Characterization of the solution structure of the M intermediate of photoactive yellow protein using high-angle solution x-ray scattering
Hironari Kamikubo 1, Nobutaka Shimizu 2, Miki Harigai 1, Yoichi Yamazaki 1, Yasushi Imamoto 1 and Mikio Kataoka 3*
1 Nara Institute of Science and Technology
2 SPring-8
3 Nara Inst. of Science & Technology
* To whom correspondence should be addressed. E-mail: kataoka{at}ms.naist.jp.
Submitted on September 12, 2006
Revised on October 16, 2006
Accepted on 2 January 2007
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Abstract |
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It is widely accepted that photoactive yellow protein (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 profile of the dark state of intact PYP exhibited two broad peaks in the high-angle region (0.3 Å-1 < Q < 0.8 Å-1). The intensities and positions of the peaks were systematically changed due to 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. Using molecular fluctuation analysis, structural models of the chromophore-binding region of PYPM were constructed to reproduce the observed profile of T23. The structure obtained by averaging 51 potential models revealed the displacement of the loop connecting 
4 and 5, and the deformation of the 
4 helix. High-angle x-ray scattering with molecular fluctuation simulation allow us to derive the structural properties of the transient state of a protein in solution.
Key Words:
PAS/LOV, fluctuation analysis, structural change, structural modeling, synchrotron radiation
