different aggregation mechanisms can occur simultaneously, our results indicate that the prefibrillar phase is mainly controlled by a simple coagulation-evaporation kinetic mechanism, in which concentration acts as a critical parameter. These experimental facts, along with the kinetic model used, suggest a critical role for thermal concentration fluctuations in the process of fibril nucleation.

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