21ENGBIO_De Novo protein scaffolds for uranium decontamination

Energy sustainability is indisputably one of the most challenging and pressing socioeconomic problems facing the world this century. Problems associated with climate change disasters from increasing CO2 emissions by the burning of fossil fuels has led to a desperate need to use energy sources that are carbon neutral. In creating a decarbonised power sector, renewable energy sources are at the forefront of most people's minds. Here, energy from uranium nuclear fission, that currently provides around 20% of the UK's electricity, is classified as a zero carbon energy source at the point of production according to the Governments' Department for Business, Energy & Industrial Strategy (BEIS). Indeed, energy utility providers such as EDF and E.ON now include nuclear in their renewable energy portfolio. However, public acceptance and the continued use of nuclear power is heavily reliant on sustained future investment in decommissioning and clean-up of generated nuclear wastes including the large stockpile of existing legacy wastes. The safe management of radioactive wastes, where uranium is the major component by mass, is thus a vital enabler for a secure nuclear energy future. Current UK policy is to dispose of its higher activity radioactive wastes in the subsurface in a geological disposal facility, and lower level radioactive wastes (including uranium and medical radioisotopes) above ground in the Low Level Waste Repository (LLWR, Cumbria). However, 8 of the UK's 15 reactors will reach the end of their lifecycle by the end of this decade and any decommissioning and new builds (currently planned) and their associated wastes need to be accompanied by rigorous safety cases. However, to achieve this, underpinning research to ensure long term waste containment is essential in order to implement whole systems solutions to this major environmental challenge.
To address these pressing issues, we propose to take advantage of synthetic biology to bioengineer new protein derived materials that self-assemble into a triple helical 'coiled coil' fluorescent structures in order to both sequester environmental levels of uranium with unprecedented selectivity, and to report on its concentration and chemical using fluorescent read out signals.
Synthetic peptide scaffolds offer an excellent approach to building preorganised, three-dimensional binding environments for metals, inspired by the highly selective coordination observed in natural metalloproteins but without the often, arduous task of creating recombinant proteins through mutagenesis. Such structures can be predictably manipulated and controlled by specific engineering of the amino acid sequence, to systematically optimise binding. In this way, uranium mobility in the natural and engineered environment from over 60 years of civil nuclear anthropogenic activities can be monitored in the field. We will first bioengineer peptide sequences, whose structures can form helices and exhibit protein type tertiary and quaternary structures, are compatible with the environmental conditions (e.g. pH fluctuations), and whose binding sites are predisposed to selectively bind uranium over other omnipresent metal ions and chemical entities such as carbonates and phosphates. We will then modify the design in an iterative fashion with help from molecular dynamics modelling simulations to optimise the binding properties before encapsulating/attaching them to materials (e.g. polymers, magnetic particles) to create dual sensor and decontamination devices. The key goal is to develop new materials, technology and spectroscopic based tools to help manage the UK's significant inventory of radioactive wastes and contaminated materials by applying a new bio-recycling and bioremediation tool kit to increase the sustainability of nuclear power as a key carbon neutral energy source in line with the 2050 net zero carbon agenda.

Using metalloproteins as a starting point for the bioinspired design of a range of new fluorescent responsive materials, a library of synthetically engineered fluorescent peptide sequences based on a repeating abcdefg heptad unit will be fabricated that selectively bind to and report on uranium concentration by forming helical coiled coils. This phenomenon will be tested in model nuclear waste environments representative of those across the Nuclear Decommissioning Authority estate and the binding sequences adjusted accordingly to maximise the strength of uranyl(VI) binding. This will be achived by translating the binding residues to determined distances along the coil, introducing non natural amino acids such as phosphoserine that will show a much higher affinity to uranyl and also by manipulating the 3D structures by modification of the outer amino acid residues. We aim to examine helix-loop-helix-loop structures in helical bundles and asymmetric binding sites to optimise metal-peptide inner and outer sphere interactions. The most viable peptides will be engineered into simple proof-of-concept sensing and decontamination materials and devices including filter paper based testing strips, sol gels and hydrogels, plasmonic Au nanoparticles, magnetic iron particles, silica and polymer beads. These can functionlised to incorporate chemical functional groups can bind strongly (covalently or electrostatically) with a given peptide sequence (eg. through thiol-maleimide coupling and amide coupling procedures). These proposed coiled coils offer distinct advantages over engineered recombinant proteins and shorter linear peptides previously reported. They are easily re-designed to incorporate other fluorescent groups outside the UV range of tryptophan chromophores, e.g. blue, green and red dyes (e.g. Alexafluor and cyanine dyes) which will enable the optimal fluorescence reporter in these coiled coils, advantageous when examining heterogeneous samples.