Final Report Summary - BIONANOPORE (Biomimetic nanopore for a mechanistic study of the nuclear pore complex) The biological cell is filled with many different types of nanometer-size pores and channels that control the exchange of ions and molecules between subcellular compartments. These passageways are of vital importance to cellular function. Examples include: ion channels at the cell surface that regulate the flow of ions; pores inserted into cell membranes upon viral infection that serve as conduits for genome transfer; and, the major focus of this project, the nuclear pore complex (NPC), which controls the transport of messenger RNA and proteins across the nuclear envelope of eukaryotic cells. The NPC is the sole connection between the nucleus and the cytosol of eukaryotic cells. By connecting the genetic material and the protein-synthesizing apparatus, this remarkable cellular machine controls all transport of proteins and RNA across the nuclear envelope. A central characteristic of the NPC is that it functions as a selective sieve: while permeable to ions and small solutes (up to ~40 kD in mass), passage of larger molecules through this ‘gatekeeper’ is reserved for transport receptors (such as Impβ) that ferry cargo. The NPC is a complex macromolecular structure ( ~120 MDa in mass) composed of ~30 distinct protein subunits (referred to as nucleoporins or Nups). Roughly one third of these Nups contain natively unfolded FG domains, which are believed to constitute the key NPC components that regulate the selective access of transport receptor-cargo complexes across the channel.Advances in nanotechnology now make it possible to study and shape matter at the nanometer scale, opening the way to imitate biological structures at the molecular level to both study and harness their ingenuity. Indeed, since their first fabrication about a decade ago, nanometer-sized pores and channels in solid-state materials have served as a scaffold for a variety of biology-inspired applications. A major direction of these efforts has been to devise molecular separation methods that exhibit selectivity based on specific biochemical properties. In other work, biologically derived pores have been employed as sensitive biosensors to detect molecules in solution. In this work, we pursue a relatively new avenue of application, combining both biological and synthetic components to recapitulate the properties of a complex biological system, the NPC, and to establish a new experimental scaffold to facilitate in vitro studies of the NPC that have not otherwise been possible. We have demonstrated a bottom-up approach that enables single-molecule transport studies on a biomimetic ‘minimalist NPC’ in vitro. By removing the complexity of the cellular environment, we are able to study the function of essential NPC components by increasing the complexity of our system in a step-wise manner. Our strategy involves constructing a biomimetic NPC by covalently tethering specifically chosen Nup proteins to a solid-state nanopore. We show that it is possible to electrically monitor the translocation of Impβ through such a biomimetic NPC at the single-molecule level with high temporal resolution. Our approach has the sensitivity to reveal subtle differences between different Nups that can be quantified and rationalized.Excitingly, our biomimetic NPC recapitulates a key transport property of the native NPC, selectivity: it allows for the transport of the transport receptor, Impβ, but blocks the passage of non-receptor protein, BSA. In light of anticipated conformational differences between the different types of Nups, it is interesting to observe that biomimetic NPCs made of either Nup153 or Nup98 show very similar Impβ transport properties with dwell times in the range of ~2.5 ms for both Nups. The Nup153-coated pores are however less selective than that of Nup98-coated pores, which agrees with our finding that Nup153 allows for a larger open channel.In summary, we have built a de novo designed ‘minimalist NPC’ that faithfully reproduces the essential feature of selectivity of the NPC. This tool is a significant step forward in experimentally studying the NPC by affording a much needed and unprecedented single-molecule in vitro assay suited for real-time, rigorous testing of key NPC components and their mechanistic role in selective transport. We have used electrophysiology as a new technique to measure the ionic conductivity as well as Impβ transport across the biomimetic pore at single-molecule resolution. We have found that translocation events through such biomimetic NPCs are indeed observed for transport receptors (Impβ) whereas the passage of non-specific proteins (BSA) is strongly inhibited. Importantly, our approach has the advantage of revealing subtle differences such as how Nup98-coated pores are more selective than Nup153-coated pores. Lastly, an artistic rendition promoting our biomimetic NPC was featured on the cover of the journal Trends in Biotechnology and is attached for illustrative purposes. Further work to investigate the mechanistic interaction between FG-Nups and nuclear transport receptors has lead the design and development of a novel fluorescence microscopy method capable of nanometer-scale axial resolution at sub-second time resolutions. We have been able demonstrated our new technique in a proof-of-concept manner and will soon publish this result. Finally, as this project has come to an end, it has evolved into an exciting new direction, undertaking a single-molecule investigation of the mechanistic details of the bacterial adaptive immune system known as CRISPR-Cas. final1-trendsbiotechcover.jpg
