Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex
Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex
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DOI：
10.1021/bi9803123
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发表时间：
1998-05-26
期刊：
影响因子：
2.9
通讯作者：
Stayton, PS
中科院分区：
文献类型：
--
作者：
Klumb, LA;Chu, V;Stayton, PS
高亲和力的链霉亲和素-生物素复合物的特点是广泛的氢键网络。通过 Asn 23、Ser 27 和 Tyr 43 的定点突变，对生物素的脲基氧的氢键能量进行了研究。开发了一种新的竞争性生物素结合测定法，以提供 K-d 变化的直接平衡测量。相对于野生型链霉亲和素，S27A、Y43F、Y43A、N23A 和 N23E 突变体在 37°C 下显示的 Delta Delta G 度分别为 2.9、1.2、2.6、3.5 和 2.6 kcal/mol。通过等温滴定量热法测量所有突变体的平衡结合焓，Y43A和N23A突变体在25℃下表现出平衡结合焓大幅降低，分别为8.9和6.9 kcal/mol。与野生型相比，S27A 和 N23E 突变体的结合焓小幅降低，分别为 1.6 和 0.9 kcal/mol，而 Y43F 突变体在 25 ℃ 时的结合焓增加了 -2.6 kcal/mol。在 37 ℃ 时，Y43A 和 N23A 突变体分别表现出 7.8 和 7.9 kcal/mol 的降低，而相对于野生型，S27A、N23E 和 Y43F 突变体显示出 4.9、3.7 和 1.2 kcal/mol 的减少。还进行了动力学分析以探讨氢键对活化势垒的贡献。野生型链霉亲和素在 37°C 下的 k(off) 为 (4.1 +/- 0.3) x 10(-5) s(-1)，保守型 Y43F、S27A 和 N23A 突变体的 k(off) 增加至 (20 +/- 1) x 10(-5) s(-1)；分别为 (660 +/- 40) x 10(-5) s(-1) 和 (1030 +/- 220) x 10(-5) s(-1)。 Y43A 和 N23E 突变体的 k(off) 分别增加了 93 倍和 188 倍。通过解离速率温度依赖性的过渡态分析确定每个突变体的活化能和焓。除 Y43F 之外的所有突变体均表现出活化焓的大幅降低。 Y43F突变体具有更正的活化焓，因此具有更有利的活化熵，这是活化势垒整体降低的基础。对于每个脲基氧氢键位置上最保守的突变体，结合态改变解释了单个过渡态模型中的大部分能量变化，表明脲基氧氢键相互作用在解离过渡态中被破坏。
The high-affinity streptavidin-biotin complex is characterized by an extensive hydrogen-bonding network. A study of hydrogen-bonding energetics at the ureido oxygen of biotin has been conducted with site-directed mutations at Asn 23, Ser 27, and Tyr 43. A new competitive biotin binding assay was developed to provide direct equilibrium measurements of the alterations in K-d. S27A, Y43F, Y43A, N23A, and N23E mutants display Delta Delta G degrees at 37 degrees C relative to wild-type streptavidin of 2.9, 1.2, 2.6, 3.5, and 2.6 kcal/mol, respectively. The equilibrium-binding enthalpies for all of the mutants were measured by isothermal titration calorimetry, and the Y43A and N23A mutants display large decreases in the equilibrium binding enthalpy at 25 degrees C of 8.9 and 6.9 kcal/mol, respectively. The S27A and N23E mutants displayed small decreases in binding enthalpy of 1.6 and 0.9 kcal/mol relative to wild-type, while the Y43F mutant displayed a -2.6 kcal/mol increase in the binding enthalpy at 25 degrees C. At 37 degrees C, the Y43A and N23A mutants display decreases of 7.8 and 7.9 kcal/mol, respectively, while the S27A, N23E, and Y43F mutants displayed decreases of 4.9, 3.7, and 1.2 kcal/mol relative to wild-type. Kinetic analyses were also conducted to probe the contributions of the hydrogen bonds to the activation barrier. Wild type streptavidin at 37 degrees C displays a k(off) of (4.1 +/- 0.3) x 10(-5) s(-1) and the conservative Y43F, S27A, and N23A mutants displayed increases in k(off) to (20 +/- 1) x 10(-5) s(-1); (660 +/- 40) x 10(-5) s(-1), and (1030 +/- 220) x 10(-5) s(-1), respectively. The Y43A and N23E mutants displayed 93-fold and 188-fold increases in k(off), respectively. Activation energies and enthalpies for each of the mutants were determined by transition-state analysis of the dissociation rate temperature dependence. All of the mutants except Y43F display large reductions in the activation enthalpy. The Y43F mutant has a more positive activation enthalpy, and thus a more favorable activation entropy that underlies the overall reduction in the activation barrier. For the most conservative mutant at each ureido oxygen hydrogen-bonding position, bound-state alterations account for most of the energetic changes in a single transition-state model, suggesting that the ureido oxygen hydrogen-bonding interactions are broken in the dissociation transition state.