Nanoscopic and Photonic Ultrastructural Characterization of Two Distinct Insulin Amyloid States

International Journal of Molecular Sciences

Katarzyna Maria Psonka-Antonczyk³  Julien Duboisset³  Bjørn Torger Stokke³  Tamotsu Zako²  Takahiro Kobayashi²  Mizuo Maeda²  Sofie Nyström¹  Jeff Mason¹  Per Hammarström¹  K. Peter R. Nilsson¹ 

[1] Department of Physics, Chemistry and Biology, Linköping University, Linköping 581 83, Sweden; E-Mails:;Bioengineering laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351 0198, Japan; E-Mails:;Department of Physics, Norwegian University of Science and Technology,7491 Trondheim, Norway; E-Mails:

关键词: amyloid proteins;    oligomeric amyloid state;    pre-fibrillar intermediate state;    oligothiophene fluorescence stains;    fluorescence assay;    TIRFM-AFM;   

DOI  :  10.3390/ijms13021461

来源: mdpi

【 摘 要 】

Two different conformational isoforms or amyloid strains of insulin with different cytotoxic capacity have been described previously. Herein these filamentous and fibrillar amyloid states of insulin were investigated using biophysical and spectroscopic techniques in combination with luminescent conjugated oligothiophenes (LCO). This new class of fluorescent probes has a well defined molecular structure with a distinct number of thiophene units that can adopt different dihedral angles depending on its binding site to an amyloid structure. Based on data from surface charge, hydrophobicity, fluorescence spectroscopy and imaging, along with atomic force microscopy (AFM), we deduce the ultrastructure and fluorescent properties of LCO stained insulin fibrils and filaments. Combined total internal reflection fluorescence microscopy (TIRFM) and AFM revealed rigid linear fibrous assemblies of fibrils whereas filaments showed a short curvilinear morphology which assemble into cloudy deposits. All studied LCOs bound to the filaments afforded more blue-shifted excitation and emission spectra in contrast to those corresponding to the fibril indicating a different LCO binding site, which was also supported by less efficient hydrophobic probe binding. Taken together, the multi-tool approach used here indicates the power of ultrastructure identification applying AFM together with LCO fluorescence interrogation, including TIRFM, to resolve structural differences between amyloid states.

【 授权许可】

CC BY   
© 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.