EUROISMAR 2019

The interaction of the N-terminal domain of huntingtin exon-1 with membrane surfaces promotes poly-glutamine mediated aggregation, and is thought to play a role in the etiology of Huntington’s disease. We investigated the kinetics of binding of two huntingtin peptides, comprising the 16-residue N-terminal amphiphilic domain alone (httNT) and with a seven residue poly-glutamine C-terminal tract (httNTQ7), to small unilamellar lipid vesicles (SUV), ~31 nm in diameter and ~4.3 MDa in molecular weight, using solution NMR experiments designed to probe interactions of NMR visible states with sparsely-populated, invisible or 'dark', high molecular weight species. Specifically, we make use of Dark state Exchange Saturation Transfer (DEST) and lifetime line broadening (dR2) supplemented with the measurements of the maximal value of the contribution of fast-relaxing magnetization component to the total NMR signal, Cfast_max. In the exchange regime where the transverse spin relaxation rates in the bound state are smaller than the strength of the DEST saturation radio-frequency field, the combination of DEST and dR2 data is not sufficient to unambiguously determine the population of the bound state (pB) and its transverse relaxation rates at the same time. We show that these exchange and relaxation parameters can be de-correlated by the measurement of Cfast_max which is directly proportional to pB. When integrated into the analysis of DEST/dR2 data, Cfast_max provides an indispensable source of information for quantitative studies of exchange involving high-molecular-weight dark states. While the population of the species bound to the SUV surface is substantial, on the order of 7-8%, the exchange between the free peptides and the SUV-bound states is slow on the relaxation time-scale (kex~200 1/s). The C-terminal regions of the peptides remain flexible even in the SUV-bound form due to transient detachment from the lipid surface that occurs on a time-scale several-fold faster than the binding proper.