Light-Induced Unfolding of Photoactive Yellow Protein Mutant M100L

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Abstract
Light-activation of the PAS domain protein photoactive yellow protein (PYP) is believed to trigger a negative phototactic response in the phototropic bacterium Halorhodospira halophila. To investigate transient conformational changes of the PYP photocycle, we utilized the PYP mutant M100L that displays an increased lifetime of the putative signaling-state photointermediate PYPM by 3 orders of magnitude, as previously reported for the M100A mutant [Devanathan, S., Genick, U. K., Canestrelli, I. L., Meyer, T. E., Cusanovich, M. A., Getzoff, E. D., and Tollin, G. Biochemistry (1998) 37, 11563−11568]. The FTIR difference spectrum of PYPM and the ground state of M100L demonstrated extensive peptide-backbone structural changes as observed in the FTIR difference spectrum of the wild-type protein and PYPM. The conformational change investigated by CD spectroscopy in the far-UV region showed reduction of the α-helical content by approximately 40%, indicating a considerable amount of changes in the secondary structure. The optical activity of the p-coumaric acid chromophore completely vanished upon PYPM in contrast to the dark state, indicating deformation of the binding pocket structure in PYPM. The tertiary structural changes were further monitored by small-angle X-ray scattering measurements, which demonstrated a significant increase of the radius of gyration of the molecule by approximately 5% in PYPM. These structural changes were reversed concomitantly with the chromophore anionization upon the dark state recovery. The observed changes of the quantities provided a more vivid view of the structural changes of the mutant PYP in going from PYPM to PYPdark, which can be regarded as a process of folding of the secondary and the tertiary structures of the “PAS” domain structure, coupled with the p-coumaric acid chromophore deprotonation and isomerization.