title: Lysine residues in the N-terminal huntingtin amphipathic α-helix play a key role in peptide aggregation.
abstract: Huntington's disease is a genetic neurodegenerative disorder caused by an expansion in a polyglutamine domain near the N-terminus of the huntingtin (htt) protein that results in the formation of protein aggregates. Here, htt aggregate structure has been examined using hydrogen-deuterium exchange techniques coupled with tandem mass spectrometry. The focus of the study is on the 17-residue N-terminal flanking region of the peptide that has been shown to alter htt aggregation kinetics and morphology. A top-down sequencing strategy employing electron transfer dissociation is utilized to determine the location of accessible and protected hydrogens. In these experiments, peptides aggregate in a deuterium-rich solvent at neutral pH and are subsequently subjected to deuterium-hydrogen back-exchange followed by rapid quenching, disaggregation, and tandem mass spectrometry analysis. Electrospray ionization of the peptide solution produces the [M + 5H](5+) to [M + 10H](10+) charge states and reveals the presence of multiple peptide sequences differing by single glutamine residues. The [M + 7H](7+) to [M + 9](9+) charge states corresponding to the full peptide are used in the electron transfer dissociation analyses. Evidence for protected residues is observed in the 17-residue N-terminal tract and specifically points to lysine residues as potentially playing a significant role in htt aggregation.
authors.affiliation: C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV, 26506, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Arndt
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Brown
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: Burke
authors.forename: Justin
authors.initials: J
authors.lastname: Legleiter
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Valentine
mesh_terms: D041961:Deuterium Exchange Measurement / Q000379:methods*; D000071058:Huntingtin Protein; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D017433:Protein Structure, Secondary; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D008239:Lysine
keywords: Huntington's disease; amphipathic alpha helix; atomic force microscopy; hydrogen-deuterium exchange; thioflavin T fluorescence assay