Troduced spin-labels that is characteristic of the inside-outside polarity of sidechains

Troduced spin-labels that is characteristic of the inside-outside polarity of sidechains in a b?strand, and (2) a characteristic distance of ,21 A MedChemExpress JI 101 between spinlabels introduced with an i to i+6 sequence spacing in a b-strand. In the EPR model CP21 biological activity strand b1 is comprised of residues L12-S19 and b2 of N31-T36. The later start of strand b1 is a result of the increased mobility of the A8-R11 segment in the EPR data [11]. Increased mobility for this segment is also observed by ssNMR [10]. The 1655472 end of strand b1 at S19 25033180 in the EPR model is consistent with the strong protection observed for H18 and the inclusion of this residue in strand b1 in the present study. Strand b2 in the EPR model (N31-T36) ends one residue earlier and starts three residues later than in the ssNMR model (S28-Y37), whereas the HX protection data in this work suggests that strand b2 begins as early as I26. In contrast to strand b1, there was only one probe of i to i+6 distances reported for strand b2, between residues G24 and ?T30. The distance between these probes was 23 A, indicating ?a conformation more extended than the expected 21 A distance[11], which seems consistent with a b-sheet conformation. The only mobility probe available between residues 25 and T30 was for residue S28, so that these data also do not rule out an earlier starting position for strand b2. The inclusion of residue Y37 as the last residue in strand b2 is supported by strong HX protection, and fluorescence data indicating restricted mobility and solvent accessibility for Y37 as well as FRET contacts to residues F15 and F23 [41].Comparison with Flexibility Predictions from Molecular Dynamics CalculationsThe beginning of strand b1 comprised of residues A8 13 shows minimal HX protection, with slowly exchanging amide protons only observed for residues N14-H18 (Fig. 3). The lack of protection for the N-terminal part of strand b1 indicates this segment is flexible. These results are consistent with ssNMR line broadening noted for residues A8 13 in 2D 13C fpRFDR (finitepulse radiofrequency-driven recoupling) spectra of amylin fibrils [10]. Line broadening in NMR spectra is typically associated with motion on ms-ms timescales. Fast motion on these ms-ms timescales would provide an avenue for amide proton exchange on the much slower hour to day timescales of the HX experiments in this work. Increased mobility of the A8 13 segment also 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 pr.Troduced spin-labels that is characteristic of the inside-outside polarity of sidechains in a b?strand, and (2) a characteristic distance of ,21 A between spinlabels introduced with an i to i+6 sequence spacing in a b-strand. In the EPR model strand b1 is comprised of residues L12-S19 and b2 of N31-T36. The later start of strand b1 is a result of the increased mobility of the A8-R11 segment in the EPR data [11]. Increased mobility for this segment is also observed by ssNMR [10]. The 1655472 end of strand b1 at S19 25033180 in the EPR model is consistent with the strong protection observed for H18 and the inclusion of this residue in strand b1 in the present study. Strand b2 in the EPR model (N31-T36) ends one residue earlier and starts three residues later than in the ssNMR model (S28-Y37), whereas the HX protection data in this work suggests that strand b2 begins as early as I26. In contrast to strand b1, there was only one probe of i to i+6 distances reported for strand b2, between residues G24 and ?T30. The distance between these probes was 23 A, indicating ?a conformation more extended than the expected 21 A distance[11], which seems consistent with a b-sheet conformation. The only mobility probe available between residues 25 and T30 was for residue S28, so that these data also do not rule out an earlier starting position for strand b2. The inclusion of residue Y37 as the last residue in strand b2 is supported by strong HX protection, and fluorescence data indicating restricted mobility and solvent accessibility for Y37 as well as FRET contacts to residues F15 and F23 [41].Comparison with Flexibility Predictions from Molecular Dynamics CalculationsThe beginning of strand b1 comprised of residues A8 13 shows minimal HX protection, with slowly exchanging amide protons only observed for residues N14-H18 (Fig. 3). The lack of protection for the N-terminal part of strand b1 indicates this segment is flexible. These results are consistent with ssNMR line broadening noted for residues A8 13 in 2D 13C fpRFDR (finitepulse radiofrequency-driven recoupling) spectra of amylin fibrils [10]. Line broadening in NMR spectra is typically associated with motion on ms-ms timescales. Fast motion on these ms-ms timescales would provide an avenue for amide proton exchange on the much slower hour to day timescales of the HX experiments in this work. Increased mobility of the A8 13 segment also 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 pr.