Characterization of the solution structure of the M intermediate of photoactive yellow protein using high-angle solution X-ray scattering

It is widely accepted that PYP undergoes global structural changes during the formation of the biologically active intermediate PYPM. High-angle solution x-ray scattering experiments were performed using PYP variants that lacked the N-terminal 6-, 15-, or 23-amino-acid residues (T6, T15, and T23, respectively) to clarify these structural changes. The scattering pro. le of the dark state of intact PYP exhibited two broad peaks in the high-angle region (0.3 angstrom(-1) < Q < 0.8 angstrom(-1)). The intensities and positions of the peaks were systematically changed as a result of the N-terminal truncations. These observations and the agreement between the observed scattering profiles and the calculated profiles based on the crystal structure confirm that the high-angle scattering profiles were caused by intramolecular interference and that the structure of the chromophore-binding domain was not affected by the N-terminal truncations. The profiles of the PYPM intermediates of the N-terminally truncated PYP variants were significantly different from the profiles of the dark states of these proteins, indicating that substantial conformational rearrangements occur within the chromophore-binding domain during the formation of PYPM. By use of molecular fluctuation analysis, structural models of the chromophore-binding region of PYPM were constructed to reproduce the observed pro. le of T23. The structure obtained by averaging 51 potential models revealed the displacement of the loop connecting b4 and b5, and the deformation of the alpha 4 helix. High-angle x-ray scattering with molecular fluctuation simulation allows us to derive the structural properties of the transient state of a protein in solution.