The effect of mutations on the structure of insulin fibrils studied by Fourier transform infrared (FTIR) spectroscopy and electron microscopy.
Fibril formation (aggregation) of human and bovine insulin and six human insulin mutants in hydrochloric acid were investigated by visual inspection, Thioflavin T fluorescence spectroscopy, transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy. The fibrillation tendencies of the wild-type insulins and the insulin mutants were (in order of decreasing fibrillation tendencies): Glu(B1) + Glu(B27) = bovine < human < des-(B1,B2)-insulin < Ser(B2) + Asp(B10) < Glu(A13) + Glu(B10) = Gln(B17) < Asp(B10). Transmission electron micrographs showed that the protofibrils of the mutants were similar to those of wild-type insulins and had a diameter of 5-10 nm and lengths varying from 50 nm to several microns. The fibrils of human insulin mutants exhibited varying degrees of lateral aggregation. The Asp(B10) mutant and human insulin had greater tendency to form laterally aggregated fibrils arranged in parallel bundles, whereas fibrils of the other mutants and bovine insulin were mainly arranged in helical filaments. FTIR spectroscopy showed that the native secondary structure of the wild-type insulins and the human insulin mutants in hydrochloric acid were identical, whereas the secondary structure of the fibrils formed by heating at 50 degrees C depended on the amino acid substitution. FTIR spectra of fibrils of the human insulin mutants exhibited different beta-sheet bands at 1,620-1,640 cm(-1), indicating that the beta-sheet interactions in the fibrils depended on variations in the primary structure of insulin.
Garriques
Liza Nielsen
LN
Department of Pharmaceutics, The Royal Danish School of Pharmacy, 2100 Copenhagen, Denmark. lngarriques@ucdavis.edu
Frokjaer
Sven
S
Carpenter
John F
JF
Brange
Jens
J
D000818:Animals; D002417:Cattle; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D008854:Microscopy, Electron / Q000379:methods; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D007328:Insulin