Early aggregation preceding the nucleation of insulin amyloid fibrils as monitored by small angle X-ray scattering.
The nucleation event of amyloid fibrils is one of the most crucial processes that dictate the timing and rate of the pathology of diseases; however, information regarding how protein molecules associate to produce fibril nuclei is currently limited. In order to explore this issue in more detail, we performed time-resolved small angle X-ray scattering (SAXS) measurements on insulin fibrillation, in combination with additional multidirectional analyses of thioflavin T fluorescence, FTIR spectroscopy, light scattering, and light transmittance, during the fibrillation process of bovine insulin. SAXS monitoring revealed that insulin molecules associated into rod-like prefibrillar aggregates in the very early stage of the reaction. After the formation of these early aggregates, they appeared to further coalesce mutually to form larger clusters, and the SAXS profiles subsequently showed the further time evolution of conformational development towards mature amyloid fibrils. Distinct types of structural units in terms of shape in a nano-scale order, cross-β content, and thioflavin T fluorescence intensity were observed in a manner that was dependent on the fibrillation pathways. These results suggest the presence of diverse substructures that characterize various fibrillation pathways, and eventually, manifest polymorphisms in mature amyloid fibrils.
Department of Chemistry, Graduate School of Science, Kobe University, Hyogo 657-8501, Japan.
Eri
E
Chatani
Research Reactor Institute, Kyoto University, Osaka 590-0494, Japan.
Rintaro
R
Inoue
College of Pharmaceutical Science, Ritsumeikan University, Shiga 525-8577, Japan.
Hiroshi
H
Imamura
Research Reactor Institute, Kyoto University, Osaka 590-0494, Japan.
Masaaki
M
Sugiyama
College of Pharmaceutical Science, Ritsumeikan University, Shiga 525-8577, Japan.
Minoru
M
Kato
Kyoto University, Kyoto 606-8501, Japan.
Masahide
M
Yamamoto
Institute for Chemical Research, Kyoto University, Kyoto 611-0011, Japan.
Koji
K
Nishida
Institute for Chemical Research, Kyoto University, Kyoto 611-0011, Japan.
Toshiji
T
Kanaya
D007328:Insulin / Q000378:metabolism*; D012542:Scattering, Radiation; D014961:X-Ray Diffraction
D007328:Insulin