The solution dynamics of normal and transforming p21-s proteins in both the GTP- and GDP-bound forms were examined with time-resolved fluorescence spectroscopy. The fluorescent 2'(3')-O-(N-methylanthraniloyl) derivatives (mant derivatives) of GTP, dGTP, and GDP and the aminocoumarin and fluorescein derivatives of GTP and GDP were synthesized and used as reporter groups. The fluorescence lifetimes at 5-degrees-C of the mant nucleotide derivatives increased from approximately 4 ns in solution to approximately 9 ns when bound to p21ras. At 30-degrees-C, there was a 7.8% difference in lifetime between normal p21ras.mantGTP and p21ras.mantGDP, but no difference between similar complexes of the [Asp-12] p21ras protein. These data are consistent with steady-state fluorescence intensity differences among p21ras.mantGTP, p21ras.mantGDP, and the free nucleotides. Rotational correlation times for the mantGTP- and mantGDP-bound p21 proteins, N-ras, K-ras, and H-ras, were similar at 26 ns (5-degrees-C), which is significantly longer than the 15-ns rotational correlation time predicted for a globular 21 000-Da protein. The p21-bound fluorescein and aminocoumarin nucleotide derivatives reported correlation times of 19 and 29 ns, respectively. Global analysis of the three fluorophore.p21 complexes with linked protein rotational correlation functions were best fit with a common rotational correlation time of 28 ns. Gel permeation chromatography of the GDP and mantGDP complexes of normal p21N-ras also showed greater apparent molecular weights than were expected in both cases, demonstrating that the high rotational correlation times obtained from time-resolved fluorescence measurements were not a result of the introduction of the fluorophore. The rotational data are discussed in terms of two mutually exclusive hypotheses: the p21 proteins are dimeric, or they are monomeric with an extended, asymmetric shape.
