Protein binding partners

PTKs can be subdivided into two large families, receptor tyrosine kinases (RTKs) and non-RTKs. The human genome encodes for a total of 90 tyrosine kinases of which 32 are nonreceptor PTKs that can be placed in 10 subfamilies (Fig. 1). All nonreceptor PTKs share a common kinase domain and usually contain several additional domains that mediate interactions with protein-binding partners, membrane lipids, or DNA (Table 1). These interactions may affect cellular localization and the activation status of the kinase or attract substrate proteins for phosphorylation reactions. 

Piggott AM, Karuso P. (2008) Rapid identification of a protein binding partner for the marine natural product kahalalide F by using reverse chemical proteomics. ChemBioChem 9 524-530. 

Figure 7.5 Emission spectra of FITC for a model protein assay run (A) at room temperature for 30 minutes and (B) with microwave heating for 20 seconds. Control experiments, where one of the protein binding partners, BSA-biotin is omitted from the assay, run (C) at room temperature and (D) with microwave heating for 20 seconds Room Temp Room Temperature. Adapted from reference 1.

Fig. 5 The principles behind the affinity capture of proteins for the identification of protein-binding partners (biomarker discovery/new biology) and drug targets, and for drug target evaluation.

Ribosome display (Figure 2) has been used to select protein-binding partners but, to our knowledge, never been used to find the unknown protein-binding partner for a small molecule.268 272-275 Fiowever, a recent paper by Pluckthun clearly indicates that this is possible. In this report, the authors show, for the first time, that it is possible to use ribosome display to select for catalytic activity based on catalytic turnover (directed evolution). In their experiment, they displayed variants of RTEM-/3-lactamase and used biotinylated ampicillin sulfone (80) to select for catalytically active variants (Figure 3)276... 

Figure 35 Glycan photocross-linking probes for fluorescent labeling. Photocross-linkers are shown in red and fluorophores are in blue, (a and b) Lactose molecules modified with a benzophenone cross-linker and a functional group that can react through click chemistry. After photocross-linking, the adducts are reacted with an azide or alkyne-functionalized fluorophore to fluorescently label the protein-binding partner, (c) Multivalent mannose or fucose (represented in green) probes bear a benzophenone cross-linker and a Cy3 or Cy5 fluorescent label.

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