Structure-Specific Effects of Protein Topology on Cross-β Assembly:  Studies of Insulin Fibrillation

Abstract

Systemic amyloidoses, an important class of protein misfolding diseases, are often due to fibrillation of disulfide-cross-linked globular proteins otherwise unrelated in sequence or structure. Although cross-beta assembly is regarded as a universal property of polypeptides, it is not understood how such amyloids accommodate diverse disulfide connectivities. Does amyloidogenicity depend on protein topology? A model is provided by insulin, a two-chain protein containing three disulfide bridges. The importance of chain topology is demonstrated by mini-proinsulin (MP), a single-chain analogue in which the C-terminus of the B chain (residue B30) is tethered to the N-terminus of the A chain (A1). The B30-A1 tether impedes the fiber-specific alpha --> beta transition, leading to slow formation of a structurally nonuniform amorphous precipitate. Conversely, fibrillation is robust to interchange of disulfide bridges. Whereas native insulin exhibits pairings [A6-A11, A7-B7, and A20-B19], metastable isomers with alternative pairings [A6-B7, A7-A11, A20-B19] or [A6-A7, A11-B7, A20-B1] readily undergo fibrillation with essentially identical alpha --> beta transitions. Respective pairing schemes are in each case retained. Isomeric fibrils and the amorphous MP precipitate are each able to seed the fibrillation of wild-type insulin, suggesting a structural correspondence between respective nuclei or modes of assembly. Together, our results demonstrate that effects of polypeptide topology on amyloidogenicity depend on structural context. Although the native structures and stabilities of single-chain insulin analogues are similar to those of wild-type insulin, the interchain tether constrains the extent of conformational distortion at elevated temperature, retards initial non-native aggregation, and is apparently incompatible with the mature structure of an insulin protofilament. We speculate that the general danger of fibrillation has imposed a constraint in protein evolution, selecting for topologies unfavorable to amyloid formation.

H ferritin silencing induces protein misfolding in K562 cells: A Raman analysis

“…Finally, all spectra of the same group are averaged and only the spectral region between 1515 and 1775 cm -1 has been selected for further analysis of the proteins secondary structure. The other spectral regions are excluded due to the fact that some solvents used for As well known (14,15), the Amide I band mainly arises from the C=O stretching vibration of proteins backbone, and it is very sensitive to secondary structure. This Raman band is composed of three main peaks, centered at 1650-1655 cm -1 for alpha helix structures, at 1665-1670 cm -1 for the beta sheet, and 1675-1685 cm -1 for more disordered structures (15)(16)(17).…”

“…The other spectral regions are excluded due to the fact that some solvents used for As well known (14,15), the Amide I band mainly arises from the C=O stretching vibration of proteins backbone, and it is very sensitive to secondary structure. This Raman band is composed of three main peaks, centered at 1650-1655 cm -1 for alpha helix structures, at 1665-1670 cm -1 for the beta sheet, and 1675-1685 cm -1 for more disordered structures (15)(16)(17). Consequently, decomposition of the Amide I band through fitting procedures permits to separate the contribution from these three different peaks, thus providing clear information about the relative content of alpha, beta and disordered structures.…”

“…Lorentzian-Gaussian curves are used to model Raman peaks in the fitting procedure, using a Levenberg-Marquardt nonlinear least-squares method. As widely reported in literature (14)(15)(16)(17), the mixed Lorentzian-Gaussian curve is the best choice for fitting Raman peaks recorded on liquid samples, even with the DCDR technique where the solvent is partly evaporated. The starting parameters have been chosen so that three peaks are in the Amide I area, while two or three peaks are outside, corresponding to ring modes from aromatic side chains (in the range 1550-1615 cm -1 ) (18).…”

Porcine islet amyloid polypeptide fragments are refractory to amyloid formation

“…Each result was the average of three measurements. The data were converted to mean residue ellipticity [h] and were further analyzed by the software package CDPro as we and others previously described [9,10]. Two reference data sets -SDP 42 (#6) and SDP 48 (#7) -were used in CDPro analysis since they both include denatured proteins [10].…”

“…The excitation and emission wavelengths were set at 450 nm and 482 nm, respectively. The seeding experiments (self-or cross-seeding) were performed as we previously described [9,11]. Briefly, freshly prepared amyloids were sonicated for 3 min and immediately applied to fresh sample solution at 5% (w/w) final concentration as seeds.…”