Bioinspired control of protein transport through polymer functionalised nanopores

Lead Research Organisation: University of Strathclyde
Department Name: Pure and Applied Chemistry

Abstract

This project will initiate a research programme in nanopore protein separation that is inspired by the nuclear pore complex (NPC).

NPCs are complex, giant assemblies constituted from more than 400 proteins that define nanoscale pores (i.e. nanopores) ~40 nm in diameter. Each NPC spans the nuclear membrane which separates the cell nucleus and the rest of the cell. NPCs are the only conduits in and out of the cell nucleus in all eukaryotic cells and they allow only a small set of specific proteins and genetic material related to the functioning of the cell nucleus to pass through. All the other thousands of species of unrelated but similar molecules in the cell are rejected.

Convenient separation of biomolecules is an enabling technology. NPC-studded nuclear membranes are effectively a highly specific and efficient molecular separation and purification membrane. They are capable of sorting through more than 1 kg of specific biomolecules in a human body per minute, far surpassing the performance of current technology. The creation of NPC-mimetic nanoporous membranes would benefit diverse biotechnology and biomedical applications, ranging from purification of protein disease markers for bedside medical diagnosis to continuous manufacturing of enzymes and protein therapeutics. Understanding the science underlying NPC function will help us achieve these applications and help us meet our 21st century challenges in healthcare and advanced manufacturing.

The immediate goal of this project is to establish the design rules for enabling the basic function of the NPC - the sorting of proteins according to size using nanopores with a "virtual" size cut off and which, unlike current technology, are not clogged by random interactions with proteins. The pore size of the NPC is virtual because it has a physical diameter much larger than the size of the protein. A random protein cannot however pass through because each NPC nanopore is filled with a semi-porous polymer plug with an as yet unidentified structure that specifically repels proteins, except for those proteins specific to nuclear function.

Biologists studying the NPC have proposed two leading theories to explain how the plug works: i) the "virtual gate" polymer brush model, and ii) the
"selective phase" meshwork model. This project will create artificial nanopores that are decorated with synthetic polymers as simplified models to mimic these two theoretical structures. Experiments will be conducted to verify whether either of the theories is in fact feasible.

The ultimate goal is to exploit these design rules for further development of the nanoporous membrane platform that incorporate increasingly advanced polymers for decorating the nanopores. This will create NPC-inspired nanoporous membranes with separation efficiency and selectivity that matches, and may eventually even surpass, native NPC function.

Planned Impact

Who are the users of the research and how will they benefit?
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ACADEMIC and INDUSTRY RESEARCH USERS, as well as the specific benefits accrued to them, are identified in the section "Academic beneficiaries". Multiple disciplines will benefit, including polymer science, nanomaterials, biointerfaces, and structural biology.

COMMERCIAL ENTERPRISES engaged in the above research areas, as well as those interested in protein separation and nanoporous membrane technologies will also be direct users. They will benefit from the new knowledge generated to create a separation technology that is significantly more functional than the state-of-the-art in terms of molecular specificity, operating life, throughput, and flow pressure and energy requirements. Importantly, the membrane format is highly scalable - the membrane area can be selected to suit either lab-on-a-chip or manufacturing scales.

THE WIDER PUBLIC will be the end-users and eventual beneficiaries. Convenient separation of biomolecules, especially proteins, will be transformative. The benefits are aligned with the EPSRC's Prosperity Outcomes in Productivity and Health. Overall, the technology is enabling in its capabilities and disruptive in its scalability, and will engender new business and technological opportunities. New and improved applications in biomedicine and biotechnology will improve the quality of life. Improvements to the continuous manufacturing of protein therapeutics and enzymes will lower the costs to medical treatment and high-value chemical products. Rapid and low cost biosensing as well as new capabilities in drug delivery may be enabled to improve and widen access to healthcare. (See Pathways to Impact for details.)

Furthermore, this research is aligned with goals identified by the EPSRC to enhance the UK economy. The broad field of users illustrates the INTERDISCIPLINARY NATURE of the research. The project will initiate a CUTTING-EDGE INTERNATIONAL COLLABORATION with Prof Théato, Hea of the Institute for Technical and Macromolecular Chemistry (ITMC) at the University of Hamburg. Also a PDRA will be trained in state-of-the-art research skills to DELIVER THE HIGHLY NUMERATE MANPOWER the UK needs.

What will be done to ensure that users will benefit?
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The project results will be disseminated through scientific journals and conferences targeted for their high international profile and scientific relevance.

The PI will undertake industrial visits to keep up to date with industry needs and to generate interest in the technology being developed. The visits will include Meissner Filtration Products Inc., USA, which has expressed an interest (see Letter of Support), and corporations invested in the areas of nanoporous membranes and molecular separation (e.g. Millipore, GE Healthcare).

In addition, a workshop on "Polymer Surfaces And Functional Nanoporous Membranes" will be organised for the academic and industry users to exchange research results and application ideas, and forge R&D opportunities.

To explain the benefits to the public, a public presentation at the Glasgow Science Centre (GSC) will be held, and the PI's website will advertise the results and impact, and Strathclyde's public relations office will issue press releases.

The PDRA will be trained through regular PI supervision as well as through the budgeted dissemination activities (presentation at conference and GSC). Moreover, a 2-week training visit to the Théato group at Hamburg is budgeted.

Publications

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Cheung DL (2019) Atomistic Study of Zwitterionic Peptoid Antifouling Brushes. in Langmuir : the ACS journal of surfaces and colloids

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Sousa AML (2018) Highly Active Protein Surfaces Enabled by Plant-Based Polyphenol Coatings. in ACS applied materials & interfaces

 
Description Current findings relate to i) the further development of novel plant-based coatings ("polyphenol coatings") that can be used to modify nanopores (https://pubs.acs.org/doi/10.1021/acsami.8b13793), and ii) further understanding of how surface grafted polymer brushes interact with water and hence proteins (https://pubs.acs.org/doi/10.1021/acs.langmuir.8b01939). Both of these findings constitute part of the fundamental work that will enable the fabrication of nanoporous materials for the project's main goal of paving the way for biomimetic selective protein transport and separation. As reported in our journal paper published in ACS Applied Materials and Interfaces, the polyphenol coatings enable the convenient immobilisation of polymer chains or proteins within nanopores. Just like natural sub-cellular systems, polymer structures immobilised within nanopores are necessary for controlling protein transport. One of the requirements for controlling protein transport is that the polymers (within nanopores) cannot attract proteins, which would then block the pores. Our recent paper published in the journal Langmuir describes new understanding derived from computer simulations about how (zwitterionic) polymer brushes interact with water, which is one of the crucial factors controlling protein attachment. Furthermore, the natural proteins lining natural nanopores controlling selective protein transport are also zwitterionic polymer chains. Thus our study will aid the understanding of these important protein-polymer interactions and pave the way to mimicking natural selective protein transport.
Exploitation Route The findings are reported in international journals for (mainly) academic researchers in chemicals, healthcare, and industrial biotechnology, to make use of. Some of the findings will also be reported by the visiting PDRA at the upcoming RSC-sponsored "Bioinspired Nanomaterials" meeting (March 18-19, 2019) organized by the PI, who has also invited a representative from Purolite Ltd to attend. Protein separation is an important market for Purolite and discussions of potential applications are anticipated. Furthermore, the PI will be presenting the journal paper results at the upcoming American Chemical Society National Meeting (Orlando, Mar 21 to Apr 4). This meeting is routinely attended by both academics and industry researchers.
Sectors Chemicals,Healthcare,Manufacturing, including Industrial Biotechology

URL https://pubs.acs.org/doi/10.1021/acsami.8b13793
 
Title Optical detection of molecular transport into and out of nanopores 
Description This technique is based on the confinement of evanescent optical illumination near a surface or within a thin layer of material. It is related to surface plasmon resonance (SPR) spectroscopy, commonly used for assaying protein binding kinetics. The new technique allows measurements of the concentration of fluorescent molecules over different depths along the nanopores, and thereby characterises the kinetics of molecules entering and exiting the nanopores. It is a physical technique that can enable assays of biomolecules like proteins that are transported through nanopores. Currently, light can only be confined on the surface or within thin layers of materials. So the measurement can only be performed if the nanopores and the biomolecular system of interest can be placed or recreated within a synthetic thin layer support such as on a glass slide in a flow cell. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact Results demonstrating the principle of the technique have been obtained and a manuscript is in preparation. 
 
Description Cheung NUI-Galway 
Organisation National University of Ireland, Galway
Country Ireland 
Sector Academic/University 
PI Contribution We provided a novel research direction for the collaborator at NUI-Galway, to study the molecular interactions of zwitterionic polymer brushes, which can mimic the natural proteins found in sub-cellular nanopores.
Collaborator Contribution The collaborator has constructed for us a novel simulation model of peptide-mimetic zwitterionic polymer brushes, which can be considered a mimic of the natural nucleoporin proteins linking natural nanopores.
Impact Multidisciiplinary: computational chemistry and biophysical soft matter Outcome in journal paper: Cheung DL, Lau KHA. (2018). Atomistic Study of Zwitterionic Peptoid Antifouling Brushes.. Langmuir, 2019, 35 (5), pp 1483-1494 DOI: 10.1021/acs.langmuir.8b01939
Start Year 2018
 
Description Collaboration with Patrick Theato 
Organisation Karlsruhe Institute of Technology
Country Germany 
Sector Academic/University 
PI Contribution We are providing training in the specialized technique of nanoporous waveguide spectroscopy to the visiting researcher from our partner at KIT.
Collaborator Contribution The visiting PDRA is providing skills in polymer synthesis and hydrogel preparation to my group. The in-kind contribution consists of the PDRA salary for the 4-month duration of the visit, estimated at EUR 2500 per month.
Impact Multidisciplinary: polymer chemistry (from research partner), biophysical chemistry and bioinspired materials (from PI lab at Strathclyde). Outcomes: the PDRA 4-month visit only started in Jan, 2019. Current results will be reported at the RSC-sponsored "Bioinspired Nanomaterials" meeting to be held March 18-19, 2019.
Start Year 2018