Sequence alignment of Ultramarine and ShadowR. (a) Amino acid sequences of Ultramarine and ShadowR are shown in blue and black letters, respectively. Extra amino acids (gray characters) were added to the N and C termini of Ultramarine to match the molecular size of ShadowR. Squares (gray, blue, red) indicate the amino acids (10–228 in Ultramarine/ShadowR) whose side chains are directed outward from the protein. To identify amino acids, we utilized the crystal structure (Protein Data Bank ID: 2H5Q) of mCherry which shares 64% homology to Ultramarine. Blue and Red squares indicate that the amino acids in Ultramarine were replaced to more hydrophilic and hydrophobic amino acids, respectively. Gray squares indicate that the hydrophobicity of the amino acids is identical between Ultramarine and ShadowR. The chromophore tripeptide is highlighted with a magenta box. (b) Hydrophobicity index of respective proteins as calculated simply by summing the hydropathy index of amino acids31 is indicated by the squares. (c) A homology model of ShadowR created by SWISS-MODEL42. The X-ray crystal structure of mCherry mutant (PDB ID 3NED)43 was used as a template to represent ShadowR. Relatively hydrophobic acids (I, V, L, F, C, M, A, G) are colored in magenta. The rest of amino acids which are the relatively hydrophilic ones (T, S, W, Y, P, N, Q, D, E, H, K, R) are colored in cyan. Blue and Red indicate the amino acids substituted to more hydrophilic or hydrophobic ones in Ultramarine, respectively. For the electrostatic surface potential of Ultramarine and ShadowR, see Supporting Fig. 2S.