Rosetta FunFolDes – A general framework for the computational design of functional proteins
Fig 6
Functionalization of the functionless de novo fold TOP7.
A) Structure of TOP7 with the insertion region highlighted in light red. B) Structural comparison between 101F and TOP7’s insertion region shows a 2.1 Å RMSD. C) TOP7_full model (in blue and red for the motif) superimposed over the TOP7 crystal structure. 101F’s insertion is structurally compensated mostly by the first pairing beta strand and a shift of the first alpha helix. D) CD spectrum shows a broad ellipticity signal between 210 nm and 222 nm as a representative of mixed alpha and beta secondary structures. E) The Tm for TOP7_full was 54.5°C. F) Binding affinity determined by SPR. TOP7_full shows a KD of 24.2 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 TOP7_full and the starting template TOP7. The largest structural differences are observed in the region downstream of the site IV epitope, the overall difference of the two structures is 1.5 Å (dashed horizontal line). The connecting loop between the strand that holds the epitope and the adjacent strand was also shortened to obtain a tighter connection between the 2 strands (dashed vertical region). Sequence divergence is evaluated by applying the BLOSUM62 score matrix to the sequences, yielding a total of 27.7% identity and 52.2% similarity. The epitope region is colored in light red. Identical positions between the TOP7_full and TOP7 are displayed as their residue types while positively scored changes according to BLOSUM62 are labeled with a plus (+).