Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds

doi:10.1073/pnas.0409035102

Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds

^*Department of Chemistry and School of Molecular Science (BK21), Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, South Korea; ^†Department of Biochemistry and Molecular Biology, ^∥Center for Advanced Radiation Sources, and ^¶¶Institute for Biophysical Dynamics, University of Chicago, 920 East 58th Street, Chicago, IL 60637; ^**Physik-Department E17, Technische Universitaet München, 85747 Garching, Germany; ^††National Institutes of Health, Bethesda, MD 20982; and ^§§European Synchrotron Radiation Facility, Grenoble Cedex 9, France

Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved March 18, 2005 (received for review December 6, 2004)

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Fig. 1.
Overview of PYP (46). (A) The room-temperature PYP photocycle (6, 9). (B) Structure of PYP. The pCA chromophore is shown in yellow; secondary structure is labeled using the notation of Rubinstenn et al. (47). (C) Structure of the chromophore binding pocket of PYP. Hydrogen bonds are shown as green dotted lines and chromophore atoms are labeled according to Borgstahl et al. (5).
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Fig. 2.
Difference electron density maps for distinct chemical states (α, β, γ, and δ) from an initial kinetic analysis of 47 time points from 1 ns to 1 s. Chromophore-binding pocket (A-E) and whole protein views (F-J) of the difference maps associated with the “α” (A and F) and “β” (B and G) states derived from the ESRF data and from the “β” (C and H), “γ” (D and I), and “δ” (E and J) states from the APS data. Difference maps are contoured at -4σ (red), -3σ (pink), +3σ (cyan), and +4σ (blue). These chemical states are in the order of occurrence in time through the photocycle of PYP. The population of the α, β, γ, and δ states are peaked around at nanoseconds, microseconds, milliseconds, and subseconds time range, respectively (see text and Figs. 3 and 4B).
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Fig. 3.
Chromophore-binding pocket views of refined intermediate structures and mechanism for the isomerization and rotation of the pCA chromophore upon absorption of blue light. Five distinct structural intermediates (I_CP, pR_CW, pR_E46Q, pB₁, and pB₂) were identified from four chemical states (α, β, γ, and δ) shown in Fig. 2. I_CP is shown twice to demonstrate the biphasic pathways to pR_CW and pR_E46Q. Isomerization and rotation about single bonds are shown by arrows; hydrogen bonds are dotted. A bicycle pedal mechanism (44), which couples trans-cis isomerization of the C2─C3 double bond with rotation about a nonadjacent single bond, is used for the dark state to I_CP transition. Further rotations about single bonds result in the pB₁ conformation. pB₂ reverts thermally to the dark state with no further detectable intermediates.
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Fig. 4.
Properties of the chemical kinetic mechanism of the wild-type PYP photocycle. (A) General chemical kinetic mechanism used to fit the data. The dashed arrow indicates the light-driven reaction from pG to the first intermediate observed here, I_CP. (B) Predicted concentrations of intermediates [I_CP (red), pR_CW (magenta), pR_E46Q (purple), pB₁ (blue), pB₂ (cyan), and the dark state (black)] after posterior analysis with rate coefficients (s^-1): k ₁ = 4.8 × 10⁷; k ₂ = 3.7 × 10⁷; k ₃ = 3.0 × 10³; k ₄ = 3.3 × 10⁴; k ₅ = 55; k ₆ = 100; and k ₇ = 7.1. The seven rate coefficients correspond to time constants of 21 ns, 27 ns, 333 μs, 30 μs, 18 ms, 10 ms, and 141 ms, respectively.