Tl. Hazlett et al., SOLUTION DYNAMICS OF P21(RAS)PROTEINS BOUND WITH FLUORESCENT NUCLEOTIDES - A TIME-RESOLVED FLUORESCENCE STUDY, Biochemistry, 32(49), 1993, pp. 13575-13583
The solution dynamics of normal and transforming p21-s proteins in bot
h the GTP- and GDP-bound forms were examined with time-resolved fluore
scence spectroscopy. The fluorescent 2'(3')-O-(N-methylanthraniloyl) d
erivatives (mant derivatives) of GTP, dGTP, and GDP and the aminocouma
rin and fluorescein derivatives of GTP and GDP were synthesized and us
ed as reporter groups. The fluorescence lifetimes at 5-degrees-C of th
e mant nucleotide derivatives increased from approximately 4 ns in sol
ution to approximately 9 ns when bound to p21ras. At 30-degrees-C, the
re was a 7.8% difference in lifetime between normal p21ras.mantGTP and
p21ras.mantGDP, but no difference between similar complexes of the [A
sp-12] p21ras protein. These data are consistent with steady-state flu
orescence 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 simila
r at 26 ns (5-degrees-C), which is significantly longer than the 15-ns
rotational correlation time predicted for a globular 21 000-Da protei
n. The p21-bound fluorescein and aminocoumarin nucleotide derivatives
reported correlation times of 19 and 29 ns, respectively. Global analy
sis of the three fluorophore.p21 complexes with linked protein rotatio
nal correlation functions were best fit with a common rotational corre
lation time of 28 ns. Gel permeation chromatography of the GDP and man
tGDP complexes of normal p21N-ras also showed greater apparent molecul
ar weights than were expected in both cases, demonstrating that the hi
gh rotational correlation times obtained from time-resolved fluorescen
ce measurements were not a result of the introduction of the fluoropho
re. The rotational data are discussed in terms of two mutually exclusi
ve hypotheses: the p21 proteins are dimeric, or they are monomeric wit
h an extended, asymmetric shape.