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.