O'Neill J W, Kim D E, Johnsen K, Baker D, Zhang K Y
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA.
Structure. 2001 Nov;9(11):1017-27. doi: 10.1016/s0969-2126(01)00667-0.
Thermodynamic and kinetic studies of the Protein L B1 domain (Ppl) suggest a folding pathway in which, during the folding transition, the first beta hairpin is formed while the second beta hairpin and the alpha helix are largely unstructured. The same mutations in the two beta turns have opposite effects on the folding and unfolding rates. Three of the four residues composing the second beta turn in Ppl have consecutive positive phi angles, indicating strain in the second beta turn.
We have determined the crystal structures of the beta turn mutants G55A, K54G, and G15A, as well as a core mutant, V49A, in order to investigate how backbone strain affects the overall structure of Ppl. Perturbation of the hydrophobic interactions at the closed interface by the V49A mutation triggered the domain swapping of the C-terminal beta strand that relieved the strain in the second beta turn. Interestingly, the asymmetric unit of V49A contains two monomers and one domain-swapped dimer. The G55A mutation escalated the strain in the second beta turn, and this increased strain shifted the equilibrium toward the domain-swapped dimer. The K54G structure revealed that the increased stability is due to the reduction of strain in the second beta turn, while the G15A structure showed that increased strain alone is insufficient to trigger domain swapping.
Domain swapping in Ppl is determined by the balance of two opposing components of the free energy. One is the strain in the second beta turn that favors the dimer, and the other is the entropic cost of dimer formation that favors the monomer. A single-site mutation can disrupt this balance and trigger domain swapping.
对蛋白L B1结构域（Ppl）的热力学和动力学研究表明，其折叠途径为：在折叠转变过程中，第一个β发夹形成，而第二个β发夹和α螺旋在很大程度上是无结构的。两个β转角中的相同突变对折叠和去折叠速率有相反的影响。构成Ppl中第二个β转角的四个残基中的三个具有连续的正φ角，表明第二个β转角存在应变。
我们确定了β转角突变体G55A、K54G和G15A以及一个核心突变体V49A的晶体结构，以研究主链应变如何影响Ppl的整体结构。V49A突变对封闭界面处疏水相互作用的扰动引发了C端β链的结构域交换，从而缓解了第二个β转角中的应变。有趣的是，V49A的不对称单元包含两个单体和一个结构域交换二聚体。G55A突变加剧了第二个β转角中的应变，这种增加的应变使平衡向结构域交换二聚体移动。K54G结构表明稳定性增加是由于第二个β转角中应变的降低，而G15A结构表明仅增加应变不足以触发结构域交换。
Ppl中的结构域交换由自由能两个相反组分的平衡决定。一个是有利于二聚体的第二个β转角中的应变，另一个是有利于单体的二聚体形成的熵成本。单点突变可以破坏这种平衡并触发结构域交换。
