Robinson C R, Sauer R T
Department of Biology, Massachusetts Institute of Technology, Cambridge MA 02139, USA.
Proc Natl Acad Sci U S A. 1998 May 26;95(11):5929-34. doi: 10.1073/pnas.95.11.5929.
Linker length and composition were varied in libraries of single-chain Arc repressor, resulting in proteins with effective concentrations ranging over six orders of magnitude (10 microM-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.
在单链Arc阻遏蛋白文库中，连接子的长度和组成有所变化，从而产生了有效浓度范围跨越六个数量级（10微摩尔至10摩尔）的蛋白质。具有生物活性需要11个或更多残基的连接子。平衡稳定性随连接子长度有很大变化，对于含有19个残基的富含甘氨酸的连接子达到最大值。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且有时相反的变化。通过将连接子长度固定为19个残基，并在一个16个残基的随机区域中改变丙氨酸/甘氨酸或丝氨酸/甘氨酸的比例，研究了连接子灵活性的影响。在这些文库中，似乎是组成而非序列决定稳定性。在连接子的随机区域中，对于含有11个丙氨酸和5个甘氨酸的蛋白质，在丙氨酸/甘氨酸文库中观察到最大稳定性。在丝氨酸/甘氨酸文库中，该区域最稳定的蛋白质有7个丝氨酸和9个甘氨酸。对折叠和去折叠速率进行分析表明，丙氨酸主要通过加速折叠起作用，而丝氨酸主要作用于减缓去折叠。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方面的重要作用，并提出了优化这些参数的策略。
