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Biophys. J. BioFAST: First Published October 20, 2005. doi:10.1529/biophysj.105.068833
© 2005 by the Biophysical Society.

A more recent version of this article appeared on January 15, 2006.
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Alessandro Podesta
Guido Tiana
Paolo Milani
Mauro Manno
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PROTEINS

Early events in insulin fibrillization studied by time-lapse atomic force microscopy

Alessandro Podesta 1, Guido Tiana 1, Paolo Milani 1 and Mauro Manno 2*

1 Universitá di Milano
2 Italian National Research Council (CNR)

* To whom correspondence should be addressed. E-mail: mauro.manno{at}pa.ibf.cnr.it.

Submitted on June 15, 2005
Revised on July 16, 2005
Accepted on 3 October 2005


  Abstract
The importance of understanding the mechanism of protein aggregation into insoluble amyloid fibrils relies not only on its medical consequences, but also on 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 the present 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 (AFM) that a steady-state distribution of protein oligomers with a quasi exponential tail is reached within few minutes after heating. This metastable phase lasts for few hours until fibrillar aggregates are observable. Although for such complex systems different aggregation mechanisms can occur simultaneously, our results indicate that the pre-fibrillar phase is mainly controlled by a simple coagulation-evaporation kinetic mechanism, in which concentration act 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.

Key Words: atomic force microscopy, fibrillation, fluctuations, insulin, oligomers




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Copyright © 2005 by the Biophysical Society.