Fragment complementation studies of protein stabilization by hydrophobic core residues
Fragment complementation studies of protein stabilization by hydrophobic core residues
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DOI：
10.1021/bi0014812
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发表时间：
2001-02-06
期刊：
影响因子：
2.9
通讯作者：
Linse, S
中科院分区：
文献类型：
--
作者：
Berggård, T;Julenius, K;Linse, S
通过测量具有和不具有特定位点残基替换的亚域片段之间的亲和力，研究了稳定蛋白质天然状态的相互作用。以Calbindin D-9k变异体为研究起点，在其两个EF-Hand亚域之间的43位具有单一的CNBR裂解位点。在这个变体中引入了Il位点特异性取代，涉及到两个EF-手之间的界面上的疏水核心残基。将突变体用CNBr切割，产生野生型和突变的单手Ef片段：EF1(残基1-43)和EF2(残基44-75)。用表面等离子体共振(SPR)技术研究了两个EF-手之间的相互作用，该技术跟踪了络合物的缔合和解离速率。将野生型EF1固定在葡聚糖基质上，以不同浓度注射野生型和突变型EF2。在另一组实验中，将野生型EF2固定并注射野生型或突变型EF1。解离速率常数在1.1×10~(-5)~1.0×10~(-2)之间，S在~(-1)之间，缔合速率常数在2×10~(-5)~4.0×10~(-6)M~(-1)S~(-1)之间。在无突变的情况下，EF1与EF2的亲和力高达3.6×10(11)M-1。对于11个疏水核心突变体，EF1对EF2的亲和力与相应完整蛋白的变性稳定性之间存在很强的相关性(r=0.999)。观察到的相关性表明，控制完整蛋白质稳定性的因素也有助于双分子EF1-EF2复合体的亲和力。此外，这里提供的数据表明，疏水核心残基之间的相互作用是两个EF-Hand亚结构域之间亲和力和完整结构域稳定性的主要贡献因素。
Interactions that stabilize the native state of a protein have been studied by measuring the affinity between subdomain fragments with and without site-specific residue substitutions. A calbindin D-9k variant with a single CNBr cleavage site at position 43 between its two EF-hand subdomains was used as a starting point for the study. Into this variant were introduced Il site-specific substitutions involving hydrophobic core residues at the interface between the two EF-hands. The mutants were cleaved with CNBr to produce wild-type and mutated single-EF-hand fragments: EF1 (residues 1-43) and EF2 (residues 44-75). The interaction between the two EF-hands was studied using surface plasmon resonance (SPR) technology, which follows the rates of association and dissociation of the complex. Wild-type EF1 was immobilized on a dextran matrix, and the wild-type and mutated versions of EF2 were injected at several different concentrations. In another set of experiments, wild-type EF2 was immobilized and wild-type or mutant EF1 was injected. Dissociation rate constants ranged between 1.1 x 10(-5) and 1.0 x 10(-2) s(-1) and the association rate constants between 2 x 10(5) and 4.0 x 10(6) M-1 s(-1). The affinity between EF1 and EF2 was as high as 3.6 x 10(11) M-1 when none of them was mutated. For the 11 hydrophobic core mutants, a strong correlation (r = 0.999) was found between the affinity of EF1 for EF2 and the stability toward denaturation of the corresponding intact protein. The observed correlation implies that the factors governing the stability of the intact protein also contribute to the affinity of the bimolecular EF1-EF2 complex. In addition, the data presented here show that interactions among hydrophobic core residues are major contributors both to the affinity between the two EF-hand subdomains and to the stability of the intact domain.