Structural characteristics of the nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein. Studies with ribose and base-modified fluorescent nucleotide analogs.
Structural characteristics of the nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein. Studies with ribose and base-modified fluorescent nucleotide analogs.
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DOI：
10.1021/bi00181a028
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发表时间：
1994-04
期刊：
影响因子：
2.9
通讯作者：
W. Bujalowski;M. Klonowska
中科院分区：
文献类型：
--
作者：
W. Bujalowski;M. Klonowska
使用碱基修饰的荧光核苷酸类似物 1、N6-乙烯基腺苷二磷酸 (ε ADP) 和核糖修饰的荧光类似物，研究了大肠杆菌初级复制解旋酶 DnaB 蛋白的高亲和力非相互作用核苷酸结合位点的碱基和核糖结合区域的结构特征3'(2')-O-(N-甲基邻苯甲酰基)腺苷5'-二磷酸(MANT-ADP)、3'-O-(N-甲基邻苯甲酰基)脱氧腺苷5'-二磷酸(MANT-dADP)、3'-O-(N-甲基邻苯甲酰基)-脱氧腺苷5'-三磷酸(MANT-dATP)，和2'(3')-O-(2,4,6-三硝基苯基)腺苷5'-二磷酸(TNP-ADP)。获得的数据表明这两个区域之间存在对比差异。 epsilon ADP 与 DnaB 解旋酶的结合仅导致核苷酸荧光强度增加约 21%，并且发射光谱最大值没有变化。结合的 epsilon ADP 的荧光特征在于单一寿命为 24.2 +/- 0.6 ns，仅略短于溶液中游离 epsilon ADP 的荧光寿命 (25.5 +/- 0.6 ns)。使用不同的猝灭剂、丙烯酰胺、I-和Tl+对结合的ε ADP进行溶质猝灭研究表明乙烯腺苷对溶剂的可及性有限。这些结果强烈表明 DnaB 核苷酸结合位点的碱基结合区域位于酶表面的极裂中。此外，与相同激发波长（lambda ex = 325 nm，lambda em = 410 nm）下完全固定的 epsilon ADP 的各向异性为 0.3 相比，结合的 epsilon ADP 的极限发射各向异性为 0.21 +/- 0.02，表明腺嘌呤在结合到碱基结合位点时保留了相当大的迁移率。相比之下，TNP-ADP 和 MANT-ADP（其核糖 2' 和/或 3' 氧原子上连接有修饰基团）在发射最大值时的荧光强度在与 DnaB 结合后分别增加约 4.7（lambda ex = 408 nm）和约 2.6（lambda ex = 356 nm）。此外，结合的TNP-ADP的发射光谱的最大值蓝移约11 nm，MANT-ADP的发射光谱的最大值蓝移约12 nm。与 DnaB 结合的 TNP-ADP 和 MANT-ADP 在不同溶剂中的光谱特性之间的比较表明，DnaB 核苷酸结合位点的核糖结合区域具有相对较低的极性。使用丙烯酰胺、I- 和 Tl+ 对 MANT-ADP 荧光进行的溶质猝灭研究表明，当与 DnaB 结合时，MANT 基团几乎无法接近溶剂。总而言之，这些结果表明核糖结合区域构成了一个疏水裂口或口袋，与溶剂的接触（如果有的话）非常有限。（摘要截断为 400 字）
Structural characteristics of the base- and ribose-binding regions of the high-affinity noninteracting nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein have been studied, using the base-modified fluorescent nucleotide analog 1, N6-ethenoadenosine diphosphate (epsilon ADP) and the ribose-modified fluorescent analogs 3'(2')-O-(N-methylantraniloyl)adenosine 5'-diphosphate (MANT-ADP), 3'-O-(N-methylantraniloyl)deoxyadenosine 5'-diphosphate (MANT-dADP), 3'-O-(N-methylantraniloyl)-deoxyadenosine 5'-triphosphate (MANT-dATP), and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP). The obtained data indicate contrasting differences between these two regions. Binding of epsilon ADP to the DnaB helicase causes only approximately 21% increase of the nucleotide fluorescence intensity and no shift of the emission spectrum maximum. The fluorescence of bound epsilon ADP is characterized by a single lifetime of 24.2 +/- 0.6 ns, only slightly shorter than the fluorescent lifetime of the free epsilon ADP in solution (25.5 +/- 0.6 ns). Solute-quenching studies of bound epsilon ADP, using different quenchers, acrylamide, I-, and Tl+, indicate limited accessibility of ethenoadenosine to the solvent. These results strongly suggest that the base-binding region of the DnaB nucleotide-binding site is located in the polar cleft on the enzyme's surface. Moreover, the limiting emission anisotropy of bound epsilon ADP is 0.21 +/- 0.02, compared to the anisotropy of 0.3 of completely immobilized epsilon ADP at the same excitation wavelength (lambda ex = 325 nm, lambda em = 410 nm), indicating that the adenine preserves substantial mobility when bound in the base-binding site. In contrast, fluorescence intensity at the emission maximum of TNP-ADP and MANT-ADP, which has modifying groups attached to the 2' and/or 3' oxygens of the ribose, increases upon binding to DnaB by factors of approximately 4.7 (lambda ex = 408 nm) and approximately 2.6 (lambda ex = 356 nm), respectively. Moreover, the maximum of emission spectrum of bound TNP-ADP is blue-shifted by approximately 11 nm and that of MANT-ADP by approximately 12 nm. Comparisons between spectral properties of TNP-ADP and MANT-ADP bound to DnaB and in different solvents suggest that the ribose-binding region of the DnaB nucleotide-binding site has relatively low polarity. Solute quenching studies of MANT-ADP fluorescence, using acrylamide, I-, and Tl+, indicate that the MANT group has very little accessibility to the solvent when bound to DnaB. Taken together, these results suggest that the ribose-binding region constitutes a hydrophobic cleft, or pocket, with very limited, if any, contact with the solvent.(ABSTRACT TRUNCATED AT 400 WORDS)