Thermodynamics of the Interaction of the Escherichia coli Regulatory Protein TyrR with DNA Studied by Fluorescence Spectroscopy
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- 作者：Michael F. Bailey ; Barrie E. Davidson ; Jim Haralambidis ; Terry Kwok ; and William H. Sawyer
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：May 19, 1998
- 年：1998
- 卷：37
- 期：20
- 页码：7431 - 7443
- 全文大小：146K
- 年卷期：v.37,no.20(May 19, 1998)
- ISSN：1520-4995
文摘
Fluorescence quenching was used to study thesite-specific binding of the Escherichia coliregulatory protein TyrR to a fluoresceinated oligonucleotide(9F30A/30B) containing a TyrR bindingsite. The equilibrium constant for the interaction(KL) was measured as a function of temperatureandsalt concentration in the presence and absence of ATP
S, a specificligand for TyrR. Fluorescence titrationsyielded a KL value of 1.20 × 107M-1 at 20 
C, which was independent of theacceptor (9F30A/30B)concentration in the range 5-500 nM, indicating that the systemexhibits true equilibrium binding. Clarkeand Glew analysis of the temperature dependence of binding revealed alinear dependence of R ln KLontemperature in the absence of ATPS. The thermodynamicparameters obtained at 20 C (
) were
ls/bichaw/37/i20/eqn/bi972854ae10001.gif">= -35.73 kJ mol-1,ls/bichaw/37/i20/eqn/bi972854ae10002.gif">= 57.41 kJ mol-1, andls/bichaw/37/i20/eqn/bi972854ae10003.gif">= 93.14 kJ mol-1. Saturating levelsofATPS (200 
M) strengthened binding at all temperatures andresulted in a nonlinear dependence of Rln KL on temperature. Thethermodynamic parameters characterizing binding under these conditionswerels/bichaw/37/i20/eqn/bi972854ae10004.gif">= -39.32 kJ mol-1,ls/bichaw/37/i20/eqn/bi972854ae10005.gif">=37.16 kJ mol-1,ls/bichaw/37/i20/eqn/bi972854ae10006.gif">= 76.40 kJ mol-1, andls/bichaw/37/i20/eqn/bi972854ae10007.gif">= -1.03 kJ mol-1K-1. Several conclusions were drawn fromthese data. First, binding is entropically driven at 20 Cinboth the presence and absence of ATPS. This can partly beaccounted for by counterions released fromthe DNA upon TyrR binding; in the absence of ATPS and divalentcations, the TyrR-9F30A/30Binteraction results in the release of two to three potassium ions.Second, the more favorablels/bichaw/37/i20/eqn/bi972854ae10008.gif">value,and hence tighter binding observed in the presence of ATPS, isprimarily due to a decrease inls/bichaw/37/i20/eqn/bi972854ae10009.gif">(-20.3 kJ mol-1), which overcomes anunfavorable decrease inls/bichaw/37/i20/eqn/bi972854ae10010.gif">(-16.7 kJ mol-1). Third,thenegativels/bichaw/37/i20/eqn/bi972854ae10011.gif">value obtained in the presence of ATPS indicates that the binding ofATPS favors aconformational change in TyrR upon binding to 9F30A/30B, yielding amore stable complex.
S, a specificligand for TyrR. Fluorescence titrationsyielded a KL value of 1.20 × 107M-1 at 20 C, which was independent of theacceptor (9F30A/30B)concentration in the range 5-500 nM, indicating that the systemexhibits true equilibrium binding. Clarkeand Glew analysis of the temperature dependence of binding revealed alinear dependence of R ln KLontemperature in the absence of ATPS. The thermodynamicparameters obtained at 20 C () wereS (200 M) strengthened binding at all temperatures andresulted in a nonlinear dependence of Rln KL on temperature. Thethermodynamic parameters characterizing binding under these conditionswereCinboth the presence and absence of ATPS. This can partly beaccounted for by counterions released fromthe DNA upon TyrR binding; in the absence of ATPS and divalentcations, the TyrR-9F30A/30Binteraction results in the release of two to three potassium ions.Second, the more favorableS, isprimarily due to a decrease inS indicates that the binding ofATPS favors aconformational change in TyrR upon binding to 9F30A/30B, yielding amore stable complex.