Fig. 4: Solid-state NMR spectra and analysis of reversible fibrils of human lysozyme demonstrating the formation of dynamic heterogenous intermolecular β-sheets as well as helical molten globule-like structures.
From: A structural rationale for reversible vs irreversible amyloid fibril formation from a single protein
a Aliphatic region of a 2D [13C, 13C]-DARR spectrum in black (Table S1) overlaid with a simulated spectrum in red of the irreversible fibrils using typical linewidths for an amyloid structure. Both helical and beta-structured Ala sidechain peaks are highlighted in the spectra. b Detection of intermolecular contacts in reversible lysozyme fibrils between a 15N-labeled protein and a 13C-labeled protein by a 2D PAIN experiment yielding a 15N-13C correlation spectrum (blue). The spectrum is overlaid with a 2D NC-CP (red) spectrum. The shaded regions show typical 13C chemical shift ranges for the indicated atom types (the * symbol indicates signals for an out-of-register inter-molecular contact). The inset shows cross-sections used to adjust the contour lines of the 2D spectra. c Measurement of bulk 13C T2′ relaxation times at different temperatures by a variable-delay spin echo and fit by a single exponential (Table S2). d T2′ relaxation measurements indicating that the observed 750 Hz peak width in the 2D [13C, 13C]-DARR spectrum at 8 °C (Table S1) is attributed to 250 Hz from dynamics and 500 Hz from structural heterogeneity. e Simulated 2D [13C, 13C]-DARR spectra of the sub-domain A residues from the native human lysozyme structure in green overlaid with that of the sub-domain B residues 37–80 in the irreversible fibril structure in purple (both spectra with a peak width of 750 Hz to match the experimental data). Ala sidechain peaks are highlighted as in (a). f An overlay of the experimental spectrum (black) from (a) with a sum of the two simulated spectra (cyan) from (e). g The primary sequence of human lysozyme showing the secondary structural elements of both the soluble monomer (purple and green) and the irreversible fibril (gray). The letters in bold indicate residues in the model of the irreversible fibril. Some residues whose absence or presence are discussed in the text are colored to highlight their positions: Met (yellow), Ile (red) and Ala (blue).
