The N-terminal region of the huntingtin protein, encoded by exon-1 (httex1) and containing an expanded polyglutamine tract, forms fibrils that accumulate in neuronal inclusion bodies, resulting in Huntington's disease. We previously showed that reversible formation of a sparsely populated tetramer of the N-terminal amphiphilic domain, comprising a dimer of dimers in a four-helix bundle configuration, occurs on the microsecond timescale and is an essential prerequisite for subsequent nucleation and fibril formation that takes place orders of magnitude slower on a timescale of hours. For pathogenic httex1, such as httex1Q35 with 35 glutamines, NMR signals decay too rapidly to permit measurement of time-intensive exchange-based experiments. Here, we show that quantitative analysis of both the kinetics and mechanism of prenucleation tetramerization and aggregation can be obtained simultaneously from a series of 1H-15N band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC) correlation spectra. The equilibria and kinetics of tetramerization are derived from the time dependence of the 15N chemical shifts and 1H-15N cross-peak volume/intensity ratios, while the kinetics of irreversible fibril formation are afforded by the decay curves of 1H-15N cross-peak intensities and volumes. Analysis of data on httex1Q35 over a series of concentrations ranging from 200 to 750 μM and containing variable (7 to 20%) amounts of the Met7O sulfoxide species, which does not tetramerize, shows that aggregation of native httex1Q35 proceeds via fourth-order primary nucleation, consistent with the critical role of prenucleation tetramerization, coupled with first-order secondary nucleation. The Met7O sulfoxide species does not nucleate but is still incorporated into fibrils by elongation.
Keywords: NMR spectroscopy; aggregation kinetics; huntingtin exon-1; nucleation; short-lived excited states.