THERMODYNAMIC STOICHIOMETRIES OF PARTICIPATION OF WATER, CATIONS AND ANIONS IN SPECIFIC AND NONSPECIFIC-BINDING OF LAC REPRESSOR TO DNA - POSSIBLE THERMODYNAMIC ORIGINS OF THE GLUTAMATE EFFECT ON PROTEIN-DNA INTERACTIONS
THERMODYNAMIC STOICHIOMETRIES OF PARTICIPATION OF WATER, CATIONS AND ANIONS IN SPECIFIC AND NONSPECIFIC-BINDING OF LAC REPRESSOR TO DNA - POSSIBLE THERMODYNAMIC ORIGINS OF THE GLUTAMATE EFFECT ON PROTEIN-DNA INTERACTIONS
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DOI：
10.1016/0022-2836(92)90504-d
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发表时间：
1992-11-05
影响因子：
5.6
通讯作者：
RECORD, MT
中科院分区：
文献类型：
--
作者：
HA, JH;CAPP, MW;RECORD, MT
本研究的目的是量化阳离子、阴离子和水对Lac阻遏物(Lac R)蛋白与强结合的对称lac操纵子(O Sym)DNA位点相互作用的稳定性和特异性的贡献。为此，用滤膜结合和≡纤维素层析分别测定了Lac R与O-sym操纵子和非操纵子DNA相互作用的结合常数Kobs及其与单价盐(MX)浓度的幂依赖关系(SK Obs DLOG LOG KOBS d Log[MX])。对于Lac R的特异性和非特异性结合，当生理阴离子谷氨酸(Glu−)取代氯化物(Cl−)时，固定盐浓度下的Kobs增加。在0.25 m-kx处。观察到O系统的Kobs增加了$̃40倍，而对于非操纵子DNA，通过外推估计Kobs增加了3̃00倍。对于非操纵子DNA，在实验不确定度范围内，SK obs Rd与盐浓度无关，而在KCl0.13m和0.18m之间，KClRd=−9.8(±1.0)和KGlu(SK Obs，KGlu Rd=−9.3(±0.7)，0.23m和0.36m-KGlu之间)相似。对于O sym DNA，SK obs RO随阴离子性质的不同而显著不同，至少在KGlu中，随着[KGlu]的增加，幅度似乎减小。Sk Obs RO的平均大小小于Sk Obs Rd，对于比结合的降低，其顺序为：KClRO？K OBS RO和SK OBS RO都不受单价阳离子M+(Na+、K+、NH+4或其混合物，均为氯盐)的选择的影响，并且SK OBS RO在所考察的范围内(0.125~0.3m)与[MCL]无关。SK OBS RO的这一行为与具有较大贡献的聚电解质效应的结合过程的预期行为一致。然而，考虑到23Na核磁共振观察到阳离子在DNA(NH4+>；K+>；Na+)附近的优先顺序，在固定的[MX]下，阳离子的性质对SK obs RO大小的影响是有些出乎意料的。对于特异性结合和非特异性结合，从DNA聚电解质释放的大量阳离子是SK OBS RO的主要贡献。为了解释这些数据，我们认为Glu−是一个惰性阴离子，而Ac和ClDNA磷酸基团与Lac抑制物竞争结合。通过分析[KGlu]在0.2 5~0.9m范围内随着[KGlu]的增加而明显降低的明显降低，确定了乳胶阻滞剂和O SYM操作符的最小释水化学计量比(2 10(±30)H2O)的热力学估算.根据这一分析，KGlu中特定结合和非特定结合的SK obS值的不同主要是由于特定结合中的水的释放.在KAc和KCl中，我们推导出阴离子竞争对Kobs和SK obs的影响程度不同，对于不同的阴离子和不同的结合方式是不同的。
The objective of this study is to quantify the contributions of cations, anions and water to stability and specificity of the interaction of lac repressor (lac R) protein with the strong-binding symmetric lac, operator (O sym) DNA site. To this end, binding constants K obs and their power dependences on univalent salt (MX) concentration (SK obs≡ d log K obs d log [MX]) have been determined for the interactions of lac R with O sym operator and with non-operator DNA using filter binding and DNA cellulose chromatography, respectively. For both specific and non-specific binding of lac R, K obs at fixed salt concentration [KX] increases when chloride (Cl−) is replaced by the physiological anion glutamate (Glu−). At 0.25 m-KX. the increase in K obs for O sym is observed to be $ ̃ 40-fold, whereas for non-operator DNA the increase in K obs is estimated by extrapolation to be 3 ̃ 00-fold. For non-operator DNA, SK obs RD is independent of salt concentration within experimental uncertainty, and is similar in KCl (SK obs, KCl RD=− 9.8 (±1.0) between 0.13 m and 0.18 m-KCl) and KGlu (SK obs, KGlu RD=− 9.3 (±0.7) between 0.23 m and 0.36 m-KGlu). For O sym DNA, SK obs RO varies significantly with the nature of the anion, and, at least in KGlu appears to decrease in magnitude with increasing [KGlu]. Average magnitudes of SK obs RO are less than SK obs RD, and, for specific binding decrease in the order¦ SK obs, KCl RO¦>¦ SK obs, KAc RO¦> SK obs, KGlu RO¦. Neither K obs RO nor SK obs RO is affected by the choice of univalent cation M+(Na+, K+, NH+ 4. or mixtures thereof, all as the chloride salt), and SK obs RO is independent of [MCl] in the range examined (0.125 to 0.3 m). This behavior of SK obs RO is consistent with that expected for a binding process with a large contribution from the polyelectrolyte effect. However, the lack of an effect of the nature of the cation on the magnitude of SK obs RO at a fixed [MX] is somewhat unexpected, in view of the order of preference of cations for the immediate vicinity of DNA (NH 4+> K+> Na+) observed by 23 Na nuclear magnetic resonance. For both specific and non-specific binding, the large stoichiometry of cation release from the DNA polyelectrolyte is the dominant contribution to SK obs RO. To interpret these data, we propose that Glu− is an inert anion, whereas Ac and Cl compete with DNA phosphate groups in binding to lac repressor. A thermodynamic estimate of the minimum stoichiometry of water release from lac repressor and O sym operator (210 (±30) H 2 O) is determined from analysis of the apparently significant reduction in¦ SK obs, KGlu RO¦ with increasing [KGlu] in the range 0.25 to 0.9 m. According to this analysis, SK obs values of specific arid non-specific binding in KGlu differ primarily because of the release of water in specific binding. In KAc and KCl, we deduce that anion competition affects K obs and SK obs to an extent which differs for different anions and for the different binding modes.