Excitation-Wavelength-Dependent Photocycle Initiation Dynamics Resolve Heterogeneity in the Photoactive Yellow Protein from Halorhodospira halophila

L. Tyler Mix, Elizabeth C. Carroll, Dmitry Morozov, Jie Pan, Wendy Ryan Gordon, Andrew Philip, Jack Fuzell, Masato Kumauchi, Ivo Van Stokkum, Gerrit Groenhof, Wouter D. Hoff*, Delmar S. Larsen

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

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Photoactive yellow proteins (PYPs) make up a diverse class of blue-light-absorbing bacterial photoreceptors. Electronic excitation of the p-coumaric acid chromophore covalently bound within PYP results in triphasic quenching kinetics; however, the molecular basis of this behavior remains unresolved. Here we explore this question by examining the excitation-wavelength dependence of the photodynamics of the PYP from Halorhodospira halophila via a combined experimental and computational approach. The fluorescence quantum yield, steady-state fluorescence emission maximum, and cryotrapping spectra are demonstrated to depend on excitation wavelength. We also compare the femtosecond photodynamics in PYP at two excitation wavelengths (435 and 475 nm) with a dual-excitation-wavelength-interleaved pump-probe technique. Multicompartment global analysis of these data demonstrates that the excited-state photochemistry of PYP depends subtly, but convincingly, on excitation wavelength with similar kinetics with distinctly different spectral features, including a shifted ground-state beach and altered stimulated emission oscillator strengths and peak positions. Three models involving multiple excited states, vibrationally enhanced barrier crossing, and inhomogeneity are proposed to interpret the observed excitation-wavelength dependence of the data. Conformational heterogeneity was identified as the most probable model, which was supported with molecular mechanics simulations that identified two levels of inhomogeneity involving the orientation of the R52 residue and different hydrogen bonding networks with the p-coumaric acid chromophore. Quantum calculations were used to confirm that these inhomogeneities track to altered spectral properties consistent with the experimental results.

Original languageEnglish
Pages (from-to)1733-1747
Number of pages15
Issue number11
Early online date21 Feb 2018
Publication statusPublished - 20 Mar 2018


■M.K.: Strategic Core research Center, Sanshin Metal Working Co., Ltd, 2-9-9 Niihama, Tadaoka-cho, Senboku-gun, Osaka 595-0814, Japan. Funding This work was supported by a grant from the National Science Foundation (CHE-1413739) to both D.S.L. and W.D.H. Additionally, W.D.H. acknowledges additional support from National Science Foundation Grants MCB-1051590 and MRI-1338097. G.G. and D.M. acknowledge support from the Academy of Finland (Grants 258806, 290677, and 304455 to G.G. and Grant 285481 to D.M.). The authors also acknowledge CSC-IT Center for Science, Finland, for computational resources. Notes The authors declare no competing financial interest.

FundersFunder number
National Science Foundation1726397, 1412500, MCB-1051590, MRI-1338097, CHE-1413739, 1413739
Academy of Finland290677, 258806, 285481, 304455
China Scholarship Council
National Science Foundation


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