Under solution conditions where the native state is destabilized, the largely helical polypeptide hormone insulin readily aggregates to form amyloid fibrils with a characteristic cross-β structure. However, there is a lack of information relating the 4.8 Å β-strand repeat to the higher order assembly of amyloid fibrils. We have used cryo-electron microscopy (EM), combining single particle analysis and helical reconstruction, to characterize these fibrils and to study the three-dimensional (3D) arrangement of their component protofilaments. Low-resolution 3D structures of fibrils containing 2, 4, and 6 protofilaments reveal a characteristic, compact shape of the insulin protofilament. Considerations of protofilament packing indicate that the cross-β ribbon is composed of relatively flat β-sheets rather than being the highly twisted, β-coil structure previously suggested by analysis of globular protein folds. Comparison of the various fibril structures suggests that very small, local changes in β-sheet twist are important in establishing the long-range coiling of the protofilaments into fibrils of diverse morphology.

Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like alpha-helical structure. On heating to 70 degrees C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from alpha-helical to beta-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of approximately 450 A. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.

We have examined the hydrogen exchange properties of bovine insulin under solution conditions that cause it to aggregate and eventually form amyloid fibrils. The results have been obtained at the residue-specific level using peptic digestion and mass spectrometry. A total of 19 peptides were assigned to regions of the protein and their exchange properties monitored for a period of 24 hours. The results of the peptic digestion show that residues A13 to A21 and B11 to B30 are more susceptible to proteolysis than the N-terminal regions of the protein. A total of 15 slowly exchanging amides were observed for insulin under these solution conditions. Location of the protected amides was carried out using a peptic-digestion protocol at low pH. Chromatographic separation was not required. This enabled a direct comparison of the peptides within the same mass spectrum. From kinetic analysis of the rates slow exchange has been located to 4(±1) backbone amides in the A13-A19 helix and 6(±1) in the B chain helix. The remaining 5(±1) are assigned to helix A2-A8. Taken together the results from digestion and hydrogen exchange show that at low pH and relatively high concentrations the C termini of both chains are susceptible to proteolysis but that the solution structure contains the native state helices. More generally the results demonstrate that mass spectrometry can be applied to study site-specific hydrogen exchange properties of proteins even under conditions where they are known to be partially folded and aggregate extensively in solution.

Evidence has suggested that the formation of amyloid fibrils may involve the structural rearrangement of native monomeric protein through partially folded intermediates. For example, the kinetics of amyloid fibril formation in vitro is most favorable for wild-type transthyretin under solution conditions that destabilize the native state tetramer of the protein. Furthermore, naturally occurring variants of human lysozyme, transthyretin, and immunoglobulin light chain VL domain have been shown to destabilize the native states of these proteins. Destabilization allows these proteins to unfold more readily, generating partially folded states that are proposed as amyloidogenic intermediates. These aggregation-prone intermediates are difficult to study by classical structural biology techniques as they are too dynamic for X-ray crystallography and are not amenable to nuclear magnetic resonance (NMR) measurements directly because the concentration necessary for their study promotes aggregation. In this article, hydrogen exchange measured by electrospray (ES) mass spectrometry (MS) is used to probe the solution dynamics of amyloidogenic proteins.

Wild-type and variant transthyretins form amyloid fibrils in two different diseases. The biologically active form of transthyretin is a tetramer but there is evidence that a monomeric species is the amyloidogenic intermediate. Using mass spectrometry we have developed an approach to monitor the proportions of monomer and tetramer in wild-type and variant transthyretins, and found a strong correlation between the instability of the tetramer in the gas phase and the amyloidogenicity of the protein variant. The presence of water molecules in the central channel has been found to be critical for maintaining intact the complex in the gas phase, with additional stability observed in the presence of excess thyroxine. The solution structure of monomeric transthyretin under fibril-forming conditions was studied using hydrogen exchange monitored by mass spectrometry. The results show that Val30Met transthyretin, the commonest amyloidogenic variant, exhibits loss of hydrogen exchange protection substantially more rapidly than the wild-type protein, suggesting partial unfolding of the β-sheet structure. These results provide new insights into the correlation between tetramer stability and amyloidogenicity as well as supporting a possible route to fibril formation via transient unfolding of the transthyretin monomer.

The extent of deuterium labeling of hen lysozyme, its three‐disulfide derivative, and the homologous α‐lactalbumins, has been measured by both mass spectrometry and NMR. Different conformational states of the proteins were produced by varying the solution conditions. Alternate protein conformers were found to contain different numbers of 2 H atoms. Furthermore, measurement in the gas phase of the mass spectrometer or directly in solution by NMR gave consistent results. The unique ability of mass spectrometry to distinguish distributions of 2 H atoms in protein molecules is exemplified using samples prepared to contain different populations of 2 H‐labeled protein. A comparison of the peak widths of bovine α‐lactalbumin in alternate solution conformations but containing the same average number of 2 H atoms showed dramatic differences due to different 2 H distributions in the two protein conformers. Measurement of 2 H distributions by ESI‐MS enabled characterization of conformational averaging and structural heterogeneity. In addition, a time course for hydrogen exchange was examined and the variation in distributions of 2 H atom compared with simulations for different hydrogen exchange models. The results clearly show that exchange from the native state of bovine α‐lactalbumin at 15°C is dominated by local unfolding events.