Characterization of the Oligomeric States of Insulin in Self-Assembly and Amyloid Fibril Formation by Mass Spectrometry (original) (raw)
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New Insights into the Self-Assembly of Insulin Amyloid Fibrils: An H−D Exchange FT-IR Study †
Biochemistry, 2006
The solvent protection of the amide backbone in bovine insulin fibrils was studied by FT-IR spectroscopy. In the mature fibrils, approximately 85 ( 2% of amide protons are protected. Of those "trapped" protons, a further 25 ( 2 or 35 ( 2% is H-D exchanged after incubation for 1 h at 1 GPa and 25°C or 0.1 MPa and 100°C, respectively. In contrast to the native or unfolded protein, fibrils do not H-D exchange upon incubation at 65°C. A complete deuteration of H 2 O-grown fibrils occurs when the -sheet structure is reassembled in a 75 wt % DMSO/D 2 O solution. Our findings suggest a densely packed environment around the amide protons involved in the intermolecular -sheet motive. In disagreement with the concept of "amyloid fibers as water-filled nanotubes" Proc. Natl. Acad. Sci. U.S.A. 99, 5591-5595], elution of D 2 O-grown fibrils with H 2 O is complete, which is reflected by the vanishing of D 2 O bending vibrations at 1214 cm -1 . This implies the absence of "trapped water" within insulin fibrils. The rigid conformations of the native and fibrillar insulin contrast with transient intermediate states docking at the fibrils' ends. Room-temperature seeding is accompanied by an accelerated H-D exchange in insulin molecules in the act of docking and integrating with the seeds, proving that the profound structural disruption is the sine qua non of forming an aggregation-competent conformation.
Journal of Molecular Modeling, 2012
Insulin is a hormone that regulates the physiological glucose level in human blood. Insulin injections are used to treat diabetic patients. The amyloid aggregation of insulin may cause problems during the production, storage, and delivery of insulin formulations. Several modifications to the C-terminus of the B chain have been suggested in order to improve the insulin formulation. The central fragments of the A and B chains (LYQLENY and LVEALYL) have recently been identified as β-sheet-forming regions, and their microcrystalline structures have been used to build a high-resolution amyloid fibril model of insulin. Here we report on a molecular dynamics (MD) study of single-layer oligomers of the fulllength insulin which aimed to identify the structural elements that are important for amyloid stability, and to suggest single glycine mutants in the β-sheet region that may improve the formulation. Structural stability, aggregation behavior and the thermodynamics of association were studied for the wild-type and mutant aggregates. A comparison of the oligomers of different sizes revealed that adding strands enhances the internal stability of the wild-type aggregates. We call this "dynamic cooperativity". The secondary structure content and clustering analysis of the MD trajectories show that the largest aggregates retain the fibril conformation, while the monomers and dimers lose their conformations. The degree of structural similarity between the oligomers in the simulation and the fibril conformation is proposed as a possible explanation for the experimentally observed shortening of the nucleation lag phase of insulin with oligomer seeding. Decomposing the free energy into electrostatic, van der Waals and solvation components demonstrated that electrostatic interactions contribute unfavorably to the binding, while the van der Waals and especially solvation effects are favorable for it. A per-atom decomposition allowed us to identify the residues that contribute most to the binding free energy. Residues in the βsheet regions of chains A and B were found to be the key residues as they provided the largest favorable contributions to single-layer association. The positive ΔΔG mut values of 37.3 to 1.4 kcal mol −1 of the mutants in the β-sheet region indicate that they have a lower tendency to aggregate than the wild type. The information obtained by identifying the parts of insulin molecules that are crucial to aggregate formation and stability can be used to design new analogs that can better control the blood glucose level. The results of our simulation may help in the rational design of new insulin analogs with a decreased propensity for self-association, thus avoiding injection amyloidosis. They may also be used to design new fast-acting and delayed-release insulin formulations.
Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy
Protein …, 2000
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 a-helical structure. On heating to 70 8C, 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 a-helical to b-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of ;450 Å. 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.
Structural Insight of Amyloidogenic Intermediates of Human Insulin
ACS omega, 2018
Engaging Raman spectroscopy as a primary tool, we investigated the early events of insulin fibrilization and determined the structural content present in oligomer and protofibrils that are formed as intermediates in the fibril formation pathway. Insulin oligomer, as obtained upon incubation of zinc-free insulin at 60 °C, was mostly spherical in shape, with a diameter of 3-5 nm. Longer incubation produced "necklace"-like beaded protofibrillar assembly species. These intermediates eventually transformed into 5-8 nm thick fibers with smooth surface texture. A broad amide I band in the Raman spectrum of insulin monomer appeared at 1659 cm, with a shoulder band at 1676 cm. This signature suggested the presence of major helical and extended secondary structure of the protein backbone. In the oligomeric state, the protein maintained its helical imprint (∼50%) and no substantial increment of the compact cross-β-sheet structure was observed. A nonamide helix signature band at 940 c...
Journal of Molecular Biology, 2006
Amyloid fibrils are typically rigid, unbranched structures with diameters of ∼10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly α-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low pH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to crossseed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of fulllength insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underlying protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression.
Cytotoxicity of Insulin within its Self-assembly and Amyloidogenic Pathways
Journal of Molecular Biology, 2007
Solvational perturbations were employed to selectively tune the aggregational preferences of insulin at 60°C in vitro in purely aqueous acidic solution and in the presence of the model co-solvent ethanol (EtOH) (at 40%(w/w)). Dynamic light scattering (DLS), thioflavin T (ThT)-fluorescence, Fourier transform infrared (FTIR) and atomic force microscopy (AFM) techniques were employed to characterize these pathways biophysically with respect to the pre-aggregational assembly of the protein, the aggregation kinetics, and finally the aggregate secondary structure and morphology. Using cell viability assays, the results were subsequently correlated with the cytotoxicity of the insulin species that form in the two distinct aggregation pathways. In the cosolvent-free solution, predominantly dimeric insulin self-assembles via the well-known amyloidogenic pathway, yielding exclusively fibrillar aggregates, whereas in the solution containing EtOH, the aggregation of predominantly monomeric insulin proceeds via a pathway that leads to exclusively nonfibrillar, amorphous aggregates. Initially present native insulin assemblies as well as partially unfolded monomeric species and low molecular mass oligomeric aggregates could be ruled out as direct and major cytotoxic species. Apart from the slower overall aggregation kinetics under amorphous aggregate promoting conditions, which is due to the chaotropic nature of high EtOH concentrations, however, both pathways were unexpectedly found to evoke insulin aggregates that were cytotoxic to cultured rat insulinoma cells. The observed kinetics of the decrease of cell viabilities correlated well with the results of the DLS, ThT, FTIR and AFM studies, revealing that the formation of cytotoxic species correlated well with the formation of large-sized, β-sheet-rich assemblies (N500 nm) of both fibrillar and amorphous nature. These results suggest that largesized, β-sheet-rich insulin assemblies of both fibrillar and amorphous nature are toxic to pancreatic β-cells. In the light of the ongoing discussion about putative cytotoxic effects of prefibrillar and fibrillar amyloid aggregates, our results support the hypothesis that, in the case of insulin, factors other than the specific secondary or quarternary structural features of the various different aggregates may define their cytotoxic properties. Two such factors might be the aggregate size and the aggregate propensity to expose hydrophobic surfaces to a polar environment.
Journal of Biological Chemistry, 2004
Insulin undergoes aggregation-coupled misfolding to form a cross-beta assembly. Such fibrillation has long complicated its manufacture and use in the therapy of diabetes mellitus. Of interest as a model for disease-associated amyloids, insulin fibrillation is proposed to occur via partial unfolding of a monomeric intermediate. Here, we describe the solution structure of human insulin under amyloidogenic conditions (pH 2.4 and 60 degrees C). Use of an enhanced sensitivity cryogenic probe at high magnetic field avoids onset of fibrillation during spectral acquisition. A novel partial fold is observed in which the N-terminal segments of the A- and B-chains detach from the core. Unfolding of the N-terminal alpha-helix of the A-chain exposes a hydrophobic surface formed by native-like packing of the remaining alpha-helices. The C-terminal segment of the B-chain, although not well ordered, remains tethered to this partial helical core. We propose that detachment of N-terminal segments makes possible aberrant protein-protein interactions in an amyloidogenic nucleus. Non-cooperative unfolding of the N-terminal A-chain alpha-helix resembles that observed in models of proinsulin folding intermediates and foreshadows the extensive alpha --> beta transition characteristic of mature fibrils.
Kinetics of different processes in human insulin amyloid formation
Journal of molecular …, 2007
Human insulin has long been known to form amyloid fibrils under given conditions. The molecular basis of insulin aggregation is relevant for modeling the amyloidogenesis process, which is involved in many pathologies, as well as for improving delivery systems, used for diabetes treatments. Insulin aggregation displays a wide variety of morphologies, from small oligomeric filaments to huge floccules, and therefore different specific processes are likely to be intertwined in the overall aggregation. In the present work, we studied the aggregation kinetics of human insulin at low pH and different temperatures and concentrations. The structure and the morphogenesis of aggregates on a wide range of length scales (from monomeric proteins to elongated fibrils and larger aggregates networks) have been monitored by using different experimental techniques: time-lapse atomic force microscopy (AFM), quasi-elastic light-scattering (QLS), small and large angle static light-scattering, thioflavin T fluorescence, and optical microscopy. Our experiments, along with the analysis of scattered intensity distribution, show that fibrillar aggregates grow following a thermally activated heterogeneous coagulation mechanism, which includes both tipto-tip elongation and lateral thickening. Also, the association of fibrils into bundles and larger clusters (up to tens of microns) occurs simultaneously and is responsible for an effective lag-time.