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2′I/Br-ATP; 2′I/Br-AppNHp; 2′I/Br-GppNHp; fluorescence spectroscopy; phase problem; X-ray crystallography
Abstract:
The solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into protein crystals. Several 2′-halogenated nucleotides have been reported as potential universal phasing tools for nucleotide binding proteins. However, only limited data are available dealing with the effect of 2′-substitution on recognition by the protein. We have determined equilibrium dissociation constants of 2′-halogenated ATP analogues for the ATP binding proteins UMP/CMP kinase and the molecular chaperone DnaK. Whereas the affinities to UMP/CMP kinase are of the same order of magnitude as for unsubstituted ATP, the affinities to DnaK are drastically decreased to undetectable levels. For 2′-halogenated GTP analogues, the kinetics of interaction were determined for the small GTPases p21ras (Y32W) (fluorescent mutant) and Rab5. The rates of association were found to be within about one order of magnitude of those for the nonsubstituted nucleotides, whereas the rates of dissociation were accelerated by factors of ˜100 (p21ras) or ˜105 (Rab5), and the resulting equilibrium dissociation constants are in the nm or μM range, respectively. The data demonstrate that 2′halo-ATP and -GTP are substrates or ligands for all proteins tested except the chaperone DnaK. Due to the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 105-fold decrease in affinity as observed for Rab5 places the equilibrium dissociation constant in the μM range, so that they are still well suited for crystallization of the G-protein:nucleotide complex.
