The photoactivatable NAD+ analogue [32P]2-azido-NAD+ defines intra- and inter-molecular interactions of the C-terminal domain of the G-protein G alpha t.
The photoactivatable NAD+ analogue [32P]2-azido-NAD+ defines intra- and inter-molecular interactions of the C-terminal domain of the G-protein G alpha t.
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可光激活的 NAD 类似物 [32P]2-叠氮基-NAD 定义了 G 蛋白 G α t C 端结构域的分子内和分子间相互作用。
DOI：
10.1042/bj3110987
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Ruoho,AE
中科院分区：
文献类型：
--
作者：
Vaillancourt,RR;Dhanasekaran,N;Ruoho,AE
最近，我们报道了合成并使用[32P]2-叠氮- nad +作为探针来研究g蛋白的结构组织。用百日毒素将[32P]2-叠氮化物- nad +的[32P]2-叠氮化物- adp核糖“系”在g蛋白Gt的α亚基Cys347上。叠氮化物部分的光激活将α t的c端含有Cys347的结构域与邻近的全转导蛋白的分子内和分子间结构域共价交联。然后用醋酸汞切断Cys347和[32P]2-叠氮- adp -核糖之间的巯基糖苷键，将[32P]2-叠氮- adp -核糖的放射性标记转移到“受体”结构域。adp核糖基化后的全导素光交联表明c端结构域与其他α t结构域的分子内相互作用以及与全导素α和γ亚基的分子间相互作用。利用2-（2 ' -硝基苯基亚苯基）-3-甲基-3 ' -溴吲哚啉（BNPS-skatole）和CNBr鉴定了由于光活性部分转移而被放射性标记的肽。结果表明，α t的c端与α t分子内的n端和c端结构域相互作用。对交联α二聚体和α三聚体相互作用位点的映射表明，α t的c端结构域参与了溶液中α t均聚物的形成。此外，我们的研究将β - γ亚基放置在α t的Cys347附近，正如[32P]2-氮化多核苷酸核糖从Cys347转移到伽马亚基所表明的那样，伽马亚基进一步定位于γ t的c端一半。本文的研究确定了全息转导蛋白α亚基的c端分子内和分子间相互作用。
Recently, we reported the synthesis and use of [32P]2-azido-NAD+ as a probe to study the structural organization of G-proteins. Pertussis toxin was used to ‘tether’ [32P]2-azido-ADP-ribose of [32P]2-azido-NAD+ to Cys347 of the alpha subunit of the G-protein Gt. Light activation of the azide moiety covalently cross-linked the domain containing Cys347 at the C-terminus of alpha t with neighbouring intra- and inter-molecular domains of holo-transducin. The radiolabel from [32P]2-azido-ADP-ribose was then transferred to the ‘acceptor’ domain by cleaving the thioglycosidic bond between Cys347 and [32P]2-azido-ADP- ribose with mercuric acetate. ADP-ribosylation followed by photocross-linking of holo-transducin indicated intramolecular interactions of the C-terminal domain with other alpha t domains and intermolecular interactions with holotransducin alpha and gamma subunits. The radiolabelled peptides, which were radiolabelled because of the transfer of the photoactive moiety, were identified by utilizing 2-(2′-nitrophenylsulphenyl)-3-methyl-3′- bromoindolenine (‘BNPS-skatole’) and CNBr. The results indicate that the C-terminus of alpha t interacts with both N-terminal and C-terminal domains within the alpha t molecular. Mapping the interacting sites between cross-linked alpha dimers and alpha trimers indicates that the C-terminal domain of alpha t is involved in the formation of alpha t homopolymers in solution. In addition, our studies place the beta gamma subunit in close proximity to Cys347 of alpha t, as indicated by the transfer of [32P]2-azido-ADP-ribose from Cys347 to the gamma subunit, which was further localized to the C-terminal half of gamma t. The studies presented here identify the C-terminal intra- and inter-molecular interactions of the alpha subunit of holo-transducin.