Design of single cross-strand ladders in the PSAM and their ThT binding properties. (a) Schemes of single Tyr-ladders of different lengths used in this work. Note that the locations of the Tyr ladder mutations were dictated by our limited ability to specifically introduce mutations at certain positions due to the redundancy of the PSAM gene. (b) Fluorescence emission spectra of 10 μM ThT in the absence and presence of 100 μM 8-YY/KE or 100 μM 8-EK/KE. (c) ThT fluorescence emission as a function of the number of contiguous cross-strand Tyr residues. Fluorescence intensity at 485 nm is shown after subtracting a blank (no protein) spectra of 10 μM ThT, and are normalized relative to the 8-YY/KE signal.

Binding of other dyes to side-by-side ladders. (a) Structures of dyes used. (b) Bis-ANS titration in the presence (solid circles) and absence (open circles) of 10 μM 5-YY/LL. Fluorescence emission intensity at 505 nm is plotted as a function of bis-ANS concentration. Spectra of 1, 5, 10, 25 and 50 μM bis-ANS in the presence of 5-YY/LL are shown on the right. (c) Competition of ThT binding by bis-ANS, conducted in the presence of 1 μM 5-YY/LL and 5 μM ThT. Fluorescence emission at 485 nm is plotted as a function of bis-ANS concentration. Spectra from this competition experiment are shown on the right. The same concentrations of bis-ANS were used as in (b). Note the gradual decrease in the ThT peak (maximum near 485 nm) and corresponding increase in the bis-ANS peak (maximum near 505 nm).

Design of side-by-side ladders and their ThT binding. (a) Schemes of the side-by-side ladders used in this work. (b) ThT binding titrations measuring ThT fluorescence emission at 485 nm. Protein concentrations were 10 μM. (c) Determination of the stoichiometry of ThT binding to the PSAM by binding site titration. ThT fluorescence emission intensity at 485 nm is plotted as a function of ThT concentration. The binding titration was performed with 25 μM 5-YY/LL PSAM, i.e. a protein concentration much higher than the K_d of the binding reaction. Extrapolation of the linear portion of the curve near the origin is shown as the black dashed line. The gray horizontal dashed line is a saturation value of ThT fluorescence estimated at ~110 A.U. The intercept of the two lines indicates that the saturating ThT concentration is ~25 μM, i.e. a 1:1 binding stoichiometry between ThT and the PSAM. (d) CD spectra of 0.5 mM ThT in the presence of 0.5 mM of the PSAM variants. (e) Crystallization conditions impede observation of bound ThT. ThT binding titrations to 10 μM 5-YY/LL in the presence (pink) and absence (blue) of 10% v/v PEG-400. Note that a 40% v/v PEG-400 solution was used in crystallization. (f) Schemes of point mutants of 5-YY/LL, termed –Y, –L, and YIYIY. On the right are shown ThT binding titrations of these mutants, measured as described in (b).

PSAM model for ThT binding. (a) Chemical structure of ThT. (b) Adjacent cross-strand side-chain ladders of a self-assembled antiparallel β-sheet. The sheet backbone is depicted as lines between α-carbons. Side-chains indicated circles inscribed with the letter R. (c) Scheme illustrating the PSAM concept. A segment of peptide self-assembly is hypothetically excised, linked, and capped. The crystal structure of the 8-EK/KE PSAM is shown as a cartoon model on the right, with the N- and C-terminal caps colored black and the SLB colored grey. (d) Amino acid sequence of the wild-type PSAM SLB shown according to the β-sheet topology. The rectangles indicate β-strand residues. The side chains of the residues enclosed in the dashed lines point toward the reader, and those of the other β-strand residues point away from the reader. The designations of the four β-hairpin repeats (“0” – “3”) are shown. Hydrogen-bonding partners are depicted with arrows pointing from donor to acceptor. Note that 3-, 4-, 5-, and 8-YY/LL all contain a V201L mutation in a turn region of β-hairpin 0 (denoted with an asterisk), which occurred as an unintended consequence of gene construction. Leu is present in the homologous positions (L132, L155, and L178) of β-hairpins 1, 2 and 3, and therefore likely has a negligible effect on the structure, stability, and dye-binding properties of the PSAM.

Crystal structures of ThT-binding PSAMs. (a) Packing of 5-YY/LL monomers in the crystal creates large solvent-channels. The location of the designed ThT-binding site is shown as the purple curves along the central β-sheet of two PSAMs. The N- and C-terminal caps are denoted. (b) Structures of 5-YY/LL and 8-YY/KE. Only the central β-sheet of each PSAM is shown, drawn to scale with ThT. The side chains of Ladder1 and Ladder2 are shown as sticks. The ladder mutations and their solvent-exposed surfaces are colored blue for Tyr and green for Leu, while wild-type side chains are in dark grey. The two PEG-400 molecules found near the putative ThT-binding site on 5-YY/LL are shown as red sticks. (c) The side chain conformations of the 5-YY/LL ladders. The ladders are shown looking down the long axis of the PSAM from C- to N-terminus (top) and looking straight down on the SLB (bottom). The backbone is depicted using ribbons, with turns omitted for clarity. At right, a model of ThT bound to the Tyr surface in an orientation consistent with our results is shown using the same depiction. The ThT ring conformation is derived from that observed in acetylcholinesterase. (d) The ThT-binding pocket of acetylcholinesterase shown in orthogonal views (PDB 2J3Q).24 All residues within 4 Å of ThT are shown as sticks, except for Tyr 121, which is omitted for clarity. The Tyr and Trp residues forming the orthogonal binding motif similar to that observed in 5-YY/LL are labeled (top). (e) The backbone conformations of PSAMs. The eight-stranded β-sheet used as the host in this work is shown looking from the C- to N-terminus, with turns omitted for clarity. The two ladders would be located above the β-sheet as shown. (f) Values of the three parameters Twist, Bend, Bend’ describing the 3-dimensional rotations of each β-hairpin in the SLB.25 The types of rotations are schematically shown above the graphs. Zero values define a perfectly flat, rectangular β-sheet.