Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein

Abstract

Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecules. We used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of excellent quality with strong features; these allowed the determination of structures of reaction intermediates to a resolution of 1.6 angstroms. Our results open the way to the study of reversible and nonreversible biological reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal.

Figure 1

Simplified PYP photocycle from the perspective of a time-resolved crystallographer (3, 4, 26). The dark state pG is activated by absorption of a blue photon (450 nm) to pG^* that begins the photocycle. The crystal structures of longer-lived intermediate states I_T, I_CT, pR₁ (pR_E46Q), pR₂ (pR_cw), pB₁ and pB₂ are known.

Figure 2

Stereo view of the light - dark 1.6 Å difference electron density map at 1μs time delay, superimposed on the dark PYP structure (cyan). Contour levels: red/blue −3σ/+3σ. Chromophore and some important chromophore pocket residues are shown in yellow and marked in A. A Red arrow: plume of structural displacements extends to Met18, close to the N-terminal helix, which may be strongly displaced at longer times (26, 34). B View rotated by ~90°. A large part of the molecule does not display significant DED features and remains structurally unaltered.

Figure 3

Comparison of electron density and difference electron density (DED) maps in the chromophore pocket obtained by time-resolved femtosecond serial crystallography (TR-SFX) and the Laue method. The dark state is shown in yellow in all maps. A and D: Electron density maps for the PYP dark state, TR-SFX and Laue, respectively (contour level 1.1 σ, 1.6Å resolution). The PCA chromophore and nearby residues are marked in A. Arrow: Double bond in the chromophore about which isomerization occurs. B TR-SFX DED map at 10 ns. Light green structure: I_CT intermediate. Features marked by dotted arrows belong to additional intermediates, not shown. C TR-SFX DED map at 1 μs. Pink and red structures: structures of pR₁ and pR₂ intermediates, respectively. E Laue 32 ns DED map correlates best to the TR-SFX 10 ns map. F Laue 1 μs DED map. Contour levels of the DED maps: red/white −3σ/−4σ, blue/cyan +3σ/+5σ, except for C where cyan is +7σ. Note, B and C are displayed in stereo in the SM.