Close proximity of tryptophan residues and ATP-binding site in Escherichia coli primary replicative helicase DnaB protein. Molecular topography of the enzyme.
Close proximity of tryptophan residues and ATP-binding site in Escherichia coli primary replicative helicase DnaB protein. Molecular topography of the enzyme.
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DOI：
10.1016/s0021-9258(18)31702-2
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发表时间：
1994-12
期刊：
影响因子：
--
通讯作者：
W. Bujalowski;Malgorzata Maria Klonowska
中科院分区：
文献类型：
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作者：
W. Bujalowski;Malgorzata Maria Klonowska
荧光核苷酸类似物与大肠杆菌初级复制解旋酶DnaB蛋白的结合导致蛋白色氨酸荧光的强烈猝灭。它是由从蛋白质聚糖到结合在核苷酸结合位点的类似物的有效荧光能量转移（E）引起的，表明蛋白质聚糖在紧密接近结合位点处“成簇”。这是相反的，缺乏可检测的能量转移到荧光单链DNA（ssDNA）衍生物，这表明一个遥远的分离之间的两个功能连接的结构元件的酶，核苷酸和ssDNA结合位点。E对结合的核苷酸/DnaB六聚体的平均数的依赖是非线性的，这意味着在低亲和力位点中，寡核苷酸和结合的核苷酸之间有较大的分离。光谱研究表明，色氨酸残基位于DnaB解旋酶的表面上的疏水裂缝，而酪氨酸的环境是异质性的，与6的10个酪氨酸残基位于解旋酶的表面上。荧光能量从酪氨酸转移到色氨酸的效率表明，残基的“质量中心”是分离的，可能反映了核苷酸和ssDNA结合位点的分离，酪氨酸构成ssDNA结合区的一部分。
The binding of fluorescent nucleotide analogs to the Escherichia coli primary replicative helicase DnaB protein causes strong quenching of protein tryptophan fluorescence. It results from the efficient fluorescence energy transfer (E) from tryptophans to analogs bound in the nucleotide-binding site, indicating that protein tryptophans are “clustered” in close proximity to the binding site. This is in contrast to the lack of detectable energy transfer to the fluorescent single-stranded DNA (ssDNA) derivative, suggesting a distant separation between two function-linked structural elements of the enzyme, the nucleotide- and ssDNA-binding sites. The dependence of E upon the average number of bound nucleotides/DnaB hexamer is nonlinear, implying a larger separation between tryptophans and the bound nucleotide in the low affinity sites. Spectroscopic studies reveal that tryptophan residues are located on the surface of the DnaB helicase in a hydrophobic cleft, whereas the environment of the tyrosines is heterogeneous, with 6 out of 10 tyrosine residues located on the surface of the helicase. The efficiency of the fluorescence energy transfer from the tyrosines to tryptophans suggests that the “centers of mass” of the residues are separated, possibly reflecting the separation of the nucleotide- and ssDNA-binding sites, with tyrosines constituting part of the ssDNA-binding region.