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Chemical footprinting has been used for over five decades now to use  solvent accessibility as a way to determine structural information on proteins and nucleic acids. The idea of using X-rays to  for footprinting, however, is relatively new.  X-ray footprinting was  first conceived and tried out at the National Synchrotron Light Source at Brookhaven National Laboratory in the late 90's, by Drs. Mark Chance and Michael Brenowitz. Since then, the technique has grown in popularity and been used to study a wide range of protein and nucleic acid systems, from small proteins to large complexes to ribosome motion within cells. Drs Sayan Gupta and Corie Ralston have now introduced the technique at the Advanced Light Source and Lawrence Berkeley National Laboratory. Read more below to learn more about how the technique works, and how it is used. 

How it works.

When X-rays impinge on a water-based buffer, hydroxyl radicals are created. These radicals will modify protein residues. However, the modifications occur only where they are accessible to water. Therefore, if you can locate the modifications, you can determine a solvent accessible map of the protein. This is useful, for instance, for watching protein folding,  or for determining protein interaction points.  In practice, after exposure to X-rays, the protein samples are digested with proteases, such as pepsin or GluC or trypsin, and then analyzed using mass spectrometry to determine locations of modifications. If you are familiar with hydrogen-deuterium exchange (HDX) then you will have noted the similarity of footprinting to HDX. The main difference is that with HDX, the protein backbone is mapped, whereas in FP, the sidechains are mapped.  In addition, with footprinting, any protease can be used, not just pepsin. 

Why use a synchrotron.

Other radiation sources (and chemical sources) can be used for footprinting. But synchrotrons  provide a very intense and focused X-ray beam.  This, it turns out, is very important for the footprinting experiment. An intense brief burst of radiation produces a high enough radical concentration in such a short amount of time such that modifications occur before other secondary damage affects the proteins. In addition, the brighter the beam, the less sample that is necessary. For our footprinting experiments, we use typically 5-10 micromolar concentrations of proteins, and about 200 microliters per sample.