Structure and mechanism of the reversible photoswitch of a fluorescent protein

Andresen et al. 10.1073/pnas.0502772102.

Supporting Information

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Fig. 6. Structures of the MYG chromophores. (a) Orthogonal views of the MYG chromophores from the four asFP595 crystal structures [asFP595 (not irradiated), asFP595-A143S (not irradiated), asFP595-A143S^h*n, asFP595-S158V (not irradiated)] after least-squares superpositioning of all Ca atoms of the protein molecules. Atoms are color coded by atom type (carbon wt, cyan; carbon A143S, salmon; carbon S158V, beige; oxygen, red; nitrogen, blue). (b) Difference Fourier maps generated with phases calculated from the final model of the asFP595 (not irradiated) structure and with structure factor differences F(A143S) – F(wt) (Left, contoured at 2.5s), F(A143S^h*n) –F(wt) (Middle, contoured at 3s) and F(A143S^h*n) – F(A143S) (Right, contoured at 3s). The depicted chromophores of the cis and trans conformantion are intended to aid orientation. The maps show positive differences for features, which are present at a higher level in A143S compared with asFP595 (not irradiated) (Left), in A143S^h*n compared with asFP595 (not irradiated), and in A143S^h*n compared with A143S (Right). These data show the increasing fractions of the chromophores, which are in the cis conformation from asFP595 (not irradiated) over A143S to A143S^h*n. Color coding is as in a.

Fig. 7. Influence of A143S and S158V on the chromophor of asFP595. Both mutations, A143S and S158V, support the cis conformation of the chromophor. The mutation A143S stabilizes the cis conformation of the chromophor, whereas the mutation S158V blocks the transconformation. Displayed are the chromophores of asFP595 ("off" state, cyan carbon), asFP595-S158V ("on" state, salmon carbon) and asFP595-A143S (on state, magenta carbon). In asFP595-A143S, the serine stabilizes the cis chromophore through an additional H bond to the terminal oxygen of the hydroxyphenyl ring (left arrow). The valine introduced by the mutation S158V blocks the trans conformation of the chromophore in asFP595-S158V. This finding is shown by the overlapping electron densities of V158 and the wt trans chromophore (right arrow).

Fig. 8. Rearrangement and fluctuations of residues near the backbone break after isomerization. Smoothed rms deviation (rmsd) of MYG (all atoms) and neighboring residues 61–62 and 66–67 (backbone atoms) during equilibration after spontaneous transcis isomerization. The residues C62 and S66 directly adjacent to MYG exhibit a larger rms deviation (0.12 and 0.09 nm, respectively, averaged over the last 650 ps) than second neighbors S61 and K67 (0.10 and 0.05 nm, respectively). Residues S61 and C62 N-terminal of the chromophore show significantly larger rms deviation values than their respective C-terminal counterparts K67 and S66.

Fig. 9. Interactions with residues affect the isomerization process. For all four simulated pathways, the same residues close to the chromophore are crucial for chromophore positioning and/or isomerization. The sum of Coulomb and vdW MYG-protein interactions are shown, averaged for 10 trajectories. (a) During HT^bot, all favorable MYG–protein interactions stay intact, and no significant energy barrier is found. (b) In contrast, along the HT^top pathway, T60 exhibits a significant energy barrier. (c and d) Both R pathways lead to unfavorable hydrophobic interactions. In particular, R^top (c) is mainly inhibited by H197. However, this residue does not affect R^bot (d), which is largely hampered by T60 instead. Irrespective of the isomerization mechanism, the final cis state is stabilized with respect to trans by H197, A143, and M160. The favorable interaction to A143 found in the simulation corresponds to the hydrogen bond to S143 in the on state of the A143S mutant. The described features are not only prominent in the shown averages but also in all individual trajectories (error of 1-3 kJ/mol).

Fig. 10. Dihedral potentials used for the free MD simulations. Solid line, t; dashed line, j. The potential energy barriers of both dihedrals were varied between 0 (dashed) and 7 kcal/mol (dotted line) in test simulations.

Movie 1. Representative trajectory of spontaneous trans-cis isomerization through HT^bot as observed in MD simulations starting from the excited asFP595 off state. To alleviate the observation of the trans-cis isomerization only selected residues are shown, whereas the underlying simulations accounted for the whole protein. Colors: cyan, MYG; asFP595 chain A, yellow; chain B, orange.

Table 1. Crystallographic data and refinement

a.u., asymmetric unit; rmsd, root-mean-square deviation. Data in parentheses are for the last 0.02Å (wt) or for the last 0.05Å (S158V and A143S).

*R_sym(I) = (S_hklS_i[½I_i(hkl) – <I(hkl)> ½] / S_hklS_i[I_i(hkl)]; I_i(hkl) – intensity of the i^th measurement of hkl; <I(hkl)> – average value of hkl for all i measurements

^†R_work = å_hkl[½½F_obs½ – k½F_calc½½] / å_hkl[½F_obs½]; R_free = å_hklÌT[½½F_obs½ – k½F_calc½½] / å _hklÌT[½F_obs½]; hklÌT – test set

^‡ESU, estimated overall coordinate error based on maximum likelihood.

Table 3. Ca root mean square deviations to asFP595 mol1

asFP595 tetramers contain two identical dimers. Each dimer, which is slightly asymmetric, contains two asFP595 molecules (molecule 1 and 2, respectively). Ca root mean square deviations are calculated for a comparison with asFP595 molecule 1. Numbers are rounded on the first position after the decimal point.

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