Figure 7. TRIR Spectra of Wild-type PixD and the F-Tyr PixD Variants Under Continuous Illumination.
TRIR spectra were obtained whilst continuously illuminating the protein samples with ~200mW of 450 nm light (L). The top spectrum is the TRIR spectrum of wild-type PixD obtained using the standard pump-probe approach (dark state, D). A key observation is the change in position of the C4=O bleach from 1701 cm−1 in the PixD dark state to 1686 cm−1 upon formation of the light state. Continuous illumination of the F-Tyr substituted PixD variants results in full conversion of 3-FY8 PixD, but only partial population of the light state in 2,3-F2Y8 PixD, and possibly in 3,5-F2Y8 PixD. In addition, there is no light state signature in 2,3,5-F3Y8 PixD.