Solvation-assisted pressure tuning of insulin fibrillation: from novel aggregation pathways to biotechnological applications.
Solvation-assisted pressure tuning has been employed to unravel unknown structural and kinetic aspects of the insulin aggregation and fibrillation process. Our approach, using fluorescence, Fourier transform infrared and atomic force microscopy techniques in combination with pressure and solvent perturbation, reveals new insights into the pre-aggregated regime as well as mechanistic details about two concurrent aggregation pathways and the differential stability of insulin aggregates. Pressure uniformly fosters the dissociation of native insulin oligomers, whereas the aggregation pathways at elevated temperatures are affected by pressure differently and in a cosolvent-dependent manner. Moderate pressures accelerate the amyloid pathway in the presence of EtOH (leading to essentially monomeric aggregating species) via relatively dehydrated transition states with negative activation volumes for nucleation and elongation. Alternatively, a novel, fast equilibrium pathway to distinct beta-sheet-rich oligomers with thioflavin T-binding capability is accessible to partially unfolded insulin monomers at pressures below approximately 200 bar in the absence of EtOH. These oligomers, probably off the normal fibrillation pathway, are stabilized mainly by electrostatic and hydrophobic interactions, lacking the precise packing of mature insulin fibrils, which renders them susceptible to quantitative pressure-induced dissociation. Due to a highly negative activation volume for dissociation (-70(+/-16)ml/mol), pressure dissociation is fast and technologically feasible at ambient temperatures and moderate pressures. Becoming kinetically very labile above 35 degrees C, the pressurized oligomers can re-enter the slower, ultimately irreversible, fibrillation pathway at higher temperatures. At pressures above approximately 1000 bar, the partial unfolding of insulin monomers, accompanied by a volumetric expansion, dominates the aggregation kinetics, which manifests in a progressive inhibition of the fibrillation. Unlike their precursors, the pressure-insensitivity of mature insulin fibrils demonstrates that an extensive hydrogen bonding network and optimized side-chain packing are crucial for their stability.
Grudzielanek
Stefan
S
University of Dortmund, Department of Chemistry, Physical Chemistry I, Biophysical Chemistry, Germany.
Smirnovas
Vytautas
V
Winter
Roland
R
D000818:Animals; D002417:Cattle; D000431:Ethanol / Q000737:chemistry; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism; D018625:Microscopy, Atomic Force; D011312:Pressure; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013816:Thermodynamics
D007328:Insulin; D012997:Solvents; D000431:Ethanol