Methods for elucidating the domain structures of proteins
Protease Accessibility Laddering (PAL)
Over the years, we have developed robust mass spectrometric methods for accurately elucidating the domain structures of recombinant proteins. These techniques have proved invaluable for X-ray crystallographic structure determinations (Cohen & Chait, 2001). Recently, we have developed a new variation of the limited proteolysis method that allows us to elucidate the domain structures of proteins expressed at endogenous levels (see Figure). We have named it Protease Accessibility Laddering (PAL) (Dokudovskaya et al. 2006). In PAL, genomically tagged proteins are purified on magnetic beads in their natively folded state, either alone or with their interacting partners. Homologous expression of the protein of interest at endogenous levels ensures that the protein folds properly. While attached to the beads, the proteins are probed with specific proteases, and washed to release the cleaved portions that do not remain attached to the beads. The remaining proteolytic fragments are eluted from the beads and detected by immunoblotting with antibodies against the tag, yielding what is in essence a "domain ladder" of the protein under study. Commonly used proteolytically resistant tags, such as Protein A, GFP and 6xHis, can be used. Currently, we determine the cleavage sites by Edman sequencing or MW estimation on the SDS-PAGE gel. Applications using this approach are given in Devos et al. 2004 and Devos et al. 2006. We are now working on determining the cleavage sites directly by mass spectrometry to further improve the speed and accuracy of PAL.