Homorepeats (HRs), protein fragments composed by the same amino repeated multiple times, are very common in eukaryotes and are involved in key biological processes and multiple pathologies. HRs are enriched in particular biophysical properties that enables them to perform very specialized functions but that can also trigger disease. Despite their interesting properties, the high-resolution structural characterization of HRs has been impaired due to their inherent flexibility and polymeric nature, which give poorly dispersed NMR spectra. Huntingtin (Htt), the causative agent of Huntington’s disease (HD), is the prototypical example of a HR hosting protein. Htt has a poly-Glutamine tract of variable length that becomes toxic when the number of consecutive glutamines exceeds 35. Moreover, Htt contains two Poly-Proline tracts with 11 and 10 consecutive prolines. The long-term aim of our study is to decipher the structural perturbations exerted by the extension of Poly-Glutamine tract beyond the pathological threshold, and the role that flanking regions, including the Poly-Proline tracts, have in the pathology.<br/>To overcome challenges posed by HRs, we have developed a chemical biology strategy to isotopically label individual glutamines and prolines within HRs by combining nonsense tRNA suppression and cell-free expression. Our method disentangles the spectroscopic complexity of the HR and has enabled the NMR investigation of huntingtin exon1 with a 16 residue-long poly-Glutamine tract. The resulting chemical shift analysis indicates the presence of a partially α-helix at its N-terminal region that encompasses part of the HR. The application to poly-Proline has allowed us to precisely explore the proline <i>cis/trans</i> isomerization in these HR regions. Implications of these observations to understand the structural bases of HDs will be discussed.

Access to atomic resolution structural information of homo-repeats by NMR: The Huntingtin case

2019 Spring National Meeting

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