Nucleation and growth of insulin fibrils in bulk solution and at hydrophobic polystyrene surfaces.
A technique was developed for studying the nucleation and growth of fibrillar protein aggregates. Fourier transform infrared and attenuated total reflection spectroscopy were used to measure changes in the intermolecular beta-sheet content of bovine pancreatic insulin in bulk solution and on model polystyrene (PS) surfaces at pH 1. The kinetics of beta-sheet formation were shown to evolve in two stages. Combined Fourier transform infrared, dynamic light scattering, atomic force microscopy, and thioflavin-T fluorescence measurements confirmed that the first stage in the kinetics was related to the formation of nonfibrillar aggregates that have a radius of 13 +/- 1 nm. The second stage was found to be associated with the growth of insulin fibrils. The beta-sheet kinetics in this second stage were used to determine the nucleation and growth rates of fibrils over a range of temperatures between 60 degrees C and 80 degrees C. The nucleation and growth rates were shown to display Arrhenius kinetics, and the associated energy barriers were extracted for fibrils formed in bulk solution and at PS surfaces. These experiments showed that fibrils are nucleated more quickly in the presence of hydrophobic PS surfaces but that the corresponding fibril growth rates decrease. These observations are interpreted in terms of the differences in the attempt frequencies and energy barriers associated with the nucleation and growth of fibrils. They are also discussed in the context of differences in protein concentration, mobility, and conformational and colloidal stability that exist between insulin molecules in bulk solution and those that are localized at hydrophobic PS interfaces.
Smith
M I
MI
School of Physics and Astronomy and Nottingham Nanotechnology and Nanoscience Centre, The University of Nottingham, Nottingham, United Kingdom.
Sharp
J S
JS
Roberts
C J
CJ
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D057927:Hydrophobic and Hydrophilic Interactions; D007328:Insulin / Q000737:chemistry*; D008027:Light; D018625:Microscopy, Atomic Force; D011137:Polystyrenes; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D012996:Solutions; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013499:Surface Properties; D013816:Thermodynamics
D007328:Insulin; D011137:Polystyrenes; D012996:Solutions