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Time-resolved spectroscopy of dye-labeled photoactive yellow protein suggests a pathway of light-induced structural changes in the N-terminal cap.
Phys Chem Chem Phys. 2009 Jul 14; 11(26):5437-44.PC

Abstract

The photoreceptor PYP responds to light activation with global conformational changes. These changes are mainly located in the N-terminal cap of the protein, which is approximately 20 A away from the chromophore binding pocket and separated from it by the central beta-sheet. The question of the propagation of the structural change across the central beta-sheet is of general interest for the superfamily of PAS domain proteins, for which PYP is the structural prototype. Here we measured the kinetics of the structural changes in the N-terminal cap by transient absorption spectroscopy on the ns to second timescale. For this purpose the cysteine mutants A5C and N13C were prepared and labeled with thiol reactive 5-iodoacetamidofluorescein (IAF). A5 is located close to the N-terminus, while N13 is part of helix alpha1 near the functionally important salt bridge E12-K110 between the N-terminal cap and the central anti-parallel beta-sheet. The absorption spectrum of the dye is sensitive to its environment, and serves as a sensor for conformational changes near the labeling site. In both labeled mutants light activation results in a transient red-shift of the fluorescein absorption spectrum. To correlate the conformational changes with the photocycle intermediates of the protein, we compared the kinetics of the transient absorption signal of the dye with that of the p-hydroxycinnamoyl chromophore. While the structural change near A5 is synchronized with the rise of the I(2) intermediate, which is formed in approximately 200 mus, the change near N13 is delayed and rises with the next intermediate I(2)', which forms in approximately 2 ms. This indicates that different parts of the N-terminal cap respond to light activation with different kinetics. For the signaling pathway of photoactive yellow protein we propose a model in which the structural signal propagates from the chromophore binding pocket across the central beta-sheet via the N-terminal region to helix alpha1, resulting in a large change in the protein conformation.

Authors+Show Affiliations

Biophysics Group, Department of Physics, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19551213

Citation

Hoersch, Daniel, et al. "Time-resolved Spectroscopy of Dye-labeled Photoactive Yellow Protein Suggests a Pathway of Light-induced Structural Changes in the N-terminal Cap." Physical Chemistry Chemical Physics : PCCP, vol. 11, no. 26, 2009, pp. 5437-44.
Hoersch D, Otto H, Cusanovich MA, et al. Time-resolved spectroscopy of dye-labeled photoactive yellow protein suggests a pathway of light-induced structural changes in the N-terminal cap. Phys Chem Chem Phys. 2009;11(26):5437-44.
Hoersch, D., Otto, H., Cusanovich, M. A., & Heyn, M. P. (2009). Time-resolved spectroscopy of dye-labeled photoactive yellow protein suggests a pathway of light-induced structural changes in the N-terminal cap. Physical Chemistry Chemical Physics : PCCP, 11(26), 5437-44. https://doi.org/10.1039/b821345c
Hoersch D, et al. Time-resolved Spectroscopy of Dye-labeled Photoactive Yellow Protein Suggests a Pathway of Light-induced Structural Changes in the N-terminal Cap. Phys Chem Chem Phys. 2009 Jul 14;11(26):5437-44. PubMed PMID: 19551213.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Time-resolved spectroscopy of dye-labeled photoactive yellow protein suggests a pathway of light-induced structural changes in the N-terminal cap. AU - Hoersch,Daniel, AU - Otto,Harald, AU - Cusanovich,Michael A, AU - Heyn,Maarten P, Y1 - 2009/03/03/ PY - 2009/6/25/entrez PY - 2009/6/25/pubmed PY - 2009/9/18/medline SP - 5437 EP - 44 JF - Physical chemistry chemical physics : PCCP JO - Phys Chem Chem Phys VL - 11 IS - 26 N2 - The photoreceptor PYP responds to light activation with global conformational changes. These changes are mainly located in the N-terminal cap of the protein, which is approximately 20 A away from the chromophore binding pocket and separated from it by the central beta-sheet. The question of the propagation of the structural change across the central beta-sheet is of general interest for the superfamily of PAS domain proteins, for which PYP is the structural prototype. Here we measured the kinetics of the structural changes in the N-terminal cap by transient absorption spectroscopy on the ns to second timescale. For this purpose the cysteine mutants A5C and N13C were prepared and labeled with thiol reactive 5-iodoacetamidofluorescein (IAF). A5 is located close to the N-terminus, while N13 is part of helix alpha1 near the functionally important salt bridge E12-K110 between the N-terminal cap and the central anti-parallel beta-sheet. The absorption spectrum of the dye is sensitive to its environment, and serves as a sensor for conformational changes near the labeling site. In both labeled mutants light activation results in a transient red-shift of the fluorescein absorption spectrum. To correlate the conformational changes with the photocycle intermediates of the protein, we compared the kinetics of the transient absorption signal of the dye with that of the p-hydroxycinnamoyl chromophore. While the structural change near A5 is synchronized with the rise of the I(2) intermediate, which is formed in approximately 200 mus, the change near N13 is delayed and rises with the next intermediate I(2)', which forms in approximately 2 ms. This indicates that different parts of the N-terminal cap respond to light activation with different kinetics. For the signaling pathway of photoactive yellow protein we propose a model in which the structural signal propagates from the chromophore binding pocket across the central beta-sheet via the N-terminal region to helix alpha1, resulting in a large change in the protein conformation. SN - 1463-9076 UR - https://www.unboundmedicine.com/medline/citation/19551213/Time_resolved_spectroscopy_of_dye_labeled_photoactive_yellow_protein_suggests_a_pathway_of_light_induced_structural_changes_in_the_N_terminal_cap_ L2 - https://doi.org/10.1039/b821345c DB - PRIME DP - Unbound Medicine ER -