Biophysics of biological transport and signaling "nanomachines": from theory to applications

活细胞的功能依赖于大量的分子运输和信号“生物机器”。这些自然生物机器的功能原理--通过进化得到优化--指导了用于纳米和生物技术应用的人工仿生装置和纳米材料的创造。该提案的重点是一种被称为核孔复合物（NPC）的“纳米机器”，以及相关的仿生装置和生物材料。NPC通过选择性地运输特定的大分子复合物同时有效地过滤其他大分子复合物来协调和门控细胞核和细胞质之间的大分子运输。同样重要的是，它参与维持细胞核和细胞质之间大分子的精确时空分配，通过与运输过程耦合的化学反应的能量输入之间的非平衡，逆着它们的浓度梯度浓缩货物。NPC是一个非常大的运输机，内部组织复杂。其功能机制的关键组成部分是聚合物样的内在无序蛋白质的组装，这些蛋白质填充其转运通道并决定转运的特异性和通量。与许多其他分子转运蛋白不同，NPC的转运是大规模平行的，它的通道被数百种不同类型的运载货物的转运蛋白从两个方向穿过。NPC如何在如此拥挤的条件下保持高通量和选择性而不堵塞仍然是一个谜。此外，到目前为止，NPC及其组分的生物物理学研究主要集中在易位的分子物理学和NPC组分的生物物理学上。相比之下，仍然不清楚复杂的纳米级结构和动力学如何与非平衡能量消耗机制协同工作，以产生相对于分子扰动和噪声稳健的分子梯度。理论和计算方法已被证明是不可缺少的在NPC的研究。尽管它的复杂性，NPC的结构和功能的许多方面可以从基本的物理原理来理解。因此，NPC功能的许多特性已经在纳米通道模拟物中重现，从而提供理论模型的验证。然而，一些困惑和开放的问题仍然存在。该提案的目标是缩小我们对NPC定量理解的这些差距，为理解各种健康和疾病过程奠定基础，并指导仿生设备的合理设计。在此过程中，本研究将解决几个基本的物理问题，如多物种相分离在纳米约束，通过复杂的分子组装的分子输运机制，以及在纳米尺度上的平衡和非平衡过程的耦合。

The functioning of living cells relies on a multitude of molecular transport and signaling "biomachines". The principles of function of these natural biomachines - that have been optimized by evolution - guide the creation of artificial biomimetic devices and nanomaterials for nano- and bio-technology applications. This proposal focuses on a "nanomachine" known as the Nuclear Pore Complex (NPC), and the related biomimetic devices and biomaterials. NPC coordinates and gates the macromolecular transport between the cell nucleus and the cytoplasm by selectively transporting specific macromolecular complexes while efficiently filtering others. Equally importantly, it is involved in the maintenance of precise spatio-temporal partitioning of macromolecules between the nucleus and the cytoplasm by concentrating cargoes against their concentration gradients via the non-equilibrium between energy input through chemical reactions coupled to the transport process. NPC is an unusually large transporter with a complex internal organization. The key component of its functional mechanism is the assembly of the polymer-like intrinsically disordered proteins that fill its transport channel and dictate the specificity and the throughput of transport. Unlike many other molecular transporters, NPC transport is massively parallel, and its channel is crowded by hundreds of cargo-carrying transport proteins of multiple types traversing it in both directions. It still remains a puzzle how NPC maintains high throughput and selectivity without clogging under such crowded conditions. Furthermore, so far much of the biophysical investigations of the NPC and its constituents focused on the molecular physics of translocation and the biophysics of the NPC constitutes. By contrast, it remains unclear how the complex nano-scale architecture and dynamics work in concert with the non-equilibrium energy consumption mechanisms to generate molecular gradients that are robust with respect to molecular perturbations and noise. Theoretical and computational methods have proven to be indispensable  in the study of the NPC. Despite its complexity, many aspects of NPC structure and function can be understood from fundamental physical principles. Accordingly, many properties of the NPC function have been recapitulated in nanochannel mimics, providing validation of theoretical models. However, a few puzzles and open questions still remain. The goal of this proposal is to close these gaps in our quantitative understanding of the NPC, to lay the foundation for the understanding of various health and disease processes and to guide the rational design of biomimetic devices. In the process, this research will address several fundamental physical questions, such as multi-species phase separation in nano-confinement, mechanisms of molecular transport through complex molecular assemblies, and the coupling of equilibrium and non-equilibrium processes on the nanoscale.