Klumb L A, Chu V, Stayton P S
Department of Bioengineering, University of Washington, Seattle 98195, USA.
Biochemistry. 1998 May 26;37(21):7657-63. doi: 10.1021/bi9803123.
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 Kd. S27A, Y43F, Y43A, N23A, and N23E mutants display DeltaDeltaG 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 koff of (4.1 +/- 0.3) x 10(-5) s-1, and the conservative Y43F, S27A, and N23A mutants displayed increases in koff 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 koff, 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.
高亲和力的链霉亲和素 - 生物素复合物的特征在于广泛的氢键网络。通过对Asn 23、Ser 27和Tyr 43进行定点突变，对生物素脲基氧处的氢键能量学进行了研究。开发了一种新的竞争性生物素结合测定法，以提供Kd变化的直接平衡测量。S27A、Y43F、Y43A、N23A和N23E突变体在37℃时相对于野生型链霉亲和素的ΔΔG°分别为2.9、1.2、2.6、3.5和2.6千卡/摩尔。通过等温滴定量热法测量了所有突变体的平衡结合焓，Y43A和N23A突变体在25℃时的平衡结合焓分别大幅下降8.9和6.9千卡/摩尔。相对于野生型，S27A和N23E突变体的结合焓小幅下降，分别为1.6和0.9千卡/摩尔，而Y43F突变体在25℃时的结合焓增加了 -2.6千卡/摩尔。在37℃时，Y43A和N23A突变体的结合焓分别下降7.8和7.9千卡/摩尔，而S27A、N23E和Y43F突变体相对于野生型的结合焓分别下降4.9、3.7和1.2千卡/摩尔。还进行了动力学分析，以探究氢键对活化能垒的贡献。37℃时野生型链霉亲和素的koff为(4.1±0.3)×10⁻⁵ s⁻¹，保守的Y43F、S27A和N23A突变体的koff分别增加到(20±1)×10⁻⁵ s⁻¹、(660±40)×10⁻⁵ s⁻¹和(1030±220)×10⁻⁵ s⁻¹。Y43A和N23E突变体的koff分别增加了93倍和188倍。通过对解离速率温度依赖性的过渡态分析，确定了每个突变体的活化能和活化焓。除Y43F外，所有突变体的活化焓都大幅降低。Y43F突变体具有更正的活化焓，因此具有更有利的活化熵，这是活化能垒总体降低的基础。对于每个脲基氧氢键位置最保守的突变体，在单一过渡态模型中，结合态变化占能量变化的大部分，这表明脲基氧氢键相互作用在解离过渡态中被打破。
