Computational analysis of Thr203 isomerization in green fluorescent protein.

Green fluorescent protein (GFP) is an extensively used fluorescent tag. Photoisomerization between two spectroscopically distinct states in wild-type GFP is responsible for its two visible absorption bands at 398 nm (A) and 475 nm (B). We have used molecular mechanics and database analysis to support the suggestion of other researchers that the anionic form of the GFP chromophore is responsible for the B absorption band, while the phenol form is responsible for band A. The anionic (-Otyr, Nimid, Glu222H) species is the only form that has a low energy pathway allowing for isomerization of Thr203 to a conformation where it stabilizes the phenolate form and is therefore the most likely species responsible for the B absorption band. The rotation of the Thr203 side-chain is restricted; this may be significant in the formation of the intermediate state which is central to the photoisomerization. Our calculations support the most commonly accepted mechanism for photoisomerization, and we have shown that the 201LSTQS205 sequence does not allow a g+ conformation for Thr203.