Rosetta FunFolDes – A general framework for the computational design of functional proteins
Fig 5
Functional design of a distant structural template.
A) Structural representation of 1kx8. The insertion region is colored in light red and the two disulfide bonds are labeled (CYD). B) Structural comparison between the insertion region of 1kx8 and the site II epitope (light red-filled silhouette). The local RMSD between the two segments is 2.37 Å. C) Superposition between 1kx8_d2 design model (blue with red motif) and the 1kx8 template (wheat and light red insertion site). Multiple conformational shifts are required throughout the structure to accommodate the site II epitope. D) CD spectrum of 1kx8_d2 showing a typical alpha-helical pattern with the ellipticity minima at 208 nm and 220 nm. E) 1kx8_d2 shows a melting temperature (Tm) of 43.4°C. F) Binding affinity determined by SPR. 1kx8_d2 shows a KD of 1.14 nM. Experimental sensorgrams are shown in black and the fitted curves in red. G) Per-position evaluation of structural (top) and sequence (bottom) divergence between the design model 1kx8_d2 and the starting template 1kx8. The largest structural differences are observed in the epitope insertion region, the overall difference of the two structures is 2.25 Å (dashed line). The sequence was evaluated using the BLOSUM62 score matrix, yielding a total of 13.5% identity and 38.5% similarity. The epitope region is colored in light red. Identical positions between the 1kx8_d2 and 1kx8 are labeled with the residue one letter code, while positively scored changes are labeled with plus (+).