Decorrelating Kinetic and Relaxation Parameters in Exchange Saturation Transfer NMR: A Case Study of N-Terminal Huntingtin Peptides Binding to Unilamellar Lipid Vesicles.

Dark state exchange saturation transfer (DEST) and lifetime line-broadening (Δ R2, the difference in the measured transverse relaxation rates for the observable species in the presence and absence of exchange with a species characterized by very large intrinsic transverse relaxation rates) have proven to be powerful NMR tools for studying exchange phenomena between a NMR visible species and a high-molecular weight, "dark", NMR invisible state. However, in the exchange regime, where the transverse spin relaxation rates in the bound state ( R2bound) are smaller than the strength of the DEST saturation radio frequency field, typically corresponding to systems below ∼6 MDa, the combination of DEST and Δ R2 data, while sufficient to define the apparent association rate constant, cannot unambiguously determine the population of the bound state pB and R2bound values independently. We show that the latter exchange and relaxation parameters can be decorrelated by the measurement of the maximal value of the contribution of the fast-relaxing magnetization component to the total NMR signal, Cfastmax, an observable that is directly proportional to pB. When integrated into the analysis of DEST/Δ R2 data, Cfastmax provides an indispensable source of information for quantitative studies of exchange involving high-molecular-weight dark states. We demonstrate the utility of this approach by investigating the binding kinetics of two huntingtin exon-1-derived peptides to small unilamellar lipid vesicles (SUV), ∼ 31 nm in diameter and 4.3 MDa in molecular weight. The interaction of the N-terminal amphiphilic domain of huntingtin exon-1 with membrane surfaces promotes polyglutamine-mediated aggregation and, as such, is thought to play a role in the etiology of Huntington's disease, an autosomal dominant fatal neurodegenerative condition. The first peptide comprises the 16-residue N-terminal amphiphilic domain (httNT) alone, while the second contains an additional seven residue polyglutamine tract at the C-terminus (httNTQ7). At a peptide-to-lipid molar ratio of 1:4, the population of peptide bound to the SUV surface is substantial, ∼ 7-8%, while exchange between the free and SUV-bound peptide is slow on the relaxation time-scale ( kex ∼ 200 s-1). The last two C-terminal residues of httNT and the last 9 of httNTQ7 remain flexible in the SUV-bound form due to transient detachment from the lipid surface that occurs on a time-scale several-fold faster than binding.