Figure 1.
1H-15N HSQC spectra illustrating hydrogen exchange in amylin fibrils.
(A) Control spectrum of unfibrillized 15N-amylin freshly dissolved in 95% d6-DMSO/5% DCA at 25°C, pH 3.5. Backbone crosspeaks are labeled according to sequence-specific assignments. Residues N3, T4, A5, and A8 are only visible at lower contours than shown. The group of crosspeaks connected by horizontal lines between 109 and 111 ppm (15N) are unassigned sidechain amide groups from the 6 Asn and 1 Gln in amylin. (B)Spectrum of a 15N-amylin after 4 days (99h) of D2O exchange in the fibril state, recorded in 95% d6-DMSO/5% d2-DCA. Strongly protected amide protons are labeled in bold type.
Figure 2.
Representative solvent exchange kinetics for amide protons in amylin fibrils.
Error bars were estimated from the average root-mean-square baseline noise of the 1H-15N HSQC spectra. The curves are fits of amide proton intensity decay data to an exponential model: y = I0•exp(-τ•x), obtained using the program KaleidaGraph v 4.1.3 (Synergy Software). The two free variables in the fits were I0, the initial amplitude and τ, the time constant for exchange.
Figure 3.
Time constants for hydrogen exchange as a function of residue position in the sequence.
The top of the figure indicates the position of the two β-strands reported for the ssNMR [10] and EPR models of the amylin fibril structure, as well as the revised secondary structure limits based on the qHX data in this work. Uncertainties in exchange time constants were estimated from standard errors of the fits of the qHX data to exponential decays (Fig. 2). The symbols ‘*’ indicate amide protons that exchange with rates too fast to measure, ‘U’ indicates that the amide proton of T6 is unassigned.
Figure 4.
The ssNMR structural model of amylin fibrils [10].
The long axis of the fibrils runs in and out of the plane of the page. (A) Backbone hydrogen bonding between two adjacent amylin monomers in the fibril. Amide protons involved in intermolecular β-sheet hydrogen bonds are labeled alternatively in the blue and gray monomers. Note that the β-sheet hydrogen bonding is continuous along the length of the fibril, so that the amide proton of T36 in the blue monomer is a hydrogen bond donor for the carbonyl of S35 in the next monomer below (not shown). (B) In the ssNMR model of amylin fibrils two columns of amylin β-hairpins stack against each other with C2 symmetry to form a protofilament [10]. The C-terminal strands (red and orange) constitute the packing interface between the two layers of β-sheets, whereas the N-terminal strands (green) are on the surface. Residues I26-L27 which were not assigned to strand β2 in the ssNMR model but which nevertheless show strong qHX protection are colored in light blue. The drawings were rendered in PyMOL [39].
Figure 5.
Comparison of experimental HX rates obtained in this work (gray symbols) with theoretical simulations of amylin fibril flexibility (black symbols).
(A) Theoretical B-factors obtained from a GNM calculation [32], [42] of protein dynamics based on the ssNMR model of amylin fibrils [10]. The B-factors were averaged over the 10 amylin monomers in the ssNMR model [10]. (B) Predicted 2DIR lineshapes (Γi) for amylin fibrils calculated from a MD simulation of the ssNMR amylin fibril structural model. The Γi data are from Fig. 9 of reference [12].