Unlocking the power of nanopores
![Unlocking the power of nanopores](http://biologicalsciences.leeds.ac.uk/images/resized/750x375-0-0-1-80-Structures_of_the_transmembrane____barrel_proteins_designed_in_this_study_permissions_Sheena_Radford_800x400.png)
Scientists take one step closer to uncovering new sensing technology.
Scientists at University of Leeds are part of an international collaboration that has described a new approach to designing proteins from scratch.
The approach uses Transmembrane β-barrel pores (TMBs), nanosized proteins which are extensively used for single-molecule DNA and RNA sequencing –an analysis method that can help detect disease.
TMBs also enable the shrinking of a wide array of sensing and sequencing applications into portable USB-size devices and point-of-care technologies, such as disease diagnostic tools.
The TMB pores generated in the study have custom shapes and properties, opening up new opportunities for the analysis of molecules which is critical to develop new treatments of disease.
The results were published in Science.
This is an exciting second step in our fruitful collaboration with David Baker and Anastassia, in which we combined our expertise to develop new protein sequences, unrelated to any natural proteins, that are able to fold to these very stable beta barrel folds. It both opens up new doors for fundamental studies of how these proteins fold, as well as new opportunities to exploit them in biotechnology.
Dr James Whitehouse, a Leeds PhD student who performed many of the experiments to test the folding and stability of the protein designs and now works at Oxford Nanopore Technologies, added:
“This is a brilliant culmination of much of the work from my PhD thesis. It was such an exciting experience to work alongside students and academics in Belgium, Switzerland, and the USA on such groundbreaking work.”
Rolling out new barrels
Protein nanopores are the holy grail in the field of analytical biology. These nanometer-sized proteins, which are a million time smaller than a millimeter in size, form regular pores in lipid membranes (the barrier around cells) and are widely used for single-molecule DNA and RNA sequencing.
They hold a considerable potential to advance a broad range of sensing and sequencing applications by taking them out of specialized labs and into portable devices.
However, current approaches to engineering nanopore sensors are limited to naturally occurring proteins, which have evolved for very different functions and are less than ideal starting points for sensor development.
These developments are very exciting. When we started with this idea a few years ago, many people thought it was impossible, because the design and folding of β-sheets is incredibly complex, let alone in lipid membranes. Now we have shown that we can successfully design nanopores with a high success rate, which have stable and reproducible conductance.
Research led by the VIB-VUB Center for Structural Biology (Belgium) and the University of Washington School of Medicine (USA) has taken on the challenge of designing these protein ‘barrels’ from scratch, with the ultimate goal of controlling the shape and chemistry on a molecular level.
With the help of computational design, the researchers developed methods to design stable nanopore channels with tunable pore shapes, sizes, and conductance.
Compared to natural pores, the signal generated by the designed TMBs was remarkably stable and quiet.
Sebastian Hiller (Biozentrum, University of Basel) also joined the project team, with his group using the latest methods in structure determination to show that the designs indeed folded into the desired, stable 3D structures.
“This collaboration is a great example of what's possible with protein design. Rather than repurposing biomolecules from nature, we can now create the functions we want from first principles,” said Dr. David Baker, Professor at the University of Washington School of Medicine and HHMI investigator.
Although there is still further research needed in this area, the researchers envision a future in which portable devices with different nanopores can sense a range of metabolites, proteins, and small molecules, or even perform biomolecular sequencing.
Finding how sequences allow folding to a stable structure in a membrane is one of the holy grails in protein research. This new study has made major inroads in this field and opens the door to exciting new biotechnologies into the future.
The positive results prove that nanopore design can complement mass spectrometry and other analytical methods that require big labs and big setups because the technology is smaller and more accessible.
Funding and collaborations
The paper, “Sculpting conducting nanopore size and shape through de novo protein design,” appears in the July 19 edition of Science. The research team included scientists from the University of Leeds (Funded by the Royal Society and an Excellence of Science award that links UK and Belgium scientists), the VUB-VIB Center for structural biology, UW Medicine, University of Virginia School of Medicine, and University of Basel.