Figure 3: Engineering of the γPFD interface for controlled connector assembly.

(a) De novo protein models of the TERM-(E-coil) and TERM-(K-coil) that are designed to associate together through an engineered heterodimer coiled-coil. (b) Helical wheel representation of the E3/K3 heterodimer, in which the coiled-coil is viewed as a cross-section (three letter amino acid code). The interhelical hydrophobic interactions (a–a′, d–d′) and electrostatic interactions (g–e′, e–g′) are denoted with arrows. (c) Native gel electrophoresis showing TERM-(E-coil) (lane 1) and TERM-(K-coil) (lane 2) individually or mixed together (lane 3). Filaments of γPFD are too large to run in the gel (lane 4); however, when mixed with TERM-(E-coil) (lane 5) and TERM-(K-coil) (lane 6), the filaments are capped and elongation is inhibited, which produces filaments that vary in the number of subunits. L, protein standard. (d) The length of γPFD filaments capped by various ratios of either TERM-(E-coil) or TERM-(K-coil). Results represent the mean±s.e. (n=500 filaments). (e) A TERM-(E-coil) capped filament attached at the termini of two TERM-(K-coil)-foldon three-way connectors through heterodimeric coiled-coils. (f) TEM image of the TERM-(K-coil)-foldon three-way connector assembled with γPFD filaments that are capped with TERM-(E-coil). (g–h) Increased concentration of the TERM-(E-coil)-foldon results in greater cross-linking between capped filaments in a controlled manner. Scale bars, 100 nm.