Time-resolved spectroscopy of dye-labeled photoactive yellow protein suggests a pathway of light-induced structural changes in the N-terminal cap

The photoreceptor PYP responds to light activation with global conformational changes. These changes are mainly located in the N-terminal cap of the protein, which is ∼20 Å away from the chromophore binding pocket and separated from it by the central β-sheet. The question of the propagation of the structural change across the central β-sheet is of general interest for the superfamily of PAS domain proteins, for which PYP is the structural prototype. Here we measured the kinetics of the structural changes in the N-terminal cap by transient absorption spectroscopy on the ns to second timescale. For this purpose the cysteine mutants A5C and N13C were prepared and labeled with thiol reactive 5-iodoacetamidofluorescein (IAF). A5 is located close to the N-terminus, while N13 is part of helix α1 near the functionally important salt bridge E12–K110 between the N-terminal cap and the central anti-parallel β-sheet. The absorption spectrum of the dye is sensitive to its environment, and serves as a sensor for conformational changes near the labeling site. In both labeled mutants light activation results in a transient red-shift of the fluorescein absorption spectrum. To correlate the conformational changes with the photocycle intermediates of the protein, we compared the kinetics of the transient absorption signal of the dye with that of the p-hydroxycinnamoyl chromophore. While the structural change near A5 is synchronized with the rise of the I2 intermediate, which is formed in ∼200 μs, the change near N13 is delayed and rises with the next intermediate I2′, which forms in ∼2 ms. This indicates that different parts of the N-terminal cap respond to light activation with different kinetics. For the signaling pathway of photoactive yellow protein we propose a model in which the structural signal propagates from the chromophore binding pocket across the central β-sheet via the N-terminal region to helix α1, resulting in a large change in the protein conformation.