Lso agrees with EPR data for amylin fibrils. Residues A8 13 show

Lso agrees with EPR data for amylin fibrils. Residues A8 13 show

Lso agrees with EPR data for Bexagliflozin manufacturer amylin fibrils. Residues A8 13 show increased EPR linewidths characteristic of increased mobility, and reduced differences in the mobility of spin-labels introduced on the inside and outside of the b-sheet in the segment spanning positions A8 13 (Fig. 2 in [11]). To test the hypothesis that the lower qHX protection observed for strand b1 is due to its position on the surface of the protofilament (Fig. 4B), GNM calculations [32,42] of protein flexibility were performed using the ssNMR model of the amylin protofilament [10]. The GNM formalism models fluctuations about a mean structure as order SR-3029 dependent on the distribution of distance contacts to nearby Ca atoms [42]. The predicted amplitudes of fluctuations at different sites can be used to calculate theoretical B-factors [42], which for native proteins have beenHydrogen Exchange in Amylin FibrilsFigure 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 b-sheet hydrogen bonds are labeled alternatively in the blue and gray monomers. Note that the b-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 b-hairpins stack against each other with C2 symmetry to form a protofilament [10]. The Cterminal strands (red and orange) constitute the packing interface between the two layers of b-sheets, whereas the N-terminal strands (green) are on the surface. Residues I26-L27 which were not assigned to strand b2 in the ssNMR model but which nevertheless show strong qHX protection are colored in light blue. The drawings were rendered in PyMOL [39]. doi:10.1371/journal.pone.0056467.gshown to be in good agreement with experimental B-factors determined by X-ray crystallography and to correlate with HX protection factors [34,42?4]. The theoretical B-factors calculated for the amylin fibril model are shown by the black symbols in Fig. 5a. The GNM calculations predict small B-factors indicative of reduced mobility for strands b1 and b2, as well as larger Bfactors for the N-terminal strand b1 compared to the C-terminal strand b2. Although the GNM calculations capture the features of the HX sequence profile (gray symbols in Fig. 5A) the quantitative correlation to the observed HX rates is poor (R-value = 0.17, r = 0.3 for n = 33).A better agreement (Fig. 5B) is seen when the HX rates are compared to theoretically predicted inhomogeneous frequency contributions to the 2DIR diagonal linewidths of amylin fibrils, Ci [45], calculated from an all-atom MD simulation [12] of the solvated 15755315 ssNMR amylin fibril model. The Ci values were obtained by taking into account the fluctuating electric fields at a given site caused by the movement of all nearby atoms in the MD simulation. The Ci and log(kHX) data in Fig. 5B are pair-wise correlated with an R-value of 0.56 (r,0.001 for n = 33). The Ci values show a gradient of decreasing flexibility from the unstructured segment ending at C7 to about residue N14 in strandHydrogen Exchange in Amylin Fibrilsthe HX data suggests that strand b1 extends by one residue to H18 and strand b2 starts two residues earlier at L26. Differences in protection are obs.Lso agrees with EPR data for amylin fibrils. Residues A8 13 show increased EPR linewidths characteristic of increased mobility, and reduced differences in the mobility of spin-labels introduced on the inside and outside of the b-sheet in the segment spanning positions A8 13 (Fig. 2 in [11]). To test the hypothesis that the lower qHX protection observed for strand b1 is due to its position on the surface of the protofilament (Fig. 4B), GNM calculations [32,42] of protein flexibility were performed using the ssNMR model of the amylin protofilament [10]. The GNM formalism models fluctuations about a mean structure as dependent on the distribution of distance contacts to nearby Ca atoms [42]. The predicted amplitudes of fluctuations at different sites can be used to calculate theoretical B-factors [42], which for native proteins have beenHydrogen Exchange in Amylin FibrilsFigure 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 b-sheet hydrogen bonds are labeled alternatively in the blue and gray monomers. Note that the b-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 b-hairpins stack against each other with C2 symmetry to form a protofilament [10]. The Cterminal strands (red and orange) constitute the packing interface between the two layers of b-sheets, whereas the N-terminal strands (green) are on the surface. Residues I26-L27 which were not assigned to strand b2 in the ssNMR model but which nevertheless show strong qHX protection are colored in light blue. The drawings were rendered in PyMOL [39]. doi:10.1371/journal.pone.0056467.gshown to be in good agreement with experimental B-factors determined by X-ray crystallography and to correlate with HX protection factors [34,42?4]. The theoretical B-factors calculated for the amylin fibril model are shown by the black symbols in Fig. 5a. The GNM calculations predict small B-factors indicative of reduced mobility for strands b1 and b2, as well as larger Bfactors for the N-terminal strand b1 compared to the C-terminal strand b2. Although the GNM calculations capture the features of the HX sequence profile (gray symbols in Fig. 5A) the quantitative correlation to the observed HX rates is poor (R-value = 0.17, r = 0.3 for n = 33).A better agreement (Fig. 5B) is seen when the HX rates are compared to theoretically predicted inhomogeneous frequency contributions to the 2DIR diagonal linewidths of amylin fibrils, Ci [45], calculated from an all-atom MD simulation [12] of the solvated 15755315 ssNMR amylin fibril model. The Ci values were obtained by taking into account the fluctuating electric fields at a given site caused by the movement of all nearby atoms in the MD simulation. The Ci and log(kHX) data in Fig. 5B are pair-wise correlated with an R-value of 0.56 (r,0.001 for n = 33). The Ci values show a gradient of decreasing flexibility from the unstructured segment ending at C7 to about residue N14 in strandHydrogen Exchange in Amylin Fibrilsthe HX data suggests that strand b1 extends by one residue to H18 and strand b2 starts two residues earlier at L26. Differences in protection are obs.

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