Extended Data Fig. 1: Computational analysis and validation of Tyr as an optimal anchor residue.
From: Time-resolved protein activation by proximal decaging in living systems
a, Surveying the mutational stability of each of the 20 amino acids. The mutation stability of each residue was obtained by the in silico site-saturated mutagenesis of these 3,500 residues and energy calculation of each resulting mutant. Distribution of folding stability for each candidate amino acid is shown in the heat map. The blue and red colours represent the stable and deleterious mutations, respectively. b, Schematic of the validation workflow. According to our computational analysis and ranking, Tyr is a suitable anchor residue, whereas the introduced Lys mutations were more likely to affect the enzyme activity. To validate the computational results, a total of 30 randomly selected sites on each of the two model enzymes—FLuc and Nluc—was mutated to either Lys or Tyr, and the activities of the resulting mutants were systematically compared by measuring the resulting luminescence intensity. c, d, Results from tested mutations on FLuc and NLuc model proteins. Tyrosine mutations showed more stability in general than the lysine mutants. The randomly selected 30 mutation sites of FLuc: N84, F89, L194, N197, T214, R218, N229, H244, H245, F247, F250, Y255, S284, L286, E311, A313, Q338, Y340, I351, E354, V362, L411, L418, I434, R437, L441, Q448, T527, G528 and L530. The randomly selected 30 mutation sites of NLuc: F8, Q14, L20, V23, L24, S31, F33, Q34, P42, Q44, I46, I56, I58, V60, I62, I78, F79, V92, L94, Y96, I109, Y111, F112, Y116, V129, L133, R143, L151, F153 and I157. Results are the representative data of two biological replicates.
