19F nuclear magnetic resonance measurement of the distance between the E-site GTP and the high-affinity Mg2+ in tubulin.
The distance separating the divalent metal ion high-affinity binding site and the exchangeable nucleotide binding site on tubulin was evaluated by using high-resolution 19F NMR. The 31P and 19F NMR spectra of guanosine 5'-(gamma-fluorotriphosphate) [GTP (gamma F)] were studied. Both the fluorine and the gamma-phosphate were split into a doublet with a coupling constant of 936 Hz. Tubulin purified according to the method of Weisenberg [Weisenberg, R.C., & Timasheff, S.N. (1970) Biochemistry 9, 4110-4116] was incubated with 1 mM Mn2+. After one cycle of assembly, Mn2+ replaced Mg2+ only partially, i.e., 60% at the high-affinity binding site. After colchicine treatment of tubulin to stabilize it, GTP(gamma F) was added, and the 254-MHz fluorine-19 relaxation rates were measured within the first 4 h. Longitudinal and transversal relaxation rates were determined at two concentrations of GTP(gamma F) and variable concentrations of colchicine-tubulin-Mn(II) (paramagnetic complex) or the ternary complex with magnesium (diamagnetic complex). The analysis of the relaxation data indicates that the rate of exchange of GTP(gamma F) from the exchangeable nucleotide site has a lower limit of 8.7 X 10(4) s-1 and the metal and exchangeable nucleotide binding sites are separated by an upper distance between 6 and 8 A. These data confirm that the high-affinity divalent cation site is situated in the same locus as that of the exchangeable nucleotide, forming a metal-nucleotide complex.
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
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Monasterio
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002417:Cattle; D005461:Fluorine; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D011485:Protein Binding; D011487:Protein Conformation; D013816:Thermodynamics; D014404:Tubulin / Q000378:metabolism*
D014404:Tubulin; D005461:Fluorine; D006160:Guanosine Triphosphate; D008274:Magnesium