Altered Substrate Recognition
TDA provides the environment needed for increased activity and proper regulation of many RTK domains. Data indicates that proteins assembled on TDA nanospheres adopt different conformations, leading to different substrate recognition profiles. In a recent collaboration with Molecular Devices (MD; an MDS Analytical Technologies company), TrkB tyrosine kinase domain was analyzed with and without TDA nanospheres on MD’s SubstrateFinder assay platform.
The substrate profile was shown to be remarkably different with the assembled TrkB versus TrkB in the conventional solution assay—nine new substrates were identified using template-assembled TrkB, while eight substrates recognized by untemplated enzyme were not recognized by template-assembled TrkB. This data reveals a common theme among proteins that are normally associated with a membrane, namely that it is critical to place these proteins back into the environment in which they evolved to function.
Complex Assembly Using TDA
TDA technology was first developed by assembling a four-protein complex from an E. coli chemosensory system. The chemosensory system, consisting of a receptor, kinase, response regulator, and adaptor protein, provided a model system to explore the usefulness of template-directed assembly.
Using TDA, it was demonstrated that one could assemble multiple proteins, the base of which was a fragment of the chemoreceptor Tar; this is analogous to displaying IGF-1R on the surface and assembling IGF-1R:IRS1 complexes. In solution these four proteins did not assemble, and thus, exhibited no activity. When templated, however, the proteins formed functional complexes and exhibited in vivo activation levels and function.
The study of heteroreceptor interactions and subsequent regulation was also made possible by TDA. This data, given the similarities between bacterial and human-receptor signaling pathways, suggested that similar complexes could be assembled using human proteins. Figure 3 depicts how a human signaling-pathway system can be constructed on TDA nanospheres. This use of TDA facilitates screening by allowing for simpler experiments using multiple purified components.
Membrane-associated proteins represent a large, yet underexploited group of drug targets. Because they have been removed from their natural membrane environment, for ease of purification and structural studies, they become less active and behave sub-optimally in standard assays. TDA provides a novel way to assemble these proteins, leading to proper function and activity, thereby providing an improved way to discover active inhibitors.