Fig 1.
VVD exhibits amorphous aggregation at standard conditions.
(A) The absorbance (A) spectra of VVD (0.56 mg/mL, 31 μM) at 25°C and standard buffer conditions (10% glycerol, 50 mM HEPES, 150 mM NaCl, 20 mM imidazole, pH 8) acquired every 30 min after a BL pulse, show a shift in absorbance. First and last spectra are shown in black. Arrow indicates time course of the absorbance shift. (B) The aggregation of VVD was quantified by measuring the absorbance at λ = 550 nm. A representative VVD aggregation kinetics record (0.56 mg/mL) is shown (mean ± SD, N = 3). (C) A Western blot against VVD´s 6×His shows that samples with an absorbance shift form VVD oligomers. Control samples in lanes 1 and 4 correspond to bovine serum albumin (BSA) and unaggregated protein, respectively. The pellet and the supernatant of a centrifuged aggregated VVD sample were loaded in lane 2 and 3, respectively. The pellet fraction presents three bands at molecular weights corresponding to monomer (19 kDa), dimer (38 kDa) and trimer (57 kDa) of VVD. (D) Transmission electron microscopy of VVD samples with absorbance shifts display amorphous aggregation.
Fig 2.
VVD becomes oxidized by self-produced singlet oxygen.
(A) Detection of oxidized proteins by Western blot against biotin-hydrazide in labeled samples. Lane1: VVD intentionally oxidized using 33 mM H2O2 and 1.5 mM NiCl2. Lane 2: VVD sample not illuminated. Lane 3: VVD sample illuminated for 5 min with BL. (B) The 3D structure of VVD in the dark state (Protein Data Bank 2PD7) highlighting the oxidized amino acids detected by mass spectrometry of aggregated samples. (C) Fluorescence intensity (F) records of AMDA in the absence and presence of free FAD and VVD. At t = 0 samples were subject to BL illumination during 5 min. In the presence of VVD (triangles) bleaching of AMDA is observed as a reduction in F immediately after illumination (arrow). In control experiments, free FAD (a known producer of 1O2) causes fluorescence bleaching (circles) and BL has no effect on AMDA alone (squares). Data were normalized with respect to the value of F before BL illumination. [VVD] = 30 μM, [FAD] = 30 μM, [AMDA] = 10 μM. Data: mean, error bars: SD; n = 3. (D) Addition of A. niger catalase (2.34 μM) avoided aggregation and shortened adduct mean lifetime to τ ≈ 1.1×103 s.
Fig 3.
The aggregation of VVD is under kinetic control and regulated by photoadduct dynamics.
(A) Aggregation kinetics of VVD initially prepared in a dark-state or lit-state. The kinetics records are well fit (solid lines) by a second-order reaction model that considers photoadduct decay as the limiting step for VVD aggregation. Both samples were diluted in standard buffer (10% glycerol, 50 mM HEPES, 150 mM NaCl, 20 mM imidazole, pH 8). The lit-state corresponds to the same conditions tested in Fig 1B. (B) Aggregation halts during the illumination cycling period and resumes only after samples are returned to the dark. Samples were initially illuminated for 30 s and their aggregation kinetics was followed. 5-s BL pulses were applied every ~5 min, over 68 min (circles) or 202 min (triangles). Aggregation kinetics of a sample subjected to only the initial BL pulse is included for reference (squares). Data: mean, error bars: SD; n = 3. (C-D), VVD lit-state is resistant to denaturant conditions whereas dark-state is not. Proteins initially prepared in a dark-state (C) or in a lit-state (D) were challenged with 0.01% SDS (black records) or left in standard buffer without SDS (gray records), and their absorption spectra were immediately acquired.
Fig 4.
Effect of various stabilizing factors on the aggregation of VVD.
(A) Addition of the osmolite glycerol fully avoids protein aggregation, with recovery of the three reported isosbestic points at wavelengths: 330 nm, 385 nm, and 413 nm (18). (B) Addition of BSA (at 242 μM) avoids VVD aggregation. The addition of chemically reducing agents DTT (C) and GSH (D) also limited aggregation. First and last absorption spectra are shown in black; data were acquired every 30 min.
Fig 5.
Summary of our findings and proposed models for oxidative damage and aggregation in VVD.
(A) Absorption of BL by dark-VVD results in an excited triplet state (TS). In the conventional photocycle (gray arrows) the TS decays to the flavin-cysteine adduct state corresponding to lit-VVD, from where spontaneous adduct decay follows. Alternatively (black arrows), the TS decays to the ground state by energy transfer to O2 with the production of 1O2, which in turn promotes internal chemical damage. This mechanism is expected to occur in other LOV domains. In addition, we discovered that the presence of A. niger catalase (CAT) accelerates photoadduct decay. (B) The aggregation pathway of self-oxidized VVD is shown. The lit-VVD state decays into dark-VVD, followed by VVD dimerization and formation of aggregates (VVD-A) that lose the flavin cofactor. The aggregation process is under kinetic control (governed by the dynamics of the FAD-Cys adduct) and limited by VVD dimer formation. Glycerol, BSA, DTT, GSH and CAT directly interact with VVD, impeding VVD dimerization and subsequent aggregation.