Huntington's disease is a genetic neurodegenerative disorder characterized by the formation of amyloid fibrils of the huntingtin protein (htt). The 17-residue N-terminal region of htt (Nt17) has been implicated in the formation of early phase oligomeric species, which may be neurotoxic. Because tertiary interactions with a downstream (C-terminal) polyproline (polyP) region of htt may disrupt the formation of oligomers, which are precursors to fibrillar species, the effect of co-incubation of a region of htt with a 10-residue polyP peptide on oligomerization and fibrillization has been examined by atomic force microscopy. From multiple, time-course experiments, morphological changes in oligomeric species are observed for the protein/peptide mixture and compared with the protein alone. Additionally, an overall decrease in fibril formation is observed for the heterogeneous mixture. To consider potential sites of interaction between the Nt17 region and polyP, mixtures containing Nt17 and polyP peptides have been examined by ion mobility spectrometry and gas-phase hydrogen–deuterium exchange coupled with mass spectrometry. These data combined with molecular dynamics simulations suggest that the C-terminal region of Nt17 may be a primary point of contact. One interpretation of the results is that polyP may possibly regulate Nt17 by inducing a random coil region in the C-terminal portion of Nt17, thus decreasing the propensity to form the reactive amphipathic α-helix. A separate interpretation is that the residues important for helix–helix interactions are blocked by polyP association.
The gas-phase conformations of electrosprayed ions of the model peptide KKDDDDIIKIIK have been examined by ion mobility spectrometry (IMS) and hydrogen deuterium exchange (HDX)-tandem mass spectrometry (MS/MS) techniques. [M+4H](4+) ions exhibit two conformers with collision cross sections of 418 Å(2) and 471 Å(2). [M+3H](3+) ions exhibit a predominant conformer with a collision cross section of 340 Å(2) as well as an unresolved conformer (shoulder) with a collision cross section of ~367 Å(2). Maximum HDX levels for the more compact [M+4H](4+) ions and the compact and partially-folded [M+3H](3+) ions are ~12.9, ~15.5, and ~14.9, respectively. Ion structures obtained from molecular dynamics simulations (MDS) suggest that this ordering of HDX level results from increased charge-site/exchange-site density for the more compact ions of lower charge. Additionally, a new model that includes two distance calculations (charge site to carbonyl group and carbonyl group to exchange site) for the computer-generated structures is shown to better correlate to the experimentally determined per-residue deuterium uptake. Future comparisons of IMS-HDX-MS data with structures obtained from MDS are discussed with respect to novel experiments that will reveal the HDX rates of individual residues.
Huntington's disease is neurodegenerative disease caused by an expanded polyglutamine-coding CAG repeat in exon 1 of the huntingtin gene. Huntingtin exon 1 forms the primary toxic amyloid structure in Huntington's disease; disease severity is directly correlated with polyglutamine length. Recent works have shown that fully formed amyloid plaques may not represent the most toxic species in Huntington's disease; the most neurotoxic species may be small, diffuse oligomer (4 - 20 monomer units) that are precursors to amyloid plaques. While the polyglutamine region is undisputed as the primary constituent of amyloid structure, aggregation kinetics and morphology are regulated by the presence of flanking sequences that are N- and C-terminal to theamyloid forming tract. The first seventeen residues of huntingtin exon 1 (Nt17) can form an amphipathic &agr;-helix depending upon solution conditions and the presence of a binding partner, and in most cases, mediates oligomer formation. C-terminal to the polyglutamine tract is a proline-rich region, or in the case of a model peptide a polyproline region (polyP), that can form a polyproline-type II (PPII) helix, which may regulate Nt17 in huntingtin protein with short polyglutamine regions. Much is unknown regarding residue-specific Nt17-Nt17 and Nt17-polyP interactions. The work described here utilized state-of-the-art deuterium exchange mass spectrometry techniques to identify critical hydrophilic residues in early stages of oligomer formation. Monomeric and multimeric conformations of Nt17, idependent og the polyglutamine domain, were then studied using ion mobility-mass spectrometry and molecular dynamics to gain insight into the earliest stages of Nt17-Nt17 association, and thus, aggregation. Monomeric and multimeric Nt17 could form extended helices in the gas phase. Key hydrophilic residues were chemically modified, which resulted in a sharp decline in multimer formation. Finally, Nt17-polyP interactions were probed using gas-phase deuterium exchange mass spectrometry, supplemented with molecular dynamics and an exchange kinetics model. The obtained gas-phase structures showed a reduction in Nt17 extended &agr;-helix, when compared to a monomeric and extended homodimeric conformation. Thus, it is hypothesized that polyP regulates Nt17 by not allowing transition to the amphipathic &agr;-helix. The results of this study examine the structural heterogeneity of a sequence thought to drive a potentially toxic aggregate morphology, pinpoint key residues in early oligomer formation, and provide strategies for regulation of oligomer formation.
Early stage oligomer formation of the huntingtin protein may be driven by self-association of the 17-residue amphipathic α-helix at the protein's N-terminus (Nt17). Oligomeric structures have been implicated in neuronal toxicity and may represent important neurotoxic species in Huntington's disease. Therefore, a residue-specific structural characterization of Nt17 is crucial to understanding and potentially inhibiting oligomer formation. Native electrospray ion mobility spectrometry-mass spectrometry (IMS-MS) techniques and molecular dynamics simulations (MDS) have been applied to study coexisting monomer and multimer conformations of Nt17, independent of the remainder of huntingtin exon 1. MDS suggests gas-phase monomer ion structures comprise a helix-turn-coil configuration and a helix-extended-coil region. Elongated dimer species comprise partially helical monomers arranged in an antiparallel geometry. This stacked helical bundle may represent the earliest stages of Nt17-driven oligomer formation. Nt17 monomers and multimers have been further probed using diethylpyrocarbonate (DEPC). An N-terminal site (N-terminus of Threonine-3) and Lysine-6 are modified at higher DEPC concentrations, which led to the formation of an intermediate monomer structure. These modifications resulted in decreased extended monomer ion conformers, as well as a reduction in multimer formation. From the MDS experiments for the dimer ions, Lys6 residues in both monomer constituents interact with Ser16 and Glu12 residues on adjacent peptides; therefore, the decrease in multimer formation could result from disruption of these or similar interactions. This work provides a structurally selective model from which to study Nt17 self-association and provides critical insight toward Nt17 multimerization and, possibly, the early stages of huntingtin exon 1 aggregation.
A new instrument that couples a low-pressure drift tube with a linear ion trap mass spectrometer is demonstrated for complex mixture analysis. The combination of the low-pressure separation with the ion trapping capabilities provides several benefits for complex mixture analysis. These include high sensitivity, unique ion fragmentation capabilities, and high reproducibility. Even though the gas-phase separation and the mass measurement steps are each conducted in an ion filtering mode, detection limits for mobility-selected peptide ions are in the tens of attomole range. In addition to ion separation, the low-pressure drift tube can be used as an ion fragmentation cell yielding mobility-resolved fragment ions that can be subsequently analyzed by multistage tandem mass spectrometry (MS(n)) methods in the ion trap. Because of the ion trap configuration, these methods can be comprised of any number (limited by ion signal) of collision-induced dissociation (CID) and electron transfer dissociation (ETD) processes. The high reproducibility of the gas-phase separation allows for comparison of two-dimensional ion mobility spectrometry (IMS)-MS data sets in a pixel-by-pixel fashion without the need for data set alignment. These advantages are presented in model analyses representing mixtures encountered in proteomics and metabolomics experiments.
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