Ng, Kingman and Getzoff, Elizabeth D. and Moffatt, Keith (1995) Optical Studies of a Bacterial Photoreceptor Protein, Photoactive Yellow Protein, in Single Crystals. Biochemistry, 34 (3). pp. 879-890. ISSN 0006-2960. doi:10.1021/bi00003a022. https://resolver.caltech.edu/CaltechAUTHORS:20180202-152924851
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Abstract
Photoactive yellow protein (PYP), isolated from Ectofhiorhodospira halophila, is a water soluble, 14 kDa photoreceptor protein with a fully reversible photocycle resembling that of sensory rhodopsin 11. We have established the presence of photoactivity in PYP crystals and defined the relaxation kinetics of spectroscopically distinguishable species in quantitative terms. The PYF' crystal has a bright yellow color and displays pronounced anisotropic absorption properties. Linear dichroism measurements show that the transition moment of the PYP chromophore makes an angle of 73° (or 107°) with respect to the six-fold crystallographic symmetry axis. The crystal absorbance can be bleached reversibly as indicated by absorption changes. A bleached photostationary state in the crystal can be established via CW laser illumination, and the extent of crystal bleaching is found to be clearly dependent on excitation laser wavelength, intensity and illumination time. These results provide the information for designing time-resolved crystallography experiments in which a minimum perturbation is applied to the PYP crystals. Global exponential fitting shows that the relaxation from the photostationary state in the crystal is biphasic at -4 °C; a slower component of 1.4 f 0.2 s^(-l) accounts for 60% of the absorbance change and a faster component of 5.2 f 0.9 s^(-l) for the other 40%. As a control, we found that the kinetics for the same relaxation in solution are well described by one exponential and agree quantitatively with previous studies. The two rate constants observed in the crystal show similar temperature dependences, with activation energies for the slow and fast components of 11.7 ± 1.2 and 5.5 ± 2.3 kcal/mol, respectively. However, the amplitudes associated with the two exponents show different and opposite temperature dependence. Our results show that the solution kinetic model is not directly applicable to crystals. A kinetic model consistent with the optical data is important to extract the underlying structural intermediates from the time-resolved X-ray diffraction data obtained in parallel with the optical data described here. We propose an alternative model for the photocycle in the crystal which contains an additional bleached intermediate in parallel with the last long-lived intermediate in the solution model.
Item Type: | Article | |||||||||
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Additional Information: | © 1995 American Chemical Society. Received October 13, 1994; Revised Manuscript Received November 22, 1994. This work is supported by grants from the National Institutes of Health (GM36452 to K.M. and GM37684 to E.D.G.) and the W. M. Keck Foundation. We thank Gloria E. O. Borgstahl, DeWight R. Williams, Patrick M. Burke, and Che-Fu Kuo for protein purification and crystallization and for providing all the crystals and protein solution used in this study, Michael Garavito, Daniel Picot, and Marvin Makinen for initial help with linear dichroism measurements, Ying Chen, Alan LeGrand, and Vukica Srajer for help with the microspectrophotometer, Claude Pradervand for help on timing and electronics components, Ying Chen, Alan LeGrand, Duncan McRee, Vukica Srajer, and Tsu-yi Teng for many helpful discussions, and Eric Gouaux and Marvin Makinen for comments on the manuscript. | |||||||||
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Issue or Number: | 3 | |||||||||
DOI: | 10.1021/bi00003a022 | |||||||||
Record Number: | CaltechAUTHORS:20180202-152924851 | |||||||||
Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20180202-152924851 | |||||||||
Official Citation: | Optical Studies of a Bacterial Photoreceptor Protein, Photoactive Yellow Protein, in Single Crystals Kingman Ng, Elizabeth D. Getzoff, and Keith Moffat Biochemistry 1995 34 (3), 879-890 DOI: 10.1021/bi00003a022 | |||||||||
Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | |||||||||
ID Code: | 84659 | |||||||||
Collection: | CaltechAUTHORS | |||||||||
Deposited By: | Ruth Sustaita | |||||||||
Deposited On: | 02 Feb 2018 23:55 | |||||||||
Last Modified: | 15 Nov 2021 20:21 |
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