Multiscale approach to the determination of the photoactive yellow protein signaling state ensemble

Abstract

The nature of the optical cycle of photoactive yellow protein (PYP) makes its elucidation challenging for both experiment and theory. The long transition times render conventional simulation methods ineffective, and yet the short signaling-state lifetime makes experimental data difficult to obtain and interpret. Here, through an innovative combination of computational methods, a prediction and analysis of the biological signaling state of PYP is presented. Coarse-grained modeling and locally scaled diffusion map are first used to obtain a rough bird's-eye view of the free energy landscape of photo-activated PYP. Then all-atom reconstruction, followed by an enhanced sampling scheme; diffusion map-directed-molecular dynamics are used to focus in on the signaling-state region of configuration space and obtain an ensemble of signaling state structures. To the best of our knowledge, this is the first time an all-atom reconstruction from a coarse grained model has been performed in a relatively unexplored region of molecular configuration space. We compare our signaling state prediction with previous computational and more recent experimental results, and the comparison is favorable, which validates the method presented. This approach provides additional insight to understand the PYP photo cycle, and can be applied to other systems for which more direct methods are impractical.

Figure 1. LSDMap of the coarse-grained DMC model of PYP.

Structures with large positive first diffusion coordinate (DC1) are globular and similar to the pG state, while structures with large negative DC1 are almost fully extended. The intermediate region near DC1∼2.5 correspond to potential structures for further consideration as candidate signaling pB-state configurations. Figures S1, S2, and S3 in Text S1 show representative configurations from each region. The free energy is shown in terms of DC1 and DC3 to allow for a clearer view of the intermediate region.

Figure 2. Diffusion Map-directed-Molecular Dynamics (DM-d-MD) and molecular dynamics (MD) results.

The initial starting configuration for the DM-d-MD is denoted by the gold circle. The DM-d-MD frontier points are shown in pink, and the minimum-energy frontier point is denoted by a gold star. The MD results from approximately 100 trajectories initiated from the minimum-energy DM-d-MD frontier point are shown in light blue. For reference, the coordinates for the experimental Δ25 (PDB ID 1XFQ) structures are shown as dark blue *, and the experimental WT-PYP (PDB ID: 2KX6) structures as red *. The underlying grey contours are those from Figure 1 of reference .

Figure 3. Configuration from a) pG state (PDB ID: 3PHY), b) experimental signaling state of WT-PYP (PDB ID: 2XK6), c) experimental signaling state of Δ25 (PDB ID: 1XFQ), d) representative configuration from our pB ensemble.

In all configurations the chromophore (residue 69) is shown explicitly, with the rest of the protein shown as cartoon. The α3 helix (residues 43–51) is in blue, the chromophore binding cap (residues 98–103) in green, and the first 25 N-terminal residues, when present, in brown.

Figure 4. Comparison of the Cα deviations from the pG configuration for the experimental Δ25 configurations (PDB ID 1XFQ) in black, experimental WT-PYP pB configurations (PDB ID: 2KX6) in green, and our pB ensemble in blue.

The horizontal axis is the index number relative to PYP (i.e. the first index of Δ25 is at 25 on the graph). For all three datasets, the regions of the sequence similar to the pG dark state are similar, as are most of the regions where the fluctuations are larger. The primary differences are near the chromophore (residue 69) and are discussed in the main text.