Adam Broerman

Protein interaction kinetics often affect the dynamic behavior of biological systems. While high affinity interactions are useful, they usually exchange slowly due to their low off-rates. We developed a general strategy for inducing rapid exchange of high affinity interactions.

蛋白质相互作用动力学通常影响生物系统的动态行为。虽然高亲和力相互作用很有用，但由于它们的解离速率低，它们通常交换得很慢。我们开发了一种通用策略来诱导高亲和力相互作用的快速交换。

Adam Broerman

We designed an effector-responsive conformational switch which can be attached to almost any binder to sterically modulate binding its partner. Binding the effector induces a strained intermediate state from which partner dissociation is accelerated by up to thousands-fold.

我们设计了一种效应子响应的构象开关，它可以附加到几乎所有的结合分子上，通过空间位阻调节其与伙伴的结合。结合效应子会诱导产生一个紧张的中间态，通过这种状态，伙伴的解离速度可以加速数千倍。

Adam Broerman

Most efficiently accessing this strained state requires that the conformational switch exerts force in a power stroke. To obtain this behavior, we used a flexible effector and designed an open cleft in the unstrained state where the effector can bind to initiate the switch.

为了最有效地达到这种紧张状态，需要构象开关施加力量来进行动力冲程。为了获得这种行为，我们使用了一种柔性效应子，并设计了一个在未受力状态下的开放裂缝，效应子可以在此结合以启动开关。

Adam Broerman

With the partner bound, we measured the base rate of conformational switching and found that the rates of effector binding and subsequent partner dissociation surpass this, confirming an induced-fit mechanism for effector association.

在伙伴结合的情况下，我们测量了构象转换的基础速率，并发现效应子结合和随后伙伴解离的速率超过了这个基础速率，从而证实了效应子结合的诱导契合机制。

Adam Broerman

By designing the magnitude and direction of the induced strain, we could control the partner dissociation kinetics over a wide range. Notably, simple geometric calculations on the AlphaFold predictions of the strained states predicted the dissociation kinetics with an R² of 0.6.

通过设计引入应变的大小和方向，我们可以在很宽的范围内控制伙伴解离动力学。值得注意的是，基于AlphaFold对紧张状态的预测进行简单的几何计算，预测了解离动力学，其决定系数R²为0.6。

Adam Broerman

We solved crystal structures of all major equilibrium states of one design, confirming accurate design of these complex multistate proteins. The structure of the strained ternary state(!) shows how the entire complex strains to resolve the designed structural clash.

我们解析了一种设计的所有主要平衡状态的晶体结构，确认了这些复杂的多状态蛋白设计的准确性。紧张的三元状态的结构（！）展示了整个复合体是如何应变以解决设计的构象冲突的。

Adam Broerman

This facilitated dissociation behavior can be used to rapidly switch split enzymes and to rapidly release kinetic traps in chain reactions, which would be potentially useful for constructing kinetically governed protein circuits.

这种促进解离的行为可以用于快速切换分裂酶，并在链式反应中快速释放动力学陷阱，这可能对于构建由动力学控制的蛋白质电路非常有用。

Adam Broerman

I also used facilitated dissociation to make biosensors which are just as modular as those built previously with our LOCKR platform, but which respond 70 times faster.

我们还利用促进解离技术制造了生物传感器，这些传感器与我们之前使用LOCKR平台构建的传感器一样模块化，但响应速度提高了70倍。

Adam Broerman

Finally, the residence time of cytokines stimulating their receptors modulates the downstream response, but native pathways for signal termination are slow, so this is difficult to control. We constructed cytokines which can be disengaged from their receptors far faster.

最终，细胞因子与其受体的结合时间调节了下游反应，但是信号终止的自然途径较慢，因此很难控制。我们构建了能够从其受体上更快解离的细胞因子。

Adam Broerman

Christoph showed that with this switchable cytokine, adding the effector completely reverses receptor dimerization on the cell surface within 10 seconds and immediately blocks accumulation of phosphoSTAT5.

Christoph展示了使用这种可切换的细胞因子，添加效应子在10秒内就能完全逆转细胞表面的受体二聚化，并立即阻断了磷脂酰肌醇信号转导子5（phosphoSTAT5）的积累。

Adam Broerman

generality of our approach: by fusing to our switch, almost any binder can be made to rapidly dissociate from its target!

我们方法的普适性：通过与我们的开关融合，几乎所有的结合分子都可以被设计成从其靶标上快速解离！

We are really excited to see the new avenues this will open up! If you have ideas that build upon this work and would like to discuss, please reach out. A few we have in mind:

我们对这将开辟的新途径感到非常兴奋！如果你有基于这项工作的构想，并希望讨论，请与我们联系。我们心中有几个想法：

We suspect that the effector folds upon binding, and that this flexibility is key for rapid conformational transitions against resisting force. If you are interested in collaborating to explore the link between flexibility and force generation in these systems, please reach out!

我们怀疑效应子在结合时会发生折叠，而这种灵活性是快速构象转变对抗阻力产生力的关键。如果你对探索这些系统中灵活性与力生成之间的联系感兴趣，请与我们联系！

Also, if you have ideas for how these switchable cytokines can modulate biological systems in new ways, we would love to discuss/collaborate! We envision these tools could open up a whole new way to explore the biology of transient cytokine signaling.

此外，如果你们有关于这些可切换的细胞因子如何以新的方式调节生物系统的想法，我们非常愿意讨论/合作！我们设想这些工具可以开辟一种全新的方法来探索瞬时细胞因子信号传导的生物学。

This was a massive collaborative effort with co-authors, Huge thanks to all of you!

感谢所有合作者，非常感谢你们！