Evidence for Polyproline II Helical Structure in Short Polyglutamine Tracts Brian W. Chellgren 1 , Anne-Frances Miller 1,2 and Trevor P. Creamer 1 ⁎ 1 Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky , 741 South Limestone Street, Lexington, KL 40536-0509, USA 2 Department of Chemistry, University of Kentucky , Lexington, KL 40506-0055, USA Nine neurodegenerative diseases, including Huntington's disease, are associated with the aggregation of proteins containing expanded poly- glutamine sequences. The end result of polyglutamine aggregation is a β- sheet-rich deposit. There exists evidence that an important intermediate in the aggregation process involves intramolecular β-hairpin structures. However, little is known about the starting state, monomeric polygluta- mine. Most experimental studies of monomeric polyglutamine have concluded that the backbone is completely disordered. However, such studies are hampered by the inherent tendency for polyglutamine to aggregate. A recent computational study suggested that the glutamine residues in polyglutamine tracts have a significant propensity to adopt the left-handed polyproline II (P II ) helical conformation. In this work, we use NMR spectroscopy to demonstrate that glutamine residues possess a high propensity to adopt the P II conformation. We present circular dichroism spectra that indicate the presence of significant amounts of P II helical structure in short glutamine tracts. These data demonstrate that the propensity to adopt the P II structure is retained for glutamine repeats of up to at least 15 residues. Although other structures, such as α-helices and β-sheets, become possible at greater lengths, our data indicate that glutamine residues in monomeric polyglutamine have a significant propensity to adopt the P II structure, although not necessarily in long contiguous helical stretches. We note that we have no evidence to suggest that the observed P II helical structure is a precursor to polyglutamine aggregation. Nonetheless, increased understanding of monomeric poly- glutamine structures will aid our understanding of the aggregation process. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: Huntington's disease; neurodegeneration; amyloid; aggregation; secondary structure Introduction There are nine neurodegenerative diseases that result from an expansion of a protein polyglutamine tract. 1 Expansion of the polyglutamine region leads to aggregation of the proteins, with the age of disease onset being correlated strongly with the length of the expansion. 2,3 Progression of these neurodegenerative diseases ultimately leads to death. 4 Polyglutamine peptides aggregate, provid- ing direct evidence that it is the expanded poly- glutamine regions that cause aggregation, 1 although there is some debate as to whether it is the ag- gregates that ultimately prove fatal. 5 Nonetheless, the oligomerization and aggregation process is clearly linked to pathogenesis, making the early stages of polyglutamine aggregation prime targets for intervention. 6,7 Currently, there is no effective therapeutic available for the prevention of aggrega- tion of polyglutamine and the potentially associated deleterious effects. Perutz and co-workers published evidence that polyglutamine aggregates consist of extensive three- dimensional β-sheet structures, 8 which may form tubular nanostructures. 9 Wetzel and co-workers have shed light on the formation of polyglutamine aggregates and presented evidence that the nucleat- ing structure is a β-hairpin or β-sheet formed by a single polyglutamine molecule. 2,10 Thus, there are Abbreviations used: NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; SVD, singular value decomposition. E-mail address of the corresponding author: Trevor.Creamer@uky.edu doi:10.1016/j.jmb.2006.06.044 J. Mol. Biol. (2006) 361, 362–371 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.