Computational protein design can be used to select sequences that are compatible with a fixed-backbone template. This strategy has been used in numerous instances to engineer novel proteins. However, the fixed-backbone assumption severely restricts the sequence space that is accessible via design. For challenging problems, such as the design of functional proteins, this may not be acceptable. Here, we present a method for introducing backbone flexibility into protein design calculations and apply it to the design of diverse helical BH3 ligands that bind to the anti-apoptotic protein Bcl-xL, a member of the Bcl-2 protein family. We demonstrate how normal mode analysis can be used to sample different BH3 backbones, and show that this leads to a larger and more diverse set of low-energy solutions than can be achieved using a native high-resolution Bcl-xL complex crystal structure as a template. We tested several of the designed solutions experimentally and found that this approach worked well when normal mode calculations were used to deform a native BH3 helix structure, but less well when they were used to deform an idealized helix. A subsequent round of design and testing identified a likely source of the problem as inadequate sampling of the helix pitch. In all, we tested 17 designed BH3 peptide sequences, including several point mutants. Of these, eight bound well to Bcl-xL and four others showed weak but detectable binding. The successful designs showed a diversity of sequences that would have been difficult or impossible to achieve using only a fixed backbone. Thus, introducing backbone flexibility via normal mode analysis effectively broadened the set of sequences identified by computational design, and provided insight into positions important for binding Bcl-xL.

译文

:计算蛋白设计可用于选择与固定骨架模板兼容的序列。这种策略已在许多情况下用于工程化新型蛋白质。但是,固定骨干网的假设严重限制了可通过设计访问的序列空间。对于具有挑战性的问题,例如功能蛋白的设计,这可能是不可接受的。在这里,我们介绍了一种在蛋白质设计计算中引入骨架灵活性的方法,并将其应用于与抗凋亡蛋白Bcl-xL(Bcl-2蛋白家族的成员)结合的各种螺旋BH3配体的设计。我们演示了如何使用正常模式分析来采样不同的BH3骨架,并表明与使用天然高分辨率Bcl-xL复杂晶体结构来获得低能解决方案相比,这导致了更大,更多样化的一组低能解决方案。模板。我们通过实验测试了几种设计的解决方案,发现当使用普通模式计算使天然BH3螺旋结构变形时,此方法效果很好,但当用于使理想化螺旋变形时效果不佳。随后的一轮设计和测试确定了问题的可能根源是螺旋螺距的采样不足。我们总共测试了17个设计的BH3肽序列,包括几个点突变体。其中,八个与Bcl-xL的结合良好,另外四个则显示较弱但可检测的结合。成功的设计表明,仅使用固定的骨架很难或不可能实现的序列多样性。因此,通过正常模式分析引入骨架灵活性有效地拓宽了通过计算设计识别的序列集,并提供了对结合Bcl-xL重要的位置的见解。

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