Hydrolysis of GTP by p21NRAS, the NRAS protooncogene product, is accompanied by a conformational change in the wild-type protein: use of a single fluorescent probe at the catalytic site.
2'(3')-O-(N-Methyl)anthraniloylguanosine 5'-triphosphate (mantGTP) is a fluorescent analogue of GTP that has similar properties to the physiological substrate in terms of its binding constant and the kinetics of its interactions with p21NRAS, the NRAS protooncogene product. There is a 3-fold increase in fluorescence intensity when mantGTP binds to p21NRAS. The rate constant for the cleavage of mantGTP complexed with the protein is similar to that of GTP, and cleavage is accompanied by a fluorescence intensity change in the wild-type protein complex. A two-phase fluorescence change also occurs when the nonhydrolyzable analogue 2'(3')-O-(N-methyl)anthraniloylguanosine 5'-[beta, gamma-imido]triphosphate (mantp[NH]ppG) binds to wild-type p21NRAS. The second phase occurs at the same rate as the second phase observed after mantGTP binding. Thus this second phase is probably a conformation change of the p21NRAS nucleotiside triphosphate complex and that the change controls the rate of GTP hydrolysis on the protein. With a transforming mutant, [Asp12]-p21NRAS, there is no second phase of the fluorescence change after mantGTP or mantp[NH]ppG binding, even though mantGTP is hydrolyzed. This shows that an equivalent conformational change does not occur and thus the mutant may stay in a "GTP-like" conformation throughout the GTPase cycle. These results are discussed in terms of the proposed role of p21NRAS in signal transduction and the transforming properties of the mutant.
National Institute for Medical Research, Mill Hill, London, United Kingdom.
S E
SE
Neal
J F
JF
Eccleston
M R
MR
Webb
D001665:Binding Sites; D005456:Fluorescent Dyes / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D011519:Proto-Oncogenes; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates / Q000378:metabolism
D005456:Fluorescent Dyes; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; D006153:Guanosine Diphosphate; C063981:2'(3')-O-(N-methyl)anthraniloylguanosine 5'-triphosphate; D006160:Guanosine Triphosphate; D011505:Protein-Tyrosine Kinases; D016283:Proto-Oncogene Proteins p21(ras)