NMR spectroscopy was used to characterize hydrophobic clusters in amyloidogenic unfolded states of a protein and their implications for amyloid formation. Three local hydrophobic clusters were observed in the amyloidogenic state of the phosphatidylinositol 3-kinase (PI3K) SH3 domain. Our NMR studies showed that residues with high average area buried upon folding (AABUF) parameter collapsed to form the clusters. Interestingly, the hydrophobic collapses were not stabilized by long-range tertiary interactions among the clusters that were typically observed in non-amyloidogenic unfolded states of various proteins. The lack of the long-range interactions may be a critical property of the amyloidogenic unfolded state. The SH3 domain was also engineered to disrupt one of the clusters by a single-point mutagenesis (W55G), which allowed us to investigate the effect of the clustering on folding and misfolding. The mutant form of the SH3 domain was not able to fold under folding conditions of the wild type protein (pH 3.6-4.0), supporting the cooperative folding hypothesis. However, aggregation properties of the mutant form were not influenced by the mutation, suggesting the SH3 domain forms amyloid via a non-cooperative process. © 2010 Elsevier Inc. All rights reserved.