Optimizing the stability of single-chain proteins by linker length and composition mutagenesis
Optimizing the stability of single-chain proteins by linker length and composition mutagenesis
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DOI：
10.1073/pnas.95.11.5929
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发表时间：
1998-05-26
影响因子：
11.1
通讯作者：
Sauer, RT
中科院分区：
文献类型：
--
作者：
Robinson, CR;Sauer, RT
在单链ARE阻遏子文库中，连接子的长度和组成都不同，导致蛋白质的有效浓度在六个数量级(10muM-10M)之间变化。生物活性需要11个或更多残基的连接子。平衡稳定性随接头长度变化很大，对于含有19个残基的富含甘氨酸的接头达到最大值。连接子长度对平衡稳定性的影响源于折叠和去折叠动力学中显著的、有时是相反的变化。通过将连接子长度固定在19个残基并改变16个残基随机区中Ala/Gly或Ser/Gly的比例，考察了连接子灵活性的影响，在这些文库中，似乎是组成而不是序列决定了稳定性。在Ala/Gly文库中观察到在连接子的随机区包含11个丙氨酸和5个甘氨酸的蛋白质的最大稳定性。在Ser/Gly文库中，最稳定的蛋白质在该区域有7个丝氨酸和9个甘氨酸。对折叠和去折叠速率的分析表明，丙氨酸主要通过加速折叠来起作用，而丝氨酸主要通过减缓去折叠来起作用。这些结果证明了连接子设计在决定单链蛋白质的稳定性和折叠动力学中的重要作用，并提出了优化这些参数的策略。
Linker length and composition were varied in libraries of single-chain Are repressor, resulting in proteins with effective concentrations ranging over six orders of magnitude (10 mu M-10 M). Linkers of 11 residues or more were required for biological activity. Equilibrium stability varied substantially with linker length, reaching a maximum for glycine-rich linkers containing 19 residues. The effects of linker length on equilibrium stability arise from significant and sometimes opposing changes in folding and unfolding kinetics. By fixing the linker length at 19 residues and varying the ratio of Ala/Gly or Ser/Gly in a 16-residue-randomized region, the effects of linker flexibility were examined, In these libraries, composition rather than sequence appears to determine stability. Maximum stability in the Ala/Gly library was observed for a protein containing 11 alanines and five glycines in the randomized region of the linker. In the Ser/Gly library, the most stable protein had seven serines and nine glycines in this region. Analysis of folding and unfolding rates suggests that alanine acts largely by accelerating folding, whereas serine acts predominantly to slow unfolding. These results demonstrate an important role for linker design in determining the stability and folding kinetics of single-chain proteins and suggest strategies for optimizing these parameters.