In vitro experiments presented in Section 3.4 showed that FrmR is able to bind Zn(II), Cu(I),
Co(II) and Ni(II) (the first two with high affinity and the last two with a significant weaker affinity, albeit the binding of Ni(II) was not further investigated). In order to test if these metals would be able to trigger the allosteric mechanism leading to a conformational change in FrmR and the subsequent disruption of protein:DNA complex, FA association analyses have been employed in the presence of metals. Figure 3. 20A shows titration of frmRAPro (10 nM) with a sample of FrmR incubated with a zinc salt in 20 % excess (1.2 molar equivalents ZnCl2). Given
FrmR Zn(II) affinity for the tightest sites is KZn(II)1-3 = 1.7 x 10 -10
M, 5 µM ZnCl2 was also added
to the reaction buffer which should be well in excess of the concentration required in order to maintain a metalled form of the protein throughout the experiment. A notable shift in the FrmR association curve reveals a significantly weakened binding to DNA, which implies that Zn(II) acts as an effector in vitro causing conformational change in FrmR which, in the metallated form, releases its operator region. A similar result, although less prominent, was obtained with CuCl (verified to be ≥ 95 % Cu(I)) (Figure 3. 20B). In this experiment no additional cuprous ions were added to the buffer since FrmR possesses an even tighter affinity for the metal (KCu(I)1- 2 = 4.90 ± 1.6 × 10 -15 M KCu(I)3-4 = 1.72 ± 0.7 × 10 -12 M; KCu(I)5-8 ≥ 8 × 10 -11
M). Data from both experiments were fitted to the same model used for apo-association (Section 3.6.2) and
KDNA Zn(II)FrmR
and KDNA Cu(I)FrmR
were calculated to be 3110 ± 400 nM (Figure 3. 20A) and 654 ± 130 nM (Figure 3. 20B) respectively.
For these experiments where DNA binding did not saturate, the average fitted Δrobs maximum
value from apo-protein experiments was used in the script as found in literature (Reyes- Caballero et al. 2011; Foster et al. 2014). Figure 3. 20C displays a representative data set from experiments discussed in this section in comparison with those for the apo-FrmR:DNA association (Section 3.6.5, Figure 3. 19A) in order to better appreciate the effect these metals have on DNA binding. The scheme in Figure 3. 21A was presented by Grossoehme and Giedroc in 2009 and represents the thermodynamic cycle describing the allostery response that a metalloregulatory protein for its DNA operator region will encounter upon metal binding (see Chapter 1). The protein, here FrmR, is in its tetrameric state (P4) and the assumption that
monomeric FrmR does not have affinity for DNA is made (Giedroc & Arunkumar 2007; Grossoehme & Giedroc 2009, 2012).
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Figure 3. 20 A frmRAPro (10 nM) was anaerobically titrated with FrmR in the presence of 5 µM ZnCl2. The protein was incubated with 1.2 molar equivalents of ZnCl2 and EDTA was omitted.
B frmRAPro (10 nM) was anaerobically titrated with FrmR incubated with 1.2 molar equivalents of CuCl (verified to be > 95% Cu(I)) and EDTA was omitted. C Comparison of the anisotropy change upon titration of frmRAPro with apo-FrmR (blue circles), Cu(I)-FrmR (pale blue circles) and Zn(II)-FrmR (green circles). DNA binding was monitored by fluorescence anisotropy. Symbol shapes represent individual experiments. Data were fit to a model describing a 2:1 FrmR tetramer (non-dissociable):DNA stoichiometry. Solid line represents simulated curves produced from the average KDNA determined across the experiment replicates shown.
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Figure 3. 21 Allosteric coupling scheme as described by Grossoehme and Giedroc (2012) applied to homotetrameric FrmR A Generalized thermodynamic cycle accounting for the four allosteric “end” states of a homotetrameric metalloregulatory protein (P4) can hypothetically
adopt: apo (P4), metal-bound (P4·Mn), DNA-bound apoprotein (P4·D), and a “ternary” protein–
metal–DNA complex ((P4·Mn)·D). Each equilibrium (K1, K2, K3, K4) describes a direct
transition from one configurational state to another as shown. Note also that P4 and thus the
entire scheme is in equilibrium with free P monomer, defined by Ktetramer, which has no affinity
for the DNA. B Ligand exchange equilibrium, defined by the adimensional parameter Kc, dictates the degree of allostery between the metal binding and DNA binding sites.
4 P P4
+ nM P4•Mn
Ktetramer
K1
+
+
D D
P4•D + nM (P4•Mn)•D
K3
K4
K2
P4•D + P4•Mn
P4
+ (P4•Mn)•D
Kc
A.
B.
104 | P a g e The values obtained from the experiments described in the current and in the previous section coincide with the constants in the vertical equilibria in Figure 3. 21A (KDNA
FrmR
is K3 and
KDNA
metal-FrmR
is K4). The ligand exchange equilibrium constant, Kc, represents the magnitude of
allosteric regulation and is expressed by the ratios K4/K3 and K2/K1 (Grossoehme & Giedroc
2009, 2012). The first ratio corresponds to the difference in metal affinity between the protein bound to DNA and in its free form, while the second can be thought of as the difference in DNA binding affinity between the metallated and the apo-protein (Grossoehme & Giedroc 2012). Having measured K3 and K4 by fluorescence anisotropy, we can now determine the coupling
equilibrium constant Kc and therefore obtain the allosteric coupling free energy ΔGc via the
standard thermodynamic function described in Section 1.3.2 (Equation 1). Mean ΔGc values
(and standard deviations) were calculated from the full set of (equally weighted) possible pair- wise permutations of Kc. This yields ΔGc
Zn(II)FrmR
= + 2.03 (± 0.08) kcal mol-1 and ΔGc Cu(I)- FrmR·DNA
= + 1.10 (± 0.10) kcal mol-1 (Table 8. 2, Appendix).
This approach revealed that Zn(II) is more allosterically effective when binding to FrmR, because ΔGc Cu(I)-FrmR·DNA is smaller. In fact for repressors in which metal binding induces
dissociation of the repressor of the DNA operator, the tertiary state (P4·Mn)·D is destabilized
compared to D and P4·Mn therefore the difference in free energy associated with the ligand
exchange reaction (Figure 3. 21B) will be positive and the reaction will not proceed spontaneously (Grossoehme & Giedroc 2012). Since in the presence of metal the promoter will not be occluded anymore, access for RNA polymerase will be possible.
The ability of Zn(II) and Cu(I) to disrupt the interaction between FrmR and its operator was also tested by titrating the preformed protein:DNA complex with metal aliquots. FrmR (2.5 µM, chosen because FA apo-FrmR curves showed complete associations at this [protein]) was incubated with 10 nM frmRAPro for 30 min before titration with ZnCl2 or CuCl. Figure 3. 22A
shows that dissociation of the protein:DNA complex occurs upon addition of 0.25 - 0.5 molar equivalents of Zn(II) suggesting that the allosteric mechanism allowing FrmR to release the DNA is mainly trigged during the filling of the second site of the tetrameric protein. Titration of
frmRAPro with Cu(I) results in dissociation after 2 molar equivalents of metal (Figure 3. 22B),
consistent with what is described in Section 3.5.1 where competition of eight atoms of Cu(I) per tetramer (2:1 stoichiometry) was observed. Thus FrmR binds specifically to frmRA operator- promoter as an inverse function of [Zn(II)] or [Cu(I)]. The weak affinities of FrmR for Co(II) (KCo(II)1-4 = 7.59 ± 0.4 x 10
-6
M) and Ni(II) (as suggested by competition experiment with mag- fura-2, Figure 3. 15) prevented us to carry out dissociation experiments with these metals.
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Figure 3. 22 Titration of pre-formed FrmR:DNA complexes with ZnCl2 and CuCl. frmRAPro (10
nM) was pre-incubated with FrmR (2.5 µM) before titration with A ZnCl2 and B CuCl.
Dissociation of protein:DNA complexes was monitored by fluorescence anisotropy. Experiments were performed anaerobically at pH 7.0.
[Zn(II)]/[FrmR]
0.3
0.15
0.0
A.
B.
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