F
luorescence quenching was used to study thesite-specific binding of the
Escherichia coliregu
latory protein TyrR to a f
luoresceinated o
ligonuc
leotide(9F30A/30B) containing a TyrR bindingsite. The equi
librium constant for the interaction(
KL) was measured as a function of temperatureandsa
lt concentration in the presence and absence of ATP
![](/images/gifchars/gamma.gif)
S, a specific
ligand for TyrR. F
luorescence titrationsyie
lded a
KL va
lue of 1.20 × 10
7M
-1 at 20
![](/images/entities/deg.gif)
C, which was independent of theacceptor (9F30A/30B)concentration in the range 5-500 nM, indicating that the systemexhibits true equi
librium binding. C
larkeand G
lew ana
lysis of the temperature dependence of binding revea
led a
linear dependence of
R ln
KLontemperature in the absence of ATP
![](/images/gifchars/gamma.gif)
S. The thermodynamicparameters obtained at 20
![](/images/entities/deg.gif)
C (
![](/images/gifchars/theta.gif)
) were
![](/isubscribe/journa<font color=)
ls/bichaw/37/i20/eqn/bi972854ae10001.gif">= -35.73 kJ mo
l-1,
![](/isubscribe/journa<font color=)
ls/bichaw/37/i20/eqn/bi972854ae10002.gif">= 57.41 kJ mo
l-1, and
![](/isubscribe/journa<font color=)
ls/bichaw/37/i20/eqn/bi972854ae10003.gif">= 93.14 kJ mo
l-1. Saturating
leve
lsofATP
![](/images/gifchars/gamma.gif)
S (200
![](/images/entities/mgr.gif)
M) strengthened binding at a
ll temperatures andresu
lted in a non
linear dependence of
Rln
KL on temperature. Thethermodynamic parameters characterizing binding under these conditionswere
![](/isubscribe/journa<font color=)
ls/bichaw/37/i20/eqn/bi972854ae10004.gif">= -39.32 kJ mo
l-1,
![](/isubscribe/journa<font color=)
ls/bichaw/37/i20/eqn/bi972854ae10005.gif">=37.16 kJ mo
l-1,
![](/isubscribe/journa<font color=)
ls/bichaw/37/i20/eqn/bi972854ae10006.gif">= 76.40 kJ mo
l-1, and
![](/isubscribe/journa<font color=)
ls/bichaw/37/i20/eqn/bi972854ae10007.gif">= -1.03 kJ mo
l-1K
-1. Severa
l conc
lusions were drawn fromthese data. First, binding is entropica
lly driven at 20
![](/images/entities/deg.gif)
Cinboth the presence and absence of ATP
![](/images/gifchars/gamma.gif)
S. This can part
ly beaccounted for by counterions re
leased fromthe DNA upon TyrR binding; in the absence of ATP
![](/images/gifchars/gamma.gif)
S and diva
lentcations, the TyrR-9F30A/30Binteraction resu
lts in the re
lease of two to three potassium ions.Second, the more favorab
le
![](/isubscribe/journa<font color=)
ls/bichaw/37/i20/eqn/bi972854ae10008.gif">va
lue,and hence tighter binding observed in the presence of ATP
![](/images/gifchars/gamma.gif)
S, isprimari
ly due to a decrease in
![](/isubscribe/journa<font color=)
ls/bichaw/37/i20/eqn/bi972854ae10009.gif">(-20.3 kJ mo
l-1), which overcomes anunfavorab
le decrease in
![](/isubscribe/journa<font color=)
ls/bichaw/37/i20/eqn/bi972854ae10010.gif">(-16.7 kJ mo
l-1). Third,thenegative
![](/isubscribe/journa<font color=)
ls/bichaw/37/i20/eqn/bi972854ae10011.gif">va
lue obtained in the presence of ATP
![](/images/gifchars/gamma.gif)
S indicates that the binding ofATP
![](/images/gifchars/gamma.gif)
S favors aconformationa
l change in TyrR upon binding to 9F30A/30B, yie
lding amore stab
le comp
lex.