Perez-Howard G M, Weil P A, Beechem J M
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
Biochemistry. 1995 Jun 27;34(25):8005-17. doi: 10.1021/bi00025a006.
A combination of steady-state, stopped-flow, and time-resolved fluorescence of intrinsic tryptophan and extrinsically labeled fluorescent DNA is utilized to examine the interaction of yeast TATA binding protein (TBP) with DNA. TBP is composed of two structural domains, the carboxy domain (residues 61-240), which is responsible for DNA binding and initiation of basal level transcription, and an amino terminal domain (residues 1-60), whose function is currently unknown. The steady-state fluorescence emission spectrum of the single tryptophan in the amino terminal domain of TBP undergoes a huge (30-40 nm) red-shift upon interaction with stoichiometric amounts of TATA box containing DNA. From time-resolved tryptophan fluorescence anisotropy studies, we demonstrate that, in the absence of DNA, the protein exists as a multimer in solution and it contains (at least) two primary conformations, one with the amino terminus associated tightly with the protein(s) in a hydrophobic environment and one with the amino terminus decoupled away from the rest of the protein and solvent-exposed. Upon binding DNA, the protein dissociates into a monomeric complex, upon which only the solvent-exposed amino terminus conformation remains. Kinetic and equilibrium binding studies were performed on TATA box containing DNA which was extrinsically labeled with a fluorescent probe Rhodamine-X at the 5'-end. This "fluorescent" DNA allowed for the collection of quantitative spectroscopic binding, kinetic on-rate, and kinetic off-rate data at physiological concentrations. Global analysis of equilibrium binding studies performed from 500 pM to 50 nM DNA reveals a single dissociation constant (Kd) of approximately 5 nM. Global analysis of stopped-flow anisotropy on-rate experiments, with millisecond timing resolution and TBP concentrations ranging from 20 to 600 nM (20 nM DNA), can be perfectly described by a single second-order rate constant of 1.66 x 10(5) M(-1) s(-1). These measurements represent the very first stopped-flow anisotropy study of a protein/DNA interaction. Stopped-flow anisotropy off-rate experiments reveal a single exponential k(off) of 4.3 x 10(-2) min-1 (1/k(off) = 23 min) From the ratio of on-rate to off-rate, a predicted Kd of 4.3 nM is obtained, revealing that the kinetic and equilibrium studies are internally consistent. Deletion of the amino terminal domain of TBP decreases the k(on) of TBP approximately 45-fold and eliminates classic second-order behavior.
利用稳态、停流以及内在色氨酸和外在标记荧光DNA的时间分辨荧光相结合的方法，来研究酵母TATA结合蛋白（TBP）与DNA的相互作用。TBP由两个结构域组成，羧基结构域（第61 - 240位氨基酸残基）负责DNA结合和基础水平转录的起始，而氨基末端结构域（第1 - 60位氨基酸残基），其功能目前尚不清楚。TBP氨基末端结构域中单个色氨酸的稳态荧光发射光谱在与化学计量的含TATA框的DNA相互作用时会发生巨大的（30 - 40纳米）红移。从时间分辨色氨酸荧光各向异性研究中，我们证明，在没有DNA的情况下，该蛋白在溶液中以多聚体形式存在，并且它包含（至少）两种主要构象，一种构象中氨基末端在疏水环境中与蛋白紧密结合，另一种构象中氨基末端与蛋白的其余部分分离并暴露于溶剂中。在结合DNA后，该蛋白解离成单体复合物，此时仅保留暴露于溶剂中的氨基末端构象。对在5'端用荧光探针罗丹明 - X进行外在标记的含TATA框的DNA进行了动力学和平衡结合研究。这种“荧光”DNA使得能够在生理浓度下收集定量光谱结合、动力学结合速率和动力学解离速率数据。对500皮摩尔到50纳摩尔DNA进行的平衡结合研究的全局分析显示，单一解离常数（Kd）约为5纳摩尔。对停流各向异性结合速率实验进行全局分析，时间分辨率为毫秒，TBP浓度范围为20到600纳摩尔（20纳摩尔DNA），可以用单一的二级速率常数1.66×10⁵ M⁻¹ s⁻¹完美描述。这些测量代表了对蛋白质/DNA相互作用的首次停流各向异性研究。停流各向异性解离速率实验显示单一指数形式的解离速率常数k(off)为4.3×10⁻² 分钟⁻¹（1/k(off) = 23分钟）。根据结合速率与解离速率的比值，得到预测的Kd为4.3纳摩尔，表明动力学和平衡研究在内部是一致的。删除TBP的氨基末端结构域会使TBP的结合速率常数k(on)降低约45倍，并消除经典的二级行为。
