Erved within each b-strand, much like in native proteins. In the

Erved within each b-strand, much like in native proteins. In the case of amylin fibrils these differences correlate with the packing of b-sheets into the higher-order protofilament structure. The N-terminal strand b1 on the surface of the protofilament, shows weak protection until the last five residues. By contrast, amide protons are protected throughout the Cterminal strand b2, which is buried in the protofilament structure. The HX studies described herein set the foundation for investigations to determine if protection in fibrils accrues through intermediates or arises in an all-or-none fashion, to look at how fibril structure changes with solution variables such as pH or when complexed with accessory molecules (e.g. metals or glycosaminoglycans) and to determine binding sites for ligands and drugs that target fibril growth.Supporting InformationFigure S1 NMR experiments demonstrate that amylin is an unfolded monomer in DMSO. (A) 1D-1H NMR spectrum of 220 mM human amylin (with an amidated Cterminus) in 95 d6-DMSO/5 d2-DCA, pH* 3.5, 25uC. The large resonances at 2.5 and 6.7 ppm are due to residual natural abundance DMSO and DCA, respectively. The methyl resonance at 0.8 ppm was used to characterize amylin diffusion. (B) Pulsefield gradient measurements of amylin translational diffusion. Experiments were carried out on a Bruker 500 MHz spectrometer with 1,4-dioxane added as an internal standard to the sample in A. From the diffusion coefficients of dioxane and the peptide we can ?calculate a hydrodynamic radius of 1561 A for amylin, using the formula Rpeptide = (Ddioxane/Dpeptide)Rdioxane and assuming a hy?drodynamic radius of 2.12 A for dioxane. The expected hydrodynamic radius for an unfolded Lixisenatide biological activity protein is given by the empirical equation Rh = (2.2161.07)N0.5760.02, where N is the ?number of residues. The predicted (17 A) and experimental ?) values are close, indicating that amylin behaves as an (1561 A unfolded monomer in DMSO. (TIF) Figure S2 Electron micrograph of amylin fibrils. FibrilsFigure 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 (Ci) for amylin fibrils calculated from a MD simulation of the ssNMR amylin fibril structural model. The Ci data are from Fig. 9 of reference [12]. 1516647 doi:10.1371/journal.pone.0056467.gb1, in good agreement with the qHX data. The biggest differences occur for residues L16-H18 where the MD calculations overpredict flexibility compared to the HX data. The turn segment between the two b-strands has large HX rates and Ci values. A spike is seen for both the theoretical Ci values and the experimental HX rates near residues G33-N35 in strand b2, before both values fall at the C-terminus of amylin. Although the origin of the disorder for residues G33-N35 is unknown, experimental support for increased flexibility has been observed by 2DIR spectroscopy [12].of recombinant 15N-amylin were formed under the same conditions as the hydrogen exchange experiments. Fibrils were transferred to a 400-mesh carbon-coated grid, rinsed with H2O, and negatively stained with 1 uranyl acetate. Images were obtained on a FEI Tecnai G2 BioTWIN Licochalcone-A site instrument that is part of the UConn elec.Erved within each b-strand, much like in native proteins. In the case of amylin fibrils these differences correlate with the packing of b-sheets into the higher-order protofilament structure. The N-terminal strand b1 on the surface of the protofilament, shows weak protection until the last five residues. By contrast, amide protons are protected throughout the Cterminal strand b2, which is buried in the protofilament structure. The HX studies described herein set the foundation for investigations to determine if protection in fibrils accrues through intermediates or arises in an all-or-none fashion, to look at how fibril structure changes with solution variables such as pH or when complexed with accessory molecules (e.g. metals or glycosaminoglycans) and to determine binding sites for ligands and drugs that target fibril growth.Supporting InformationFigure S1 NMR experiments demonstrate that amylin is an unfolded monomer in DMSO. (A) 1D-1H NMR spectrum of 220 mM human amylin (with an amidated Cterminus) in 95 d6-DMSO/5 d2-DCA, pH* 3.5, 25uC. The large resonances at 2.5 and 6.7 ppm are due to residual natural abundance DMSO and DCA, respectively. The methyl resonance at 0.8 ppm was used to characterize amylin diffusion. (B) Pulsefield gradient measurements of amylin translational diffusion. Experiments were carried out on a Bruker 500 MHz spectrometer with 1,4-dioxane added as an internal standard to the sample in A. From the diffusion coefficients of dioxane and the peptide we can ?calculate a hydrodynamic radius of 1561 A for amylin, using the formula Rpeptide = (Ddioxane/Dpeptide)Rdioxane and assuming a hy?drodynamic radius of 2.12 A for dioxane. The expected hydrodynamic radius for an unfolded protein is given by the empirical equation Rh = (2.2161.07)N0.5760.02, where N is the ?number of residues. The predicted (17 A) and experimental ?) values are close, indicating that amylin behaves as an (1561 A unfolded monomer in DMSO. (TIF) Figure S2 Electron micrograph of amylin fibrils. FibrilsFigure 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 (Ci) for amylin fibrils calculated from a MD simulation of the ssNMR amylin fibril structural model. The Ci data are from Fig. 9 of reference [12]. 1516647 doi:10.1371/journal.pone.0056467.gb1, in good agreement with the qHX data. The biggest differences occur for residues L16-H18 where the MD calculations overpredict flexibility compared to the HX data. The turn segment between the two b-strands has large HX rates and Ci values. A spike is seen for both the theoretical Ci values and the experimental HX rates near residues G33-N35 in strand b2, before both values fall at the C-terminus of amylin. Although the origin of the disorder for residues G33-N35 is unknown, experimental support for increased flexibility has been observed by 2DIR spectroscopy [12].of recombinant 15N-amylin were formed under the same conditions as the hydrogen exchange experiments. Fibrils were transferred to a 400-mesh carbon-coated grid, rinsed with H2O, and negatively stained with 1 uranyl acetate. Images were obtained on a FEI Tecnai G2 BioTWIN instrument that is part of the UConn elec.