Zeitaufgelöste, makromolekulare Röntgenstrukturanalyse
Zusammenfassung der Projektergebnisse
Photo-Optical Switches. Phycoerythrocyanin contains the photo-active chromophore phycoviolobilin. Upon absorption of a green or yellow photon this chromophore can be switched reversibly between two stable isomeric forms, the Z- and E-configurations. The absorption spectra of both configurations are distinct with a shift of their absorption maxima by ~60nm from 567 nm to 504 nm, respectively. This makes this protein an ideal candidate to learn how to engineer photo-optic switches from biological and nonbiological polymers. We determined, for the first time, the molecular basis of an optical switch of this kind by investigating the Z- to E-isomerization in a biliprotein using X-ray structure determination. Crystals were prepared containing phycoerythrocyanin in the pure Z- and pure E-configuration. We compared the atomic structures the Z- as well as the E-configuration. The comparison revealed insight in the mechanism of the isomerization. A network of hydrogen bonds stabilizes the Z- as well as the Econfiguration. The molecular switch is kept locked in either position at ambient temperatures. 5-Dimensional Crystallography. In order to determine a unique chemical, kinetic mechanism from time-resolved X-ray data, an additional 5th parameter needs to be varied, the most convenient of which is the temperature. The beamline 14-IDB of BioCARS, Advanced Photon Source, Argonne Nat’l lab has been upgraded from 2005 to the beginning of 2008. It is now the hottest beamline for hard X-rays in the world. We were among the first test-users during commissioning of the beam line. We collected data on crystals of the photoactive yellow protein (PYP) at 0 deg. C. The temperature has a profound effect of the cycling time in the crystals. An entire time course spanning 9 orders of magnitude in time (25 data sets) can be collected in one shift. These experiments show that the upgraded beamline delivers a polychromatic X-ray flux high enough for fast data acquisition, which is sufficient to make 5-D crystallography feasible. In addition single exposure experiments become possible, where an entire reflection pattern can be collected using only one polychromatic, ultra-short X-ray pulse. Software. In order to analyze time-resolved X-ray data we developed new, user friendly software. At the base of this software lies our well established, well tested program SVD4TX. The “new” SVD4TX implements a user friendly “graphic user interface”, back and forth Fast Fourier Transforms as well as linear algebra routines to model chemical kinetic mechanism in general. This program will be very useful to analyze the amount of data expected from 5-dimensional crystallography.
Projektbezogene Publikationen (Auswahl)
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(2006) Protein Dynamics on Different Time Scales. Journal of Non-Crystalline Solids, 352 4371-4378
F. Parak, K. Achterhold, M. Schmidt, V. Prusakov, S. Croci
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(2007) Structural Basis for the photochemistry of a-phycoerythrocyanin. Biochemistry, 46, 416-423
M. Schmidt, A. Patel, Y. Zhao, W. Reuter
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(2008) Structural dynamics and kinetics of myoglobin-CO binding: lessons from time-resolved X-ray diffraction and four-wave mixing spectroscopy, in: Biochemical Applications of Nonlinear Optical Spectroscopy (V. Yakovlev ed.), series: optical science and engineering, vol. 138, CRC press
V. Rajcu, M. Schmidt, M. Stoneman
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(2008) Structure Based Enzyme Kinetics by Time-Resolved X-ray crystallography, in: Ultrashort Laser Pulses in Medicine and Biology (eds: W. Zinth, M. Braun, P. Gilch). Springer Verlag, pp 201-241
M. Schmidt
