De novo protein design is said to have come of age (Huang et al., (2016) Nature 537 320). This is because a wide variety of synthetic protein structures can now be made from scratch, i.e., from first principles, or by applying computational design. The question is: what do we do with these new-found abilities? One challenge is to take protein design from a largely in vitro endeavour to in vivo applications in cells and whole organisms. This PhD with address this challenge by taking a set of de novo peptide modules developed in the Woolfson lab into bacterial cells and using them to install new de novo protein assemblies and functions in these cells.
Modules for protein design and assembly in cells. The Woolfson lab has made a range of helical peptide assemblies that vary in oligomeric state, helix orientation, and partner selection (Figure 1; Fletcher et al., (2012) ACS Synth Biol 1 240; Thomson et al., (2014) Science 346 485). For this new PhD project, we propose to use these designed peptides to generate a range of single-chain de novo proteins. This will be done using our computational design platform, ISAMBARD (Wood et al., (2017) Bioinformatics 33 3043) and virtual reality (VR) tools that we are developing with David Glowacki (Chemistry). The designs will be made by peptide synthesis and the recombinant expression of synthetic genes. They will be characterised by biophysical methods and structural biology.
Towards protein design in cells. Once characterised, the single-chain designs will be cloned, expressed and studied directly in E. coli. Their ability to fold and function in cells will be tested by transcriptional assays in the Savery lab (Smith et al., (2019) ACS Synth Biol 8 1284). Next, designed modules that operate fully in E. coli will be engineered to form larger, more-complex de novo assemblies in E. coli, such as de novo cytoskeletons and protein-based organelles. This will be done by designing de novo protein-protein interfaces into the modules using ISAMBARD and porting the design into E. coli. Assembly of the modules will be examined by light and electron microscopy in the Verkade lab (Figure 2, Lee et al., (2018) Nat Chem Biol 14 142). We aim to make these protein-protein interactions tunable and switchable through the addition of sites for enzymatic post-translational modifications.
Skills and training This PhD will provide training and academic development in the areas of protein design, bacterial molecular cell biology, and advanced microscopy. On the practical side the project will combine state-of-the-art methods in rational and computational protein design, peptide and protein production, biophysical characterisation and structural biology (protein X-ray crystallography), molecular biology and transcriptional assays, and advanced light and electron microscopy.