Role of an N-Terminal Loop in the Secondary Structural Change of Photoactive Yellow Protein

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Abstract
Photoactive yellow protein (PYP) is photoconverted to its putative active form (PYPM) with global conformational change(s). The changes in the secondary structure were studied by far-UV circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy using PYP, which lacks N-terminal 6, 15, or 23 amino acid residues (T6, T15, and T23, respectively). Irradiation of truncated PYPs induced the loss of the CD signal, where the maximal difference was located at 222 nm. The reduction of the CD signal was significantly larger than the calculated CD of the N-terminal helices, indicating that it is mainly accounted for by the unfolding and/or structural change of the helices located outside the N-terminal region. The difference FTIR spectra between dark and photosteady states recorded using the solution samples demonstrated that large absorbance changes in the amide mode of the β-sheet were reduced and downshifted by truncation. The structural change of the β-sheet is therefore closely correlated with the N-terminal loop. NaCl decelerates the decay of intact PYPM and T6M at low concentrations (1000 mM). For T15M and T23M, NaCl accelerates their decay at >100 mM but never decelerates their decay, suggesting that the electrostatic interaction, which plays an important role for the recovery of PYP from PYPM, is lost by removing positions 7−15. The electrostatic interaction between this region and the β-scaffold is likely to promote the conformational change of PYPM for recovery of PYP.