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Figure 4.
Fig. 4. CREB-DNA affinity and its divalent cation
enhancement. A, representative fluorescence anisotropy-derived
DNA binding isotherm of the CREB bZIP-SSCRE measured in the
presence of 10 m M MgCl[2] (diamonds) or 10 mM BaCl[2] (boxes)
in buffer A or buffer A alone (circles). Plotted are the
normalized measured millipolarization units (mP) versus the CREB
bZIP concentration (nM). B, representative divalent cation
binding isotherm. Measurements were made by titrating CaCl[2]
into a 0.990-ml reaction buffer of 25 mM Tris, pH 7.5, 5%
glycerol, 50 mM NaCl, 6 µg of bovine serum albumin, 10
µg of poly d(I-C) containing 1 nM fluoresceinated DNA and
20 nM CREB bZIP. Plotted are the measured millipolarization
units (mP) versus the Ca^2+ ion concentration. Essentially
identical binding isotherms are generated by titration with
MgCl[2] or BaCl[2]. C, CREB bZIP:SSCRE phosphate contacts.
Residues making phosphate contacts are shown as light blue
sticks and labeled in black. The DNA is shown as sticks. The
carbon atoms are colored white, oxygen atoms are red, nitrogen
atoms are blue, and phosphate atoms are shown as yellow. Arg289,
which does not make any direct phosphate contacts, is also
shown. This residue is in position to make potential
water-mediated base contacts to bases outside the consensus
SSCRE or, alternatively, may contribute to binding through a
general electrostatic effect. The two identical DNA strands are
differentiated as strand 1 (S1) and strand 2 (S2) to distinguish
cross-strand phosphate contacts made by each CREB bZIP subunit.
D, specific interactions between CREB and the SSCRE DNA. Only
DNA base pairs 1 to 4 of the consensus SSCRE, are shown. DNA
atoms are colored as in C. CREB residues involved in base
specific contacts are shown as light blue sticks. Only residue
Arg301' from the second subunit is shown as the remaining
base-specific contacts are identical between subunits. Two water
molecules (W1 and W2) and the hexahydrated magnesium ion are
shown as ball-and-sticks. Oxygen atoms are colored red and the
magnesium atom is colored light blue. Hydrogen bonds are
indicated by black dashed lines and hydrophobic contacts by
light blue dashed lines.
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