Local dynamics of the photo-switchable protein PYP in ground and signalling state probed by 2D-IR spectroscopy of -SCN labels
Schmidt-Engler, JM (Schmidt-Engler, Julian M.); Blankenburg, L (Blankenburg, Larissa); Zangl, R (Zangl, Rene); Hoffmann, J (Hoffmann, Jan); Morgner, N (Morgner, Nina); Bredenbeck, J (Bredenbeck, Jens)
Physical Chemistry Chemical Physics, 2020, 22, pp 22963-22972
Incorporation of minimally perturbative vibrational probes into proteins allows combination of the femtosecond time resolution of two dimensional infrared (2D-IR) spectroscopy with a spatial resolution on the level of single side chains. Here, we apply the thiocyanate (-SCN) label introduced by the cyanylation of cysteine to probe local dynamics in the photo-switchable protein PYP. We incorporated the -SCN label into five positions of the protein structure including PYP's core region, its solvent exposed surface and the chromophore-binding pocket. The analysis of -SCN's time dependent 2D-IR lineshape provides insight into the timescales and amplitudes of the dynamics in the label's protein and solvent microenvironment. We present a detailed analysis of the local protein dynamics found at all five labelling positions in PYP's dark state (pG). Absorption of a blue photon triggers the isomerisation of PYP's chromophore and eventually leads to an overall reorganisation of the protein structure, where PYP ends up in a less structured signalling state pB. Employing 2D-IR spectroscopy also on the signalling state allows assessment of the change of local dynamics compared to the pG state. |
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