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 R, 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 ( R) 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 R data, while sufficient to define the apparent association rate constant, cannot unambiguously determine the population of the bound state p and R 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, an observable that is directly proportional to p. When integrated into the analysis of DEST/Δ R data, C 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) alone, while the second contains an additional seven residue polyglutamine tract at the C-terminus (httQ). 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 ∼ 200 s). The last two C-terminal residues of htt and the last 9 of httQ 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.

Membrane interactions accelerate the self-aggregation of huntingtin exon 1 fragments in a polyglutamine length-dependent manner

“…The N-terminal 17-residue sequence has been demonstrated to adopt a largely helical conformation when being membranes-associated (41,42,77,78), when part of a htt17-polyQ fiber (45,46,79) or in aggregation intermediates (51,73). Here we have shown that the membrane interactions of the htt17 flanking region of the polyQ domain promotes the aggregation process of huntingtin exon 1.…”

“…Recent structural investigations reveal a highly dynamic behaviour of htt17 and the subsequent polyQ domain where htt17 association (23), its interactions with the membrane (39)(40)(41)(42)(43) or with other polypeptide domains are associated with random coilhelix structural transitions (44,45). Interestingly, the htt17 and the polyQ domains mutually influence each other and their conformational properties are coupled (46,47).…”

“…Another huntingtin domain mediating membrane interactions has been identified within residues 171-371, a region bearing an overall high cationic character (10). Furthermore, biochemical and cell biological assays demonstrate the potential role of these anchoring domains in disease development (10,21,41,42,55,56).…”

“…glutamines the slower aggregation kinetics allow for a more controlled, quantitative evaluation of the processes involved in aggregation and fibril formation similar to the use of htt17-polyQ constructs used in previous biophysical studies (e.g. (41,42,48,56,65)). The polyglutamine was then successively extended in small steps to quantitatively measure the effect of polyQs on the aggregation dynamics.…”

Protein Interactions with Nanoparticle Surfaces: Highlighting Solution NMR Techniques

“…For rapidly exchanging nanoparticle surfaces, conditions are chosen so that only a small fraction of the total protein concentration interacts with the nanoparticle. Then, relaxation behavior and other properties are used to study the bound state, offering the possibility of performing numerous experiments that can address a variety of features in studying NP‐protein interactions.…”