Thermodynamics of Fab-ssDNA interactions: contribution of heavy chain complementarity determining region 3.
Thermodynamics of Fab-ssDNA interactions: contribution of heavy chain complementarity determining region 3.
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Fab-ssDNA 相互作用的热力学：重链互补决定区 3 的贡献。
DOI：
10.1021/bi991347l
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发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Deutscher,SL
中科院分区：
文献类型：
--
作者：
Komissarov,AA;Deutscher,SL
选择重组抗ssDNA Fab、DNA-1和16重链互补决定区3（HCDR 3）突变体变体用于ssDNA结合的热力学表征。平衡荧光猝灭滴定法测定不同温度和阳离子浓度下Fab与（dT）15的亲和力。测定了标准吉布斯自由结合能（ΔG°）、焓（ΔH°）、熵（ΔS°）和相互作用时形成的离子对数量（Z）的变化。所有Fab都具有与ssDNA相互作用的亲核性质，这与先前报道的熵驱动的与dsDNA的结合相反[Tanha，J.，和Lee，J.S.（1997）Nucleic Acids Res.25，1442 - 1449]。研究了HCDR 3的单独残基对ssDNA相互作用的贡献。对HCDR 3中取代引起的ΔH°和T ΔS°变化的分析表明，HCDR 3中存在完全的熵/焓补偿。HCDR 3环末端的突变R98 A和D108 A分别使T ΔS°增加10.4和15.9 kcal/mol。在HCDR 3的顶部用脯氨酸取代精氨酸导致与（dT）15的新的静电接触。在抗ssDNA结合位点处观察到的Z和非静电相互作用的ΔG°（ΔG°nonel）的线性相关性被用于估计该Fab家族的特定ΔG°nonel[−20至−25 cal/（mol·mol 2）]、平均接触面积（450−550 μ mol 2）、最大Z（6−7）和标准阳离子浓度下的亲和力极限[（0.5−1）× 108 M-1]。结果表明，HCDR 3的合理工程改造可以用来控制抗体-DNA相互作用的亲和力和可能的特异性。
The recombinant anti-ssDNA Fab, DNA-1, and 16 heavy chain complementarity determining region 3 (HCDR3) mutant variants were selected for thermodynamic characterization of ssDNA binding. The affinity of Fab to (dT)15under different temperatures and cation concentrations was measured by equilibrium fluorescence quenching titration. Changes in the standard Gibbs free binding energy (ΔG°), enthalpy (ΔH°), entropy (ΔS°), and the number of ionic pairs (Z) formed upon interaction were determined. All Fab possessed an enthalpic nature of interaction with ssDNA, that was opposite to the previously reported entropically driven binding to dsDNA [Tanha, J., and Lee, J. S. (1997)Nucleic Acids Res. 25, 1442−1449]. The contribution of separate residues of HCDR3 to ssDNA interaction was investigated. Analysis of the changes in ΔH° andTΔS°, induced by substitutions in HCDR3, revealed a complete entropy/enthalpy compensation. Mutations R98A and D108A at the ends of the HCDR3 loop produced increases inTΔS°by 10.4 and 15.9 kcal/mol, respectively. Substitution of proline for arginine at the top of HCDR3 resulted in a new electrostatic contact with (dT)15. The observed linear correlation ofZand ΔG°of nonelectrostatic interactions (ΔG°nonel) at the anti-ssDNA combining site was used for the estimation of the specific ΔG°nonel[−20 to −25 cal/(mol·Å2)], the average contact area (450−550 Å2), the maximalZ(6−7), and the limit in affinity under standard cation concentrations [(0.5−1) × 108M-1] for this family of Fab. Results suggested that rational engineering of HCDR3 could be utilized to control the affinity and likely the specificity of Ab−DNA interactions.