We have developed several high-resolution assays for the nucleotide state of a rho-subfamily low molecular weight GTP-binding protein, Cdc42Hs. The first involves the use of the fluorescent N-methylanthraniloyl derivative of GDP (mant-GDP). As has been shown for the ras protein, mant-dGDP fluorescence is significantly enhanced (~20%) upon binding to Cdc42Hs. It was further found that the binding of mant-nucleotides results in an efficient energy transfer between the single tryptophan residue of Cdc42Hs and the mant moiety. The exchange of mant-dGDP for GDP bound to Cdc42Hs, as read-out either by the enhancement of the mant fluorescence or by energy transfer, is inhibited by physiological (mM) Mg2+ concentrations and correlates exactly to the rate of [3H]GDP exchange observed in filterbinding assays. Moreover, changes in the fluorescence of mant-dGDP are also sensitive to nucleotide dissociation induced by the dbl-oncogene product, a known nucleotide exchange factor for Cdc42Hs. A second fluorescence read-out for the nucleotide-bound state of Cdc42Hs involves the measurements of intrinsic fluorescence of a single tryptophan residue (W97) which is highly sensitive to whether GDP or GTP is bound in the nucleotide pocket. The hydrolysis of GTP to GDP by Cdc42Hs results in an ~ 30% enhancement of the protein fluorescence. The rate of this fluorescence change corresponds well to the rate of conversion of [γ-32P]GTP to GDP plus [32P]Pi as measured by filter-binding assays. This, taken together with the observation that a time-dependent enhancement of tryptophan fluorescence does not occur in the presence of a nonhydrolyzable GTP analog (GMP-PCP), suggests that the tryptophan enhancement that occurs subsequent to the binding of GTP is a direct outcome of GTP hydrolysis. Further support for this conclusion comes from the finding that the fluorescence enhancement is accelerated by the recombinant form of the recently cloned catalytic domain of Cdc42Hs-GAP (GTPase activating protein). Thus, changes in the tryptophan fluorescence of Cdc42Hs provide a real-time monitor for both the intrinsic- and GAP-stimulated GTPase activities.
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