SsNMR chemical shifts of amylinResults and Discussion Amylin Fibrils Show Variable

SsNMR chemical shifts of amylinResults and Discussion Amylin Fibrils Show Variable

SsNMR chemical shifts of amylinResults and Discussion Amylin Fibrils Show Variable Amide Proton Exchange ProtectionFigure 1 compares spectra of fully protonated amylin (Fig. 1A) with amylin partially exchanged in fibrils grown from an aqueous solution containing 10 (v/v) acetonitrile (Fig. 1B). NMR assignments for amylin in 95 DMSO/5 DCA were obtained for all 36 of the expected 1H-15N backbone amide correlations, except residue T6. The first eight residues show weaker 1H-15N crosspeaks than the rest of the peptide (Fig. 1A). Weaker correlations from this region were also seen for 15N-amylin in H2O [31] and SDS micelles [33], suggesting NMR linebroadening associated with an intrinsic dynamic process such as conformational exchange involving the C2 7 disulfide bond. Figure 1B shows the spectrum of 15N-amylin in DMSO after 4 days of D2O exchange in the fibrils. The spectrum is plotted at contour levels that emphasize residues with the strongest amide proton protection, which are labeled in bold type. Most of the strongly protected amide protons are within the two b-strands identified in the ssNMR model. The protected residues that lie immediately outside of the b-strands, H18 and I26 27, suggest that the b-strand limits extend beyond those identified for the ssNMR model. Residues labeled in plain type show intermediate amide proton occupancy. Most of these residues also fall within the two b-strands, pointing to variability in protection within a given element of secondary structure. The residues with the SIS 3 chemical information weakest protection are either not seen, or close to the baseline noise in the spectrum after 4 days of D2O exchange. These include residues in the N21-A25 turn between the b-strands and residues C2 7, which are disordered in the ssNMR model of amylin. Interestingly, the segment A8 13 that forms the N-terminal portion of strand b1 in the ssNMR model is also weakly protected. Note that in the fibril the b-strands form two intermolecular b-sheets [10], with possibly independent stabilities. Hydrogen exchange in amylin fibrils was characterized at seven time points ranging from 5 min to 356 h (,14 days). FigureHydrogen Exchange in Amylin FibrilsFigure 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 25uC, 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 15900046 fibril state, recorded in 95 d6-DMSO/5 d2-DCA. Strongly protected amide protons are labeled in bold type. doi:10.1371/journal.pone.0056467.gFigure 2. Representative solvent exchange kinetics for amide protons in amylin fibrils. Error bars were estimated from the order PHCCC 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(-t 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 t, the time constant for exchange. doi:10.1371/journal.pone.0056467.gN Nfibrils suggest that L27 is not in a b-sheet conformation but otherwise support b-sheet structure for all resid.SsNMR chemical shifts of amylinResults and Discussion Amylin Fibrils Show Variable Amide Proton Exchange ProtectionFigure 1 compares spectra of fully protonated amylin (Fig. 1A) with amylin partially exchanged in fibrils grown from an aqueous solution containing 10 (v/v) acetonitrile (Fig. 1B). NMR assignments for amylin in 95 DMSO/5 DCA were obtained for all 36 of the expected 1H-15N backbone amide correlations, except residue T6. The first eight residues show weaker 1H-15N crosspeaks than the rest of the peptide (Fig. 1A). Weaker correlations from this region were also seen for 15N-amylin in H2O [31] and SDS micelles [33], suggesting NMR linebroadening associated with an intrinsic dynamic process such as conformational exchange involving the C2 7 disulfide bond. Figure 1B shows the spectrum of 15N-amylin in DMSO after 4 days of D2O exchange in the fibrils. The spectrum is plotted at contour levels that emphasize residues with the strongest amide proton protection, which are labeled in bold type. Most of the strongly protected amide protons are within the two b-strands identified in the ssNMR model. The protected residues that lie immediately outside of the b-strands, H18 and I26 27, suggest that the b-strand limits extend beyond those identified for the ssNMR model. Residues labeled in plain type show intermediate amide proton occupancy. Most of these residues also fall within the two b-strands, pointing to variability in protection within a given element of secondary structure. The residues with the weakest protection are either not seen, or close to the baseline noise in the spectrum after 4 days of D2O exchange. These include residues in the N21-A25 turn between the b-strands and residues C2 7, which are disordered in the ssNMR model of amylin. Interestingly, the segment A8 13 that forms the N-terminal portion of strand b1 in the ssNMR model is also weakly protected. Note that in the fibril the b-strands form two intermolecular b-sheets [10], with possibly independent stabilities. Hydrogen exchange in amylin fibrils was characterized at seven time points ranging from 5 min to 356 h (,14 days). FigureHydrogen Exchange in Amylin FibrilsFigure 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 25uC, 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 15900046 fibril state, recorded in 95 d6-DMSO/5 d2-DCA. Strongly protected amide protons are labeled in bold type. doi:10.1371/journal.pone.0056467.gFigure 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(-t 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 t, the time constant for exchange. doi:10.1371/journal.pone.0056467.gN Nfibrils suggest that L27 is not in a b-sheet conformation but otherwise support b-sheet structure for all resid.

Proton-pump inhibitor

Website: