RESUMO
Photoactive Yellow Protein (PYP) is a bacterial blue light receptor that enters a photocycle after excitation. The intermediate states are formed on time scales ranging from femtoseconds up to hundreds of milliseconds, after which the signaling state with a lifetime of about 1 s is reached. To investigate structural changes and dynamics, we incorporated the SCN IR label at distinct positions of the photoreceptor via cysteine mutation and cyanylation. FT-IR measurements of the SCN label at different sites of the well-established dark state structure of PYP characterized the spectral response of the label to differences in the environment. Under constant blue light irradiation, we observed the formation of the signaling state with significant changes of wavenumber and lineshape of the SCN bands. Thereby we deduced light-induced structural changes in the local environment of the labels. These results were supported by molecular dynamics simulations on PYP providing the solvent accessible surface area (SASA) at the different positions. To follow protein dynamics via the SCN label during the photocycle, we performed step-scan FT-IR measurements with a time resolution of 10 µs. Global analysis yielded similar time constants of τ1 = 70 µs, τ2 = 640 µs, and τ3 > 20 ms for the wild type and τ1 = 36 µs, τ2 = 530 µs, and τ3 > 20 ms for the SCN-labeled mutant PYP-A44C*, a mutant which provided a sufficiently large SCN difference signal to measure step-scan FT-IR spectra. In comparison to the protein (amide, E46) and chromophore bands the dynamics of the SCN label show a different behavior. This result indicates that the local kinetics sensed by the label are different from the global protein kinetics.