A Photoactive Phosphonamide Derivative of GTP for the Identification of the GTP-Binding Domain in ݭTubulin

A GTP photoaffinity probe (¹²⁵I-APTG) was developed that incorporated an [¹²⁵I]-N-(4-azidophenyl)-2-amino-3-(4-hydroxy-3-iodophenyl)propionamide group at the ?-position of GTP through a phos-phonamide linkage. A combination of saturation and GTP protection studies (90% protection at 25 µM GTP with an apparent of 5 µM) validated the use of this new probe as a satisfactory GTP mimic. This probe offered the advantage of possessing an ¹²⁵I radiolabel external to the GTP moiety, in contrast to the previously reported [?³²P]-8-N3GTP that possessed an internal ³²P radiolabel. This novel feature accommodated the purification of photolabeled peptides using a combination of ion-exclusion, gel filtration, and HPLC techniques. [¹²⁵I]APTG was used to identify a peptide (ݺ65-79) in the exchangeable GTP-binding domain of the ݭsubunit of tubulin.