Instantaneous fibrillation of egg white proteome with ionic liquid and macromolecular crowding
Pankaj BharmoriaDibyendu MondalMatheus M. PereiraMárcia C. NevesMafalda R. AlmeidaMaria C. GomesJoão F. ManoIgor BdikinRute A. S. FerreiraJoão A. P. CoutinhoMara G. Freire
11
Citation
70
Reference
10
Related Paper
Citation Trend
Abstract:
Abstract The wide application of protein fibrils as functional materials has been restricted by the limited scalability of fibrillation methods, slow kinetics, and use of expensive purified proteins. Herein, inspired by the biological cooperativity of proteins in macro-molecularly crowded environments, these restrictions have been overcome. Using ionic liquid cholinium tosylate that acts as a fibrillation agent, instantaneous production of protein fibrils is shown directly from a real and low-cost matrix, i.e. egg white. The fibrillation of egg white proteome is confirmed by microscopy, whereas the fibrillation kinetics is monitored by fluorescence changes of the thioflavin T dye and secondary structural transitions. Spectroscopic and molecular docking studies are used to identify the proteins involved and to appraise the molecular-level mechanisms ruling the proteins structural changes upon fibrillation. The obtained fibrils have enhanced mechanical stiffness and cytocompatibility, demonstrating their potential to act as improved enzyme supports.Keywords:
Fibrillation
Thioflavin
Proteome
Docking (animal)
Deposits of fibrils formed by disease-specific proteins are the molecular hallmark of such diverse human disorders as Alzheimer's disease, type II diabetes, or rheumatoid arthritis. Amyloid fibril formation by structurally and functionally unrelated proteins exhibits many generic characteristics, most prominently the cross β-sheet structure of their mature fibrils. At the same time, amyloid formation tends to proceed along one of two separate assembly pathways yielding either stiff monomeric filaments or globular oligomers and curvilinear protofibrils. Given the focus on oligomers as major toxic species, the very existence of an oligomer-free assembly pathway is significant. Little is known, though, about the structure of the various intermediates emerging along different pathways and whether the pathways converge towards a common or distinct fibril structures. Using infrared spectroscopy we probed the structural evolution of intermediates and late-stage fibrils formed during in vitro lysozyme amyloid assembly along an oligomeric and oligomer-free pathway. Infrared spectroscopy confirmed that both pathways produced amyloid-specific β-sheet peaks, but at pathway-specific wavenumbers. We further found that the amyloid-specific dye thioflavin T responded to all intermediates along either pathway. The relative amplitudes of thioflavin T fluorescence responses displayed pathway-specific differences and could be utilized for monitoring the structural evolution of intermediates. Pathway-specific structural features obtained from infrared spectroscopy and Thioflavin T responses were identical for fibrils grown at highly acidic or at physiological pH values and showed no discernible effects of protein hydrolysis. Our results suggest that late-stage fibrils formed along either pathway are amyloidogenic in nature, but have distinguishable structural fingerprints. These pathway-specific fingerprints emerge during the earliest aggregation events and persist throughout the entire cascade of aggregation intermediates formed along each pathway.
Thioflavin
Oligomer
Amyloid (mycology)
Cite
Citations (28)
Thioflavin
Amyloid (mycology)
Congo red
Cite
Citations (121)
Thioflavin
Oligomer
Amyloid (mycology)
Amyloid disease
Cite
Citations (7)
Thioflavin
Amyloid (mycology)
Oligomer
Beta sheet
Cite
Citations (107)
Thioflavin
Isothermal Titration Calorimetry
Amyloid (mycology)
Cite
Citations (0)
Thioflavin
Cite
Citations (49)
Thioflavin
Amyloid (mycology)
Cite
Citations (0)
Thioflavin
Amyloid (mycology)
Cite
Citations (29)
Thioflavin
Amyloid (mycology)
Cite
Citations (10)
Abstract Fibrillation of differently modified amyloid β peptides and deposition as senile plaques are hallmarks of Alzheimer’s disease. N-terminally truncated variants, where the glutamate residue 3 is converted into cyclic pyroglutamate (pGlu), form particularly toxic aggregates. We compare the molecular structure and dynamics of fibrils grown from wildtype Aβ(1–40) and pGlu 3 -Aβ(3–40) on the single amino acid level. Thioflavin T fluorescence, electron microscopy, and X-ray diffraction reveal the general morphology of the amyloid fibrils. We found good agreement between the 13 C and 15 N NMR chemical shifts indicative for a similar secondary structure of both fibrils. A well-known interresidual contact between the two β-strands of the Aβ fibrils could be confirmed by the detection of interresidual cross peaks in a 13 C- 13 C NMR correlation spectrum between the side chains of Phe 19 and Leu 34. Small differences in the molecular dynamics of residues in the proximity to the pyroglutamyl-modified N-terminus were observed as measured by DIPSHIFT order parameter experiments.
Thioflavin
Amyloid (mycology)
Residue (chemistry)
Cite
Citations (15)