THE PURIFICATION AND CRYSTALLIZATION OF MYXOBACTERIAL PHYTOCHROME FOR TIME RESOLVED X-RAY CRYSTALLOGRAPHY
Location
SU-217
Department
Biology
Abstract
Phytochromes are red light photoreceptors found in plants, fungi, and bacteria. They regulate a variety of important physiological responses such as seed germination and shade avoidance in plants and synthesis of light-harvesting complexes in photosynthetic bacteria. Their role in non-photosynthetic bacteria is not well-understood. Phytochromes are composed of a photosensory core module (PCM) covalently attached to the C-terminal effector domain, usually histidine kinase. The photoactive phytochrome structure is unknown at several intervals of its photocycle. The aim of the experiment was to purify and crystallize bacteriophytochrome from non-photosynthetic myxobacterium Stigmatella aurantiaca, denoted SaBphP2 for time-resolved X-ray crystallography at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The process begins with the growth of E. coli cells expressing PCM of SaBphP2. The cells are sonicated and the protein is extracted using Ion Metal Affinity Chromatography (IMAC). The absorbance spectra were measured to determine the concentration of the purified protein and photoactivity upon illumination with red light (700 nm) and far red light (750 nm). The protein was crystallized using the batch method where protein and crystallization solution are mixed directly in large quantities to produce thousands of microcrystals. The SaBphP2 microcrystals diffracted optimally at the ESRF beamline, enabling the determination of the photoactive phytochrome structure 700ms post illumination with red-light diode. This experiment is important because it gives the proper techniques to purify and crystallize photoreceptor proteins for time-resolved X-ray crystallography, enabling determination of the phytochrome structure at various late intermediates of its photocycle.
Faculty Sponsor
Emina Stojikovic
Faculty Sponsor
Marius Schmidt
THE PURIFICATION AND CRYSTALLIZATION OF MYXOBACTERIAL PHYTOCHROME FOR TIME RESOLVED X-RAY CRYSTALLOGRAPHY
SU-217
Phytochromes are red light photoreceptors found in plants, fungi, and bacteria. They regulate a variety of important physiological responses such as seed germination and shade avoidance in plants and synthesis of light-harvesting complexes in photosynthetic bacteria. Their role in non-photosynthetic bacteria is not well-understood. Phytochromes are composed of a photosensory core module (PCM) covalently attached to the C-terminal effector domain, usually histidine kinase. The photoactive phytochrome structure is unknown at several intervals of its photocycle. The aim of the experiment was to purify and crystallize bacteriophytochrome from non-photosynthetic myxobacterium Stigmatella aurantiaca, denoted SaBphP2 for time-resolved X-ray crystallography at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The process begins with the growth of E. coli cells expressing PCM of SaBphP2. The cells are sonicated and the protein is extracted using Ion Metal Affinity Chromatography (IMAC). The absorbance spectra were measured to determine the concentration of the purified protein and photoactivity upon illumination with red light (700 nm) and far red light (750 nm). The protein was crystallized using the batch method where protein and crystallization solution are mixed directly in large quantities to produce thousands of microcrystals. The SaBphP2 microcrystals diffracted optimally at the ESRF beamline, enabling the determination of the photoactive phytochrome structure 700ms post illumination with red-light diode. This experiment is important because it gives the proper techniques to purify and crystallize photoreceptor proteins for time-resolved X-ray crystallography, enabling determination of the phytochrome structure at various late intermediates of its photocycle.