TY - JOUR
T1 - Islet amyloid
T2 - Phase partitioning and secondary nucleation are central to the mechanism of fibrillogenesis
AU - Padrick, Shae B.
AU - Miranker, Andrew D.
PY - 2002/4/9
Y1 - 2002/4/9
N2 - Islet amyloid polypeptide (IAPP) contributes to the pathogenesis of type II diabetes by depositing as cytotoxic amyloid fibers in the endocrine pancreas. Fiber formation occurs with a marked conformational change from an unstructured precursor. Using real-time quantitative kinetic methods, fibrillogenesis was characterized as a function of protein, denaturant, and seed concentration. Several observations are in sharp contrast to the expectations for nucleation-dependent polymerization. First, the half-time of conversion for both de novo and seeded kinetics were found to be independent of protein concentration. Second, while elongation kinetics scale linearly with protein concentration, they are relatively insensitive to changes in the total seed concentration. Third, seeded bypass of de novo fiber formation kinetics shows a lag phase. The seeded lag phase is eliminated by a time delay before the introduction of seed to a de novo reaction. Last, conversion is highly cooperative, with the time required for 10-90% conversion occurring much faster than the lag time. At a minimum, four kinetic steps are required to describe these observations: activation, fiber independent nucleation, fiber-dependent nucleation, and elongation. Furthermore, we invoke a phase transition in which protein initially forms an off-pathway dispersion. This single construct allows us to model both the concentration independence of the de novo reaction time and the first-order concentration dependence of the elongation kinetics. Marked acceleration of this reaction by hexafiuoro-2-propanol reinforces this view by altering the relative solubility of the two phases and/or by stabilizing hydrogen-bonded structures in the transition states of the reaction pathway.
AB - Islet amyloid polypeptide (IAPP) contributes to the pathogenesis of type II diabetes by depositing as cytotoxic amyloid fibers in the endocrine pancreas. Fiber formation occurs with a marked conformational change from an unstructured precursor. Using real-time quantitative kinetic methods, fibrillogenesis was characterized as a function of protein, denaturant, and seed concentration. Several observations are in sharp contrast to the expectations for nucleation-dependent polymerization. First, the half-time of conversion for both de novo and seeded kinetics were found to be independent of protein concentration. Second, while elongation kinetics scale linearly with protein concentration, they are relatively insensitive to changes in the total seed concentration. Third, seeded bypass of de novo fiber formation kinetics shows a lag phase. The seeded lag phase is eliminated by a time delay before the introduction of seed to a de novo reaction. Last, conversion is highly cooperative, with the time required for 10-90% conversion occurring much faster than the lag time. At a minimum, four kinetic steps are required to describe these observations: activation, fiber independent nucleation, fiber-dependent nucleation, and elongation. Furthermore, we invoke a phase transition in which protein initially forms an off-pathway dispersion. This single construct allows us to model both the concentration independence of the de novo reaction time and the first-order concentration dependence of the elongation kinetics. Marked acceleration of this reaction by hexafiuoro-2-propanol reinforces this view by altering the relative solubility of the two phases and/or by stabilizing hydrogen-bonded structures in the transition states of the reaction pathway.
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U2 - 10.1021/bi0160462
DO - 10.1021/bi0160462
M3 - Article
C2 - 11926832
AN - SCOPUS:0037046151
SN - 0006-2960
VL - 41
SP - 4694
EP - 4703
JO - Biochemistry
JF - Biochemistry
IS - 14
ER -