Amyloid formation is associated with structural changes of native polypeptides to monomeric intermediate states and their self-assembly into insoluble aggregates. Characterizations of the amyloidogenic intermediate state are, therefore, of great importance in understanding the early stage of amyloidogenesis. Here, we present NMR investigations of the structural and dynamic properties of the acid-unfolded amyloidogenic intermediate state of the phosphatidylinositol 3-kinase (PI3K) SH3 domain-a model peptide. The monomeric amyloidogenic state of the SH3 domain studied at pH 2.0 (35°C) was shown to be substantially disordered with no secondary structural preferences.15 N NMR relaxation experiments indicated that the unfolded polypeptide is highly flexible on a subnanosecond timescale when observed under the amyloidogenic condition (pH 2.0, 35°C). However, more restricted motions were detected in residues located primarily in the b-strands as well as in a loop in the native fold. In addition, nonnative long-range interactions were observed between the residues with the reduced flexibility by paramagnetic relaxation enhancement (PRE) experiments. These indicate that the acid-unfolded state of the SH3 domain adopts a partly folded conformation through nonnative longrange contacts between the dynamically restricted residues at the amyloid-forming condition.Keywords: amyloids; PI3K SH3; NMR; dynamics; amyloidogenic intermediate; long-range interactions; PRE Interest in the unfolded and partly folded states of proteins has been growing because of their important roles in a variety of biological processes, including cellular signaling and transcriptional activation (Shortle 1993;Dyson and Wright 2005). In addition, it is widely accepted that partly structured states of proteins are involved in early events in the formation of amyloid, which underlies a number of debilitating human diseases such as Alzheimer's and Parkinson's diseases (Dobson 2003). Thus, characterization of the unfolded and partially folded states of proteins is of central importance in understanding the molecular mechanism of the biological processes as well as the amyloid diseases. Multinuclear, multidimensional NMR spectroscopy has proven to be a powerful technique to investigate site-specific structural and dynamic properties of nonnative states of proteins (Shortle 1996;Dyson and Wright 2004;Redfield 2004 Abbreviations: NMR, nuclear magnetic resonance; HSQC, heteronuclear single-quantum coherence; PI3K SH3, phosphatidylinositol 3-kinase Src homology 3; R 2 , transverse relaxation rate; NOE, nuclear Overhauser effect; R 1r , relaxation rate in the rotating frame; H/D, hydrogen/deuterium; PRE, paramagnetic relaxation enhancement; MTSL, (1-oxyl-2,2,5,5-tetramethyl-D3-pyrroline-3-methyl) methanethiosulfonate; UV, ultraviolet; CD, circular dichroism.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi
