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

Abstract

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 ( R₂^bound) 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 p_B and R₂^bound 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, C_fast^max, an observable that is directly proportional to p_B. When integrated into the analysis of DEST/Δ R₂ data, C_fast^max 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 (htt^NT) alone, while the second contains an additional seven residue polyglutamine tract at the C-terminus (htt^NTQ₇). 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 ( k_ex ∼ 200 s^-1). The last two C-terminal residues of htt^NT and the last 9 of htt^NTQ₇ 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.