Chemical modification of bovine transducin: probing the GTP-binding site with affinity analogs

Abstract

The structure of the GTP-binding site of transducin, a signal-transducing G-protein involved in the visual excitation process, was studied by affinity labeling. Radioactive GTP analogues with reactive groups attached to different moieties of the GTP molecule were obtained and include 8-azido-GTP, P3-(4-azidoanilino)-P1-5'-GTP (AA-GTP), 5'-[p-(fluorosulfonyl)benzoyl]guanosine (FSBG), 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)-GTP (ANPAP-GTP), the 2',3'-dialdehyde derivative of GTP (oGTP), and a bifunctional cross-linking analogue, 8-azido-P3-(4-azidoanilino)-P1-5'-GTP (8-azido-AA-GTP). With the exception of FSBG, all of the analogues were found to bind to transducin specifically and serve as a cofactor to activate the retinal cGMP cascade or act as a competitive inhibitor for the GTPase activity of transducin. The labeling sites of these analogues were localized by tryptic peptide mapping. ANPAP-GTP and oGTP were unable to covalently modify transducin, suggesting that the 2'- and 3'-hydroxy groups on the ribose ring of GTP are not in direct contact with the protein. AA-GTP only labeled the T alpha subunit of transducin and was localized on the 21-kDa tryptic fragment of T alpha. This indicates that the phosphate moiety of the bound GTP is in direct contact with this peptide. On the other hand, 8-azido-GTP labeled both the T alpha and T beta gamma subunits of transducin. The labeling on T alpha was on the 12-kDa tryptic fragment, suggesting that the guanine ring binding site is composed of a different peptide fragment than the phosphate binding region. Treatment with the bifunctional analogue 8-azido-AA-GTP generated the cross-linked products of T alpha and T beta gamma. This observation implies that the guanine ring of the bound GTP on T alpha could be in close proximity with T beta gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of Tubulin‐Gi Protein Interaction in Rat Cerebral Cortex with Aging

“…Recent evidence indicates that G proteins may form polymers with the same or different subspecies (Hatta et al ., 1986;Hingorani et al, 1989;Nakamura and Rodbell, 1990 ;Vaillancourt et al, 1990) . Moreover, guanine nucleotide transfer between G, and G, has been demonstrated (Hatta et al, 1986) .…”

m-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: synthesis and application

“…On the other hand, a similar GTP derivative was unable to label the visual G-protein transducin [8]. In the present work, m-aminoacetophenone was attached to GTP via the y-phosphate, as previous work [4] suggested that modification at this position does not disturb binding to G-proteins, the coupling of the anilide to the Assay of adenylate cyclase activity Turkey erythrocytes [10] and rat parotid [11] membranes were prepared as previously described.…”

Two-dimensional infrared spectroscopy of azido-nicotinamide adenine dinucleotide in water

“…Consequently, nicotinamide-analogs with vibrational probes attached to the nicotinamide ring are likely to be able to report on the dynamics of the active sites of a wide variety of nicotinamide-dependent enzymes. Analogs of NAD + are widely used as mechanistic probes to understand enzymatic pathways, [42][43][44][45][46][47] and the idea of using a NAD-analog as a spectroscopic reporter holds great potential as a probe with widespread applications to many important enzymatic systems.…”