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1.
Figure 10

Figure 10. From: An Alternate Proton Acceptor for Excited State Proton Transfer in Green Fluorescent Protein: Rewiring GFP.

Comparison of the proposed H+ transfer routes in wtGFP and S65T/H148D upon excitation.

Deborah Stoner-Ma, et al. J Am Chem Soc. ;130(4):1227-1235.
2.
Figure 2

Figure 2. From: An Alternate Proton Acceptor for Excited State Proton Transfer in Green Fluorescent Protein: Rewiring GFP.

Absorbance and excitation spectra in D2O of S65T and S65T/H148D at pD 7.1 (normalized at 280 nm).

Deborah Stoner-Ma, et al. J Am Chem Soc. ;130(4):1227-1235.
3.
Figure 8

Figure 8. From: An Alternate Proton Acceptor for Excited State Proton Transfer in Green Fluorescent Protein: Rewiring GFP.

Polarization-resolved TRIR data for S65T/H148D pD 7.1 at 100 ps after excitation.

Deborah Stoner-Ma, et al. J Am Chem Soc. ;130(4):1227-1235.
4.
Figure 6

Figure 6. From: An Alternate Proton Acceptor for Excited State Proton Transfer in Green Fluorescent Protein: Rewiring GFP.

Fluorescence decay at 515 and 490 nm of S65T/H148D and E222Q/H148D in both D2O (pD 6.1) and H2O (pH 6.5).

Deborah Stoner-Ma, et al. J Am Chem Soc. ;130(4):1227-1235.
5.
Figure 5

Figure 5. From: An Alternate Proton Acceptor for Excited State Proton Transfer in Green Fluorescent Protein: Rewiring GFP.

Time-resolved fluorescence emission (normalized at 508 nm) of wt GFP pD 7.1, S65T pD 5.1, S65T/H148D pD 6.1 and E222Q/H148D pD 6.1 in D2O.

Deborah Stoner-Ma, et al. J Am Chem Soc. ;130(4):1227-1235.
6.
Figure 1

Figure 1. From: An Alternate Proton Acceptor for Excited State Proton Transfer in Green Fluorescent Protein: Rewiring GFP.

Model for the interconversion of the A and B forms of the chromophore via the I form. Adapted from Brejc et al.18

Deborah Stoner-Ma, et al. J Am Chem Soc. ;130(4):1227-1235.
7.
Figure 3

Figure 3. From: An Alternate Proton Acceptor for Excited State Proton Transfer in Green Fluorescent Protein: Rewiring GFP.

Normalized steady state fluorescence emission of S65T/H148D (pH 7.5) with 410 nm and 484 nm excitation.

Deborah Stoner-Ma, et al. J Am Chem Soc. ;130(4):1227-1235.
8.
Figure 9

Figure 9. From: An Alternate Proton Acceptor for Excited State Proton Transfer in Green Fluorescent Protein: Rewiring GFP.

Photoconversion of S65T/H148D using a combination of wavelengths (333, 351, and 364 nm) followed for 2 hours. WtGFP photoconversion with unfiltered Hg lamp is shown in inset.

Deborah Stoner-Ma, et al. J Am Chem Soc. ;130(4):1227-1235.
9.
Figure 7

Figure 7. From: An Alternate Proton Acceptor for Excited State Proton Transfer in Green Fluorescent Protein: Rewiring GFP.

Time-resolved difference IR spectra of a) wt GFP, b) S65T pD 5.1, c) S65T/H148D pD 7.1, and d) E222Q/H148D pD 7.1 following excitation at 400 nm.

Deborah Stoner-Ma, et al. J Am Chem Soc. ;130(4):1227-1235.
10.
Figure 4

Figure 4. From: An Alternate Proton Acceptor for Excited State Proton Transfer in Green Fluorescent Protein: Rewiring GFP.

TRF of S65T/H148D, E222Q/H148D and S65T at pD 5.1 in D2O with times delays noted. The short rise times seen in the first 6 ps for S65T/H148D and S65T simply reflect the convolution of the 4 ps instrument response with the emission. The apparent vibronic structure in the S65T emission may reflect interference effects in the collection optics.

Deborah Stoner-Ma, et al. J Am Chem Soc. ;130(4):1227-1235.
11.
Figure 11

Figure 11. From: An Alternate Proton Acceptor for Excited State Proton Transfer in Green Fluorescent Protein: Rewiring GFP.

A representation of the proposed potential energy surfaces for wtGFP (A) and S65T/H148D GFP (B). In wtGFP the phenolic OH bond is shorter and the acceptor remote. In S65T/H148D the donor and acceptor are close yielding the low barrier and flat potential surface. On excitation, wtGFP forms A* which converts to I* via a barrier on the picosecond time scale. In S65T/H148D the transfer is barrierless and occurs in < 1 ps. The I* state so formed is vibrationally hot and cools to form the emissive state. The hot state may also access a nonradiative part of the potential energy surface.

Deborah Stoner-Ma, et al. J Am Chem Soc. ;130(4):1227-1235.

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