Human profilin I reduces aggregation and concomitant toxicity of the polyglutamine-containing N-terminal region of the huntingtin protein encoded by exon 1 (httex1) and responsible for Huntington’s disease. Here, we investigate the interaction of profilin with httex1using NMR techniques designed to quantitatively analyze the kinetics and equilibria of chemical exchange at atomic resolution, including relaxation dispersion, exchange-induced shifts, and lifetime line broadening. We first show that the presence of two polyproline tracts in httex1, absent from a shorter huntingtin variant studied previously, modulates the kinetics of the transient branched oligomerization pathway that precedes nucleation, resulting in an increase in the populations of the on-pathway helical coiled-coil dimeric and tetrameric species (τex≤ 50 to 70 μs), while leaving the population of the off-pathway (nonproductive) dimeric species largely unaffected (τex∼750 μs). Next, we show that the affinity of a single molecule of profilin to the polyproline tracts is in the micromolar range (Kdiss∼ 17 and ∼ 31 μM), but binding of a second molecule of profilin is negatively cooperative, with the affinity reduced ∼11-fold. The lifetime of a 1:1 complex of httex1with profilin, determined using a shorter huntingtin variant containing only a single polyproline tract, is shown to be on the submillisecond timescale (τex∼ 600 μs andKdiss∼ 50 μM). Finally, we demonstrate that, in stable profilin–httex1complexes, the productive oligomerization pathway, leading to the formation of helical coiled-coil httex1tetramers, is completely abolished, and only the pathway resulting in “nonproductive” dimers remains active, thereby providing a mechanistic basis for how profilin reduces aggregation and toxicity of httex1.
Assessment of chemical exchange in tryptophan–albumin solution through 19F multicomponent transverse relaxation dispersion analysis
