Fig 1.
Ribbon representations of PpSB1-LOV dimer.
(A) dark state (PDB ID: 5J3W), the N and C termini of a single protein chain are labeled that shows intertwined N-terminus. Protein chains A and B are shown in pink and salmon colors, respectively. Each protein chain is bound to an FMN cofactor shown as stick models (colored by element: carbon, yellow; nitrogen, blue; oxygen, red; phosphorus, pink). The twofold axis runs from top to bottom. Inset to the figure shows the arginine cluster in FMN binding pocket of PpSB1-LOV where dotted lines represent the hydrogen bonds between the FMN and the arginine residues. (B) Superposition of dark (salmon, PDB ID: 5J3W) and light (cyan, PDB ID: 3SW1) states showing structural differences. The LOV core domains of protein chains shown on the left (transparent) were structurally aligned as described previously [28]. In order to superpose the second protein chain (shown on right), a (~ 29°) rotation and translation is required. Arrows indicate the rotation shifts between the respective secondary structure elements.
Fig 2.
UV/vis spectrum recorded for the PpSB1-LOV-R66I in the light- and dark-states in solution.
The inset shows the characteristic absorption maxima at 447 nm after the dark state recovery. The same sample was measured in light- and dark-state. Please note that the difference in absorbance at ˜280 nm is insignificant as this is usually due to the flavin chromophore involved in the adduct formation.
Fig 3.
(A) and (B) Small-angle scattering data of PpSB1-LOV-R66I in the light- and dark-states. Distance distribution function P(r) is shown as inset in (A).
Fig 4.
Guinier plots of the merged SAXS data of PpSB1-LOV-R66I in the dark- and light-states.
Straight lines are linear fits to the measured data.
Table 1.
Physical parameters determined from merged SAXS data.
Guinier radii RG determined from real and reciprocal space, volume of correlation Vc, ratio QR = Vc2/RG, and molecular mass Mm determined from QR of PpSB1-LOV-R66I in the light- and dark-states.
Fig 5.
Comparison of SAXS data of PpSB1-LOV-R66I in fully populated (A, B, C) dark-state and (D, E, F) light-state. Experimental SAXS data was recorded during two independent experiments on beamlines P12 at EMBL and BM29 at ESRF. (A, D) Log-log plots, (B, E) Kratky-plots of SAXS data. (C, F) SAXS intensities measured on P12 divided by intensities measured on BM29 in the low q-regions that have been used for further analysis.
Fig 6.
Small-angle scattering data of PpSB1-LOV-R66I in the light- and dark-states and structural models obtained by CORAL.
(A) and (B) Experimental data and theoretical scattering curves (solid lines) using the program CORAL of the light- and dark-state crystal structures (PDB ID’s: 3SW1 and 5J3W) including flexible ends that are not seen in the crystal structures. Data are shifted for clarity. The lower panel shows the residuals of the models. (C) CORAL structural models of light (cyan) and dark (salmon) states.
Fig 7.
Small-angle scattering data of PpSB1-LOV-R66I in the light- and dark-states structural models obtained by EOM.
(A) and (B) Experimental data and theoretical scattering curves (solid lines) using the program EOM of the light- and dark-state crystal structures (PDB ID’s: 3SW1 and 5J3W) including flexible ends that are not seen in the crystal structures. Data are shifted for clarity. The lower panel shows the residuals of the models. (C). EOM structural models of light (cyan) and dark (salmon) states.
Fig 8.
Distribution of (A) RG and (B) Dmax of PpSB1-LOV-R66I in dark- and light-states as obtained by EOM. Dashed and solid lines show values of generated pool and selected structures, respectively.
Fig 9.
Ab initio shape reconstructions of PpSB1-LOV-R66I.
(A) Dark (salmon mesh) and (B) light- (cyan mesh) states determined by SAXS. Crystal structures of the dimers of dark- (PDB ID 5J3W) and light (PDB ID 3SW1) are shown as ribbon structures and are aligned in the respective envelopes in cyan and salmon with bound FMN molecules as space filled. Extra density at the top and bottom of the ab initio models correspond to residues that are not resolved in the crystal structures. Black arrow heads show the location of Arg66.
Fig 10.
Structural transition from light- to dark-state in PpSB1-LOV-R66I followed by time-course SAXS.
(A) Experimental SAXS data measured at selected time-points. Solid lines are fits to the data assuming a two-state population of known PpSB1-LOV-R66I light- and dark-state structures based on the CORAL models with the only free fit parameter ϕ that represents the population of the light-state (Eq 1). Data and fits are shifted for clarity. (B) Difference SAXS patterns ΔI(q,t) of data measured for the time points shown in (A) minus the SAXS pattern recorded at t = 0 min. The solid lines are theoretical fits to the data as in (A) based on the CORAL models.
Fig 11.
Dark- minus light-state measured under steady-state conditions (red line) and the difference of time-resolved SAXS (black circles) data recorded at 98.5 min and 0 min.
Fig 12.
Light-state fraction of PpSB1-LOV-R66I as a function of time.
Determined from the integral of ΔI(q,t) up to 0.2 Å-1 and a structure-based fit of I(q,t) assuming a mixed population of known light- and dark-states CORAL models. The solid line is a single-exponential fit to the data points of the integral of ΔI(q,t). For clarity error bars are shown for selected points.