Metals like copper, zinc and iron are important nutrients to all life. Their special properties which make them so useful to us in things like batteries and catalysts also make them useful to living organisms. Using hard x-ray fluorescence microprobes at the Advanced Photon Source, we have been able to see, often for the first time, where the metals themselves are inside cells and tissues. Yet, many of the images we acquire lead us to new questions. Are these metals required for the activity of proteins? Which proteins are binding which metals inside the cell? With over a third of all proteins thought to bind metals, knowing which metals are bound and how that binding changes in response to the environment could have big implications. For instance, the mismanagement of metals is involved in many diseases, including Lou Gehrig's disease, Wilson and Menkes disease, and possibly even Alzheimer's disease. Metals are also an environmental toxin, such as hexavalent chromium, and they are used in drugs, like the platinum in cisplatin that treats prostate cancer. In another vein, hexavalent uranium is an environmental contaminant of concern at several U.S. Department of Energy sites. The metal-binding proteins in microbes, such as those in the genus Shewanella, which can reduce uranium mobility in groundwater, play a major role in the beneficial activity of these organisms � and knowing which metal is in which protein at a given point in time could lead to new insights into how they do their work. We have developed a new tool to investigate this, combining native two-dimensional gel electrophoresis and x-ray fluorescence imaging, to quantitatively measure the amount of sulfur, iron, zinc, and other metals at every point of the 2-D separation of proteins. By coupling this with mass-spectrometry, we have identified a novel protein (PA5217) as a zinc-binding protein in P. aeruginosa. Our finding highlights how this method not only determines changes in metal occupancy, but also identifies the associated protein.

X-ray fluorescence imaging and metalloproteomics: Tying images of metals in cells to the proteins that bind them

247th National Meeting (2014)

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