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Biophys. J. BioFAST: First Published June 9, 2006. doi:10.1529/biophysj.105.077438
© 2006 by the Biophysical Society.

A more recent version of this article appeared on September 1, 2006.
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SUPRAMOLECULAR ASSEMBLIES

Amyloid Fibrils of Glucagon Characterized by High-Resolution Atomic Force Microscopy

Kathy L. De Jong 1, Bev Incledon 2, Christopher M. Yip 3 and Michael R DeFelippis 1*

1 Eli Lilly and Company
2 Eli Lilly Canada
3 University of Toronto

* To whom correspondence should be addressed. E-mail: defelippis_michael_r{at}lilly.com.

Submitted on November 8, 2005
Revised on January 3, 2006
Accepted on 31 May 2006


  Abstract
Glucagon solutions at pH 2.0 were subjected to mechanical agitation at 37 °C in the presence of a hydrophobic surface to explore the details of aggregation and fiber formation. High-resolution intermittent-contact atomic force microscopy (AFM) performed in solution revealed the presence of aggregates after 0.5 hours; however, longer agitation times resulted in the formation of fibrillated structures with varying levels of higher-order assembly. Height, periodicity and amplitude measurements of these structures allowed the identification of four distinct fiber types. The most elementary fiber form, designated a filament, self-associates in a specific wound fashion to produce protofibrils composed of two filaments. Subsequent self-assembly of these filaments and protofibrils leads to two well-defined fibrillar motifs, termed Type I and Type II. AFM imaging of pH 2.8 glucagon solutions not agitated nor exposed to elevated temperature revealed the presence of amorphous aggregates prior to the formation of fibrillar structures similar to those seen at pH 2.0. Time-course solution ATR-FTIR spectroscopy and thioflavin T binding studies suggested that glucagon aggregation and fibril formation was associated with the development of {beta}-sheet structure. The results of these studies are used to describe a possible mechanism for glucagon aggregation and fibrillation that is consistent with a hierarchical assembly model proposed for amyloid fibril formation.

Key Words: aggregation, denaturation, hydrophobic surfaces, mechanism, physical stability




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Copyright © 2006 by the Biophysical Society.