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. Author manuscript; available in PMC: 2009 Aug 5.
Published in final edited form as: Biochemistry. 2008 Jul 11;47(31):8058–8069. doi: 10.1021/bi800443k

Table 2.

Operator binding by LLhP variantsa.

lacO1 lacOsym lacOdisC
Low affinitybKd (x 10−11 M) High affinitycKd (x 10−11 M) Allosteric ratiod Low affinity Kd (x 10−11 M) High affinity Kd (x 10−11 M) Allosteric ratio Low affinity Kd (x 10−11 M) High affinity Kd (x 10−11 M) Allosteric ratio
Lacle >10000 1.5 ± 0.4 >1000 >10000 0.18 ± 0.06 >10000 >10000 380 ± 80 >100
LLhP 220 ± 20 0.98 ± 0.09 220 ± 30 61 ± 15 1.0 ± 0.10 60 ± 15 1200 ± 300 12 ± 3 100 ± 35
I48S 3700 ± 220 32 ± 2.0 110 ± 10 500 ± 34 6.0 ± 0.20 83 ± 7 ∼14000±4600 260 ± 36 ∼50
I48V 1700 ± 660 10 ± 1.4 170 ± 70 260 ± 18 3.1 ± 0.20 82 ± 8 5300 ± 560 51 ± 11 100 ± 25
Q55T 660 ± 86 6.5 ± 0.94 100 ± 20 260 ± 43 2.2 ± 0.25 119 ± 24 2700 ± 250 35 ± 5.3 79 ± 14
Q55V 2200 ± 260 23 ± 1.6 93 ± 13 350 ± 37 6.4 ± 0.80 56 ± 9 13000 ± 3400 140 ± 20 92 ± 27
G58L >10000 >10000 NRf >10000 >10000 NR >10000 >10000 NR
G58T >10000 >10000 NR >10000 >10000 NR >10000 >10000 NR
S61C ≥20000 15 ± 4.3 ∼1000 >10000 >10000 NR NBDg 66 ± 5.1 >1000
S61M
 ≥30000
 28 ± 3.4
 ∼1000
 >10000
 >10000
 NR
 NBD
 126 ± 9.8
 >1000
LLhP 1780 ± 110 13 ± 1.9 136 ± 12
Hepes bufferh
a

DNA binding data were determined from 3−4 independent measurements, using two different preparations of protein. Reported errors represent one standard deviation. Buffer used for DNA-binding measurements was 10 mM Tris-HCl, pH 7.4, 150 mM KCl, 5% DMSO, 0.1 mM EDTA, and 0.3 mM DTT. DNA concentration was 1.5 × 10−12 M.

b

For LLhP variants, low affinity conditions were in the absence of hypoxanthine. For Lacl, low affinity conditions were in the presence of 1mM IPTG.

c

For LLhP variants, high affinity conditions included a saturating concentration of hypoxanthine (see Materials and Methods). For Lacl, no effector was present.

d

Low affinity binding divided by high affinity binding.

e

Values from (9). Lacl measurements were also repeated in parallel with LLhP and showed similar results.

f

NR: No response to the addition of hypoxanthine.

g

NBD: Very little, if any, binding detected.

h

The same buffer as solution scattering experiments: 0.12 mM Hepes-KOH, pH 7.6, 200 mM KCl, 5% glycerol, 1 mM EDTA, and 0.3 mM DTT.