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Functional Assay of GUN4 by Team:Macquarie Australia 2015

The assay undertaken involved adding protoporphyrin IX to cell lysates overexpressing GUN4 and exposing these samples to light at a wavelength of 280nm. This excites tryptophan residues in GUN4 and, when bound, the the resulting 340nm fluorescence will excite protoporphyrin IX, resulting in an emission peak at 635nm that can be detected by a spectrophotometer.

For this assay, cells were lysed through use of a French press and these lysates were diluted ten-fold to approximately 0.2mg/ml concentration of protein. Protoporphyrin was prepared to a concentration of 2µM and added to samples of lysate containing 0µg, 5µg, 10µg, 15µg and 20µg of GUN4. A non-specific protein control (Bovine Serum Albumin) and heat treated GUN4 cell lysates (80 degrees Celsius for ten minutes) were used as controls.

When excited at 280nm, emission peaks at 635nm were detected through fluorescence spectrophotometry for each GUN4 lysate, with increased intensity as GUN4 concentration rose. BSA showed no fluorescent activity when excited at this wavelength and the heat treated lysates displayed a much lower level of fluorescence compared to the unheated samples.

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This demonstrates that GUN4 is biologically active, binding to protoporphyrin IX. Further, the linear increase in emission at 635nm with greater concentration of GUN4 is indicative of higher rates of binding. This corresponds with the modelling conducted by the Macquarie University team of 2014 that suggests a linear pattern of protoporphyrin IX/GUN4 complex formation. Our functional assay builds upon this framework as we found that this linear relationship only occurs at low protein concentrations when the lysates were diluted to a 0.2mg/ml.

This assay is a fast and efficient method for detecting GUN4 functionality and represents a useful new tool for the study of the chlorophyll a biosynthesis pathway.