Solution dynamics of p21ras proteins bound with fluorescent nucleotides: a time-resolved fluorescence study.
The solution dynamics of normal and transforming p21ras 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Hazlett
T L
TL
Department of Physics, University of Illinois at Urbana, Champaign 61801.
Moore
K J
KJ
Lowe
P N
PN
Jameson
D M
DM
Eccleston
J F
JF
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D003374:Coumarins; D003848:Deoxyguanine Nucleotides; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D046911:Macromolecular Substances; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000378:metabolism; D012399:Rotation; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D003374:Coumarins; D003848:Deoxyguanine Nucleotides; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D012996:Solutions; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; C029603:deoxyguanosine triphosphate; D016283:Proto-Oncogene Proteins p21(ras); D016650:Fluorescein-5-isothiocyanate