The sacrificial inactivation of the blue-light photosensor cryptochrome from Drosophila melanogaster

Photoactivation of Drosophila melanogaster cryptochrome results in tryptophan decomposition, conformational changes, and final FAD release.

where Δ ∥ , and Δ , are the difference absorptions probed parallel and perpendicular to the excitation polarisation, respectively. Δ T , is the total amount of difference absorption along all three space coordinates, so that the perpendicular contribution needs to be counted twice.
In none rigid media, e.g. liquid solution, rotational diffusion is the main cause of the disappearance of the anisotropy induced by polarised excitation. For a particular absorption transition of a spherical molecule, the decay of anisotropy is given by Jabłoński's equation [1] , eqn. (2).
where is the anisotropy at = 0 given by eqn. (3). In case of a multicomponent system, i.e. several species contribute to a particular absorption band in the time-resolved absorption experiment, the total anisotropy total is given as the S3 sum of all 1 individual anisotropies arising from each individual species weighted by the fractional contribution to the observed absorption signal according to eqn. (5) total λ, t = 3 4  In terms of rotational diffusion of a globular protein, one can consider an Einstein sphere.
Using Einstein's law the rate constant of rotational correlation is given in eqn. (6) where ; is the viscosity (in kg(dm) −1 s −1 ), ? is the temperature (in °K), > is the gas constant (in kg(dm) 2 s −2 mol −1 K −1 ), < m is the volume of one mole of the rotating unit (in (dm) 3 mol −1 ), A is the molecular mass of the rotating unit (in mol), B̅ is the specific volume of the rotating unit (in (dm) 3 g −1 ), and ℎ is the hydration of the rotating unit (in (dm) 3 g −1 ). A typical value of B̅ for proteins is close to 0.73 mL g −1 . Typically, ℎ can be estimated via the observation that 1 g of protein binds ca. 0.23 g H2O [2] . Thus, for DmCRY with a molecular mass of 66716 g mol −1 and the used buffer conditions, i.e. 10% glycerol in aqueous buffer yielding a viscosity of ca.
(32 ns) −1 . Since total depolarisation is reached in ca. 5  and Methods for more details) and from the orientations given by the crystal structure the corresponding anisotropies were evaluated. However, recently we have shown that the crystal structure might not resemble the energetic minimum on the potential energy surface [6] potentially due to artificial packing effects and since proteins are in general 'dynamic' structures, we performed molecular dynamics simulations on DmCRY in its 'dark' ground state and in its four 'light' radical pair states in order to account for a more realistic   −0.20 Tab. S1 Expected anisotropies and corresponding angles between the transition moment for flavin excitation, I JK , to the transition moments of the most prominent transition dipole moments of all potentially contributing species of the eT pathway based on the DmCRY crystal structure (pdb code: 4GU5) [5] and based on relaxed structures from MD simulations.  2 Tyr°, based on the DmCRY crystal structure (pdb code: 4GU5) [5] and based on relaxed structures from MD simulations.

Estimation of product quantum yields
FAD release requires unfolding of the peptide following Trp decomposition. The chance for FAD release should increase with the number of decomposed Trp's, since this will eventually cause destabilisation of the secondary/tertiary protein structure. However, with increasing number of decomposed Trp's the probability of RPR increases (due to decreasing distances within the corresponding RPs) so that the chance for further Trp decomposition decreases limiting the rate of FAD release. Thus, the total yield of free FAD is expected to be low, since several subsequent photo-reactions are required for a high probability of FAD release, i.e. three photo-reactions (no photo-cycle). According to our time-resolved data, the QY of FAD°− should be ca. 80% for intact DmCRY, since we have 20% loss due to RPR in the 1st RP. In general, an exact determination of a quantum yield is difficult per se. However, we can make a reasonable estimate for the QY for FAD°− via the following formula: where:  DmCRY (pdb code: 4GU5) [5] secondary structure (transparent cartoon) with highlighted FAD in stick representation and four potential tunnels allowing entry of small molecules into the FAD binding pocket. The tunnels were calculated by the Caver 3.0 program. [12] S25  [5] and 4I6J (orange, MmCRY2/FBXL3) [13] ).
DmCRY is aligned to MmCRY2 (not shown). The C-terminal tails (CTT) of DmCRY and FBXL3 are highlighted in cyan and red, respectively. b: Zoom into the FAD binding pocket and the adjacent eT pathway along the tryptophan tetrad and interaction between helix 22 and the surface exposed Trp FGE E . FAD, tryptophan tetrad, and positively charged amino acids are shown in stick representation.