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

A GTP photoaffinity probe (I-125-APTG) was developed that incorporated an [I-125]-N-(4-azidophenyl)-2-amino-3-(4-hydroxy-3-iodophenyl)propionamide group at the gamma-position of GTP through a phosphonamide linkage. A combination of saturation and GTP protection studies (90% protection at 25 mu M GTP with an apparent K-d of 5 mu M) validated the use of this new probe as a satisfactory GTP mimic. This probe offered the advantage of possessing an I-125 radiolabel external to the GTP moiety, in contrast to the previously reported [gamma P-32]-8-N(3)GTP that possessed an internal P-32 radiolabel. This novel feature accommodated the purification of photolabeled peptides using a combination of ion-exclusion, gel filtration, and HPLC techniques. [I-125]APTG was used to identify a peptide (beta:65-79) in the exchangeable GTP-binding domain of the beta-subunit of tubulin.