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"Fluorescent Timer": Protein That Changes Color with Time
Alexey Terskikh, Arkady Fradkov, Galina Ermakova, Andrey Zaraisky, Patrick Tan, Andrey V. Kajava, Xiaoning Zhao, Sergey Lukyanov, Mikhail Matz, Stuart Kim, Irving Weissman, and Paul Siebert

Supplementary Material

A structural model of drFP583 from Anthozoa species built on the basis of the 3D structure of GFP from Aequorea Victoria. (A) Stereoview of the model (blue trace) superimposed on the GFP structure (dotted trace). Fragments in green indicate regions of drFP583 with different (as compared with GFP) backbone conformations due to insertions/deletions in the alignment of these proteins. Mutated residues are in red. Tyr67 (numbering corresponds to drFP583) is in yellow. The initial structure was constructed using the HOMOLOGY module of the Insight II program (1). The model had standard tyrosine and glycine residues instead of the fluorophore in positions 67 and 68. The resulting structure was subjected to the 300 steps of minimization based on the steepest descent algorithm, with the backbone atoms of α-helical segments restrained to their starting positions with force constant K = 100. The next 500 steps of the refinement were performed without any restrictions, using conjugate gradients algorithm. The CHARMM force field (2) and the distance-dependent dielectric constant were used for the energy calculations. The program PROCHECK (3) was used to check the quality of the modeled structure. The stereo figure was generated with the Molscript program (4). The atomic coordinates are available on the Web at (B) A sequence alignment of drFP583 from Anthozoa species and GFP from Aequorea Victoria. The stretches of ( strands are underlined. The box indicates the ( helix. Mutated residues are shown in red, and the fluorophore is shown in yellow. The alignment was obtained as follows: First, a multiple sequence alignment of several GFP sequences and drFP583 was constructed using ClustalW (5); second, manual alignment corrections were applied whenever necessary, based on the 3D structure of GFP; and finally, the locations of gaps and insertions were defined more precisely by inspection of the rigid-body superposition of the drFP583 model and the GFP structure. The final alignment is similar to the one presented by Matz et al. (6), with several minor modifications in the gap locations.

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Supplemental Figure 1.

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