Figure 4. Site-specific mutagenesis of noteworthy motifs in the R-coil subdomain.

(A) Diagram indicating the design of the constructs. Translucent bars indicate continuity of three conserved motifs along the length of the central repetitive domain. Neg. charge: negative residues present in positions 11, 12, and 14 of the repeat. TxT: Thr–Xaa–Thr motif in positions 6–8 of the repeat. Y-ladder: an entirely conserved Tyr in position 3 of the motif. Three mutants were created in which these motifs were extended into the R-coils. K-coils: all arginines in the R-coils were replaced with lysines. (B) Freezing curves with T₅₀ and number of unfrozen droplets indicated where applicable.