Abstract
Human islet amyloid polypeptide (hIAPP) aggregates into fibrils through oligomers that have been postulated to contain α-helices as well as β-sheets. We employ a site-specific isotope labeling strategy that is capable of detecting changes in dihedral angles when used in conjunction with 2D IR spectroscopy. The method is analogous to the chemical shift index used in NMR spectroscopy for assigning protein secondary structure. We introduce isotope labels at two neighbouring residues, which results in an increased intensity and positive frequency shift if those residues are α-helical versus a negative frequency shift in β-sheets and turns. The 2D IR dihedral index approach is demonstrated for hIAPP in micelles for which the polypeptide structure is known, using pairs of 13C18O isotope labels L12A13 and L16V17, along with single labeled control experiments. Applying the approach to aggregation experiments performed in buffer, we show that about 27-38% of hIAPP peptides adopt an α-helix secondary structure in the monomeric state at L12A13, prior to aggregation, but not at L16V17 residues. At L16V17, the kinetics are described solely by the monomer and fiber conformations, but at L12A13 the kinetics exhibit a third state that is created by an oligomeric intermediate. Control experiments performed with a single isotope label at A13 exhibit two-state kinetics, indicating that a previously unknown change in dihedral angle occurs at L12A13 as hIAPP transitions from the intermediate to fiber structures. We propose a mechanism for aggregation, in which helices seed oligomer formation via structures analogous to leucine rich repeat proteins.
Highlights
Human islet amyloid polypeptide aggregates into fibrils through oligomers that have been postulated to contain a-helices as well as b-sheets
We introduce isotope labels at two neighbouring residues, which results in an increased intensity and positive frequency shift if those residues are a-helical versus a negative frequency shift in b-sheets and turns
The 2D IR dihedral index approach is demonstrated for Human islet amyloid polypeptide (hIAPP) in micelles for which the polypeptide structure is known, using pairs of 13C18O isotope labels L12A13 and L16V17, along with single labeled control experiments
Summary
The abnormal aggregation of peptides and proteins into amyloid brils is associated with more than 20 human diseases, which include metabolic disorders like type 2 diabetes[1] as well as various neurodegenerative disorders such as Alzheimer's and Parkinson's diseases.[2]. When an isotope label becomes incorporated into a parallel b-sheet, like many residues do when bers form, coupling between strands produce a negative shi in the labelling frequency.[32] single labels are very sensitive to amyloid b-sheet formation. The inter-strand coupling for in-register residues of a parallel b-sheet, such as forms in hIAPP bers, is negative (Fig. 1B). Spectra of singly isotope labelled peptides are used for control experiments to separate vibrational shi s due to a change in electrostatic environment from shi s caused by vibrational coupling.[32,33,34,35] double labels provide a qualitative and quantitative measure of the secondary structure, with a positive frequency shi indicating an a-helix and a negative shi a b-sheet, and an intensity enhancement over random coils. We propose a structure, based on a known protein fold, for the oligomer that appears consistent with many experimental observations
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