A systematic screen of beta(2)-microglobulin and insulin for amyloid-like segments

Abstract

Identifying sequence determinants of fibril-forming proteins is crucial for understanding the processes causing >20 proteins to form pathological amyloid depositions. Our approach to identifying which sequences form amyloid-like fibrils is to screen the amyloid-forming proteins human insulin and beta(2)-microglobulin for segments that form fibrils. Our screen is of 60 sequentially overlapping peptides, 59 being six residues in length and 1 being five residues, covering every noncysteine-containing segment in these two proteins. Each peptide was characterized as amyloid-like or nonfibril-forming. Amyloid-like peptides formed fibrils visible in electron micrographs or needle-like microcrystals showing a cross-beta diffraction pattern. Eight of the 60 peptides (three from insulin and five from beta(2)-microglobulin) were identified as amyloid-like. The results of the screen were used to assess the computational method, and good agreement between prediction and experiments was found. This agreement suggests that the pair-of-sheets, zipper spine model on which the computational method is based is at least approximately correct for the structure of the fibrils and suggests the nature of the sequence signal for formation of amyloid-like fibrils.

Fig. 1.

Electron micrographs showing fibrils (A–C and E) and needle-shaped microcrystals (D and F) formed by insulin and β₂m and hexameric peptides from their sequences. (A) β₂m91:KIVKWD. (B) β₂m83:NHVTLS. (C) Full-length β₂m. (D) β₂m62:FYLLYY. (E) Full-length insulin. (F) IB12:VEALYL. The number following the protein name is the position in the protein sequence of the first residue. The sequence of the hexameric peptide follows. The length of the calibration bars is 100 nm.

Fig. 2.

Ribbon diagrams of β₂m (Protein Data Bank ID code 1LDS) (A) and insulin (Protein Data Bank ID code 1GUJ) (B) structures showing the positions of the predicted segments by the 3D profile method and the segments that were experimentally determined to form fibrils or needle-like crystals. Note that there is good, but not perfect, correlation between the predicted and experimentally determined segments, as discussed in the text.