GTP analogues

The hydrolysis of mant-GTP bound to Ras can be monitored by a slight decrease in fluorescence. Binding experiments of N-Ras with the non-hydro-lyzable GTP-analogue mant-GppNHp showed a biphasic increase in fluorescence. The slow phase had the same amplitude as the decrease observed for the hydrolysis of Ras mant-GTP which led to the hypothesis that a conformational change in the Ras protein proceeds GTP-hydrolysis [170] and represents the rate limiting step ... 

In studying G-proteins, several tools were developed. For example, certain toxins are able to modify covalently the G-proteins, others inhibit the GTPase activity, which allows a permanent activation. PAL can provide the same perspective, whereas photolabile agonists or GTP analogues covalently modify the receptor or G-proteins. 

Photoreactive GTP analogues have proven beneficial for studies of G-proteins, since using such probes can not only lead to the right G-protein being identified, activated by the agonist, but also information about the environment of the GTP binding site can be assessed. 

Leukotriene B4 has been implicated in inflammatory processes and chemo-taxis. The G-protein-coupled receptor in human myeloid cells was postulated to possess two distinct binding sites. Photoaffinity studies using unmodified tritiated LTB4 as photoreactive species labeled two different proteins in the presence (53 kDa) and in the absence of a non-cleavable GTP analogue (56 kDa). This means that stabilization of the G-protein in the GTP-bound state resulted in an interconversion of the first high affinity binding site to an alternative low-affinity binding site [99]. 

Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and WiTTiNGHOFER, A., The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-RafL in complex with RaplA and a GTP analogue. Nature, 1995, 375, 554. 

Interaction of the receptor with Gs also alters the receptor to a state with high affinity for agonist [37,47-49], whereas addition of nonhydrolyzable GTP analogues disrupts the association and converts the receptor to low affinity state [47-49]. Thus the increased binding of GTP to Gs induced by occupancy of the receptor by hormone [45] leads secondarily to dissociation of the receptor from Gs and a return of the receptors to a low affinity state [37,49]. Due to the GTPase activity of Gs, as GTP is also converted to c GDP which reassociates with the jBy complex to form inactive GS-GDP. As a result of the conversion of as-GTP to as GDP, activation of adenylate cyclase ceases unless hormone continues to be present to initiate another cycle of activation. 

Some other aspects of G protein chemistry should be noted. Thus A1F,C (fluoraluminate) can bind to G proteins forming an activated Ga GDP AIF4 complex that mimics the active Ga GTP complex. Similarly, various non-hydrolysable GTP analogues, notably guanosine-5 -[y-thio] triphosphate (GTPf y-S]), bind to Gas and cause persistent activation. [ S GTl Iy-S has been very useful for radioactively labelling Ga proteins and hence establishing that particular hormones or NTs act via a G protein-coupled mechanism. 

We have also demonstrated the utility of analytical DEAR coupled with ESI-MS in the determination of bound nucleotide. This technique may be useful in evaluation of K-ras protein that has been loaded with GTP analogues in order to activate it (7). 

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