A photoactive phosphonamide derivative of GTP for the identification of the GTP-binding domain in beta-tubulin.
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 gamma-position of GTP through a phosphonamide linkage. A combination of saturation and GTP protection studies (90% protection at 25 microM GTP with an apparent Kd of 5 microM) 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 [gamma 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 (beta:65-79) in the exchangeable GTP-binding domain of the beta-subunit of tubulin.
Chavan
A J
AJ
Department of Chemistry, University of Kentucky, Lexington 40506.
Kim
H
H
Haley
B E
BE
Watt
D S
DS
D000345:Affinity Labels / Q000138:chemical synthesis*; D000595:Amino Acid Sequence; D001386:Azides / Q000138:chemical synthesis* / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D007457:Iodine Radioisotopes; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010777:Photochemistry; D013053:Spectrophotometry; D014404:Tubulin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D014404:Tubulin; C069406:guanosine 5'-(trihydrogen diphosphate)-N-(4-azidophenyl)-2-phosphoramido-3-(4-hydroxy-3-iodophenyl)propionamide monoanhydride; D006160:Guanosine Triphosphate