University of Bristol
Augmenting Biology Through De Novo Protein Design in Cells
Protein design, that is, the construction of entirely new protein sequences that fold into prescribed structures—has come of age: it is possible now to generate a wide variety stable protein folds from scratch using rational and/or computational approaches. A challenge for the field is to move from what have been largely in vitro exercises to protein design in living cells and, in so doing, to augment biology. This talk will illustrate what is currently possible in this nascent field using de novo α-helical coiled-coil peptides as building blocks.1
Coiled coils are bundles of 2 or more α helices that wrap around each other to form rope-like structures. They are one of the dominant structures that direct natural protein-protein interactions. Our understanding of coiled coils provides a strong basis for building new proteins from the bottom up. The first part of this talk will survey this understanding,1 our design methods,2, 3 and our current “toolkit” of de novo coiled coils.4, 5, 6
Next, I will describe how the toolkit can be used to direct protein-protein interactions and build complex protein assemblies in bacterial cells. First, in collaboration with the Savery lab, Bristol, we have used homo- and hetero-oligomeric coiled coils as modules in engineered and de novo transcriptional activators and repressors.7 Secondly, with the Warren, Kent, and the Verkade, Bristol, labs, we have engineered hybrids of a de novo heterodimer and a natural component of bacterial microcompartments to form a “cytoscaffold” that permeates E. coli cells, see figure.8 This can be used to support the co-localisation of functional enzymes.
1. Coiled-coil design: updated and upgraded.
DN Woolfson. Subcellular Biochemistry 82, 35-61, 2017
2. CCBuilder: an interactive web-based tool for building, designing and assessing coiled-coil-protein assemblies.
CW Wood et al. Bioinformatics 30, 3029-3035, 2014
3. ISAMBARD: an open-source computational environment for biomolecular analysis, modelling and design
CW Wood et al. Bioinformatics 33, 3043–3050, 2017
4. A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology
JM Fletcher et al. ACS Synth Biol 1, 240-250, 2012
5. A set of de novo designed parallel heterodimeric coiled coils with quantified dissociation constants in the micromolar to sub-nanomolar regime
F Thomas et al. J Am Chem Soc 135, 5161-5166, 2013
6. Computational design of water-soluble α-helical barrels.
AR Thomson et al., Science 346, 485-488, 2014
7. Guiding biomolecular interactions in cells using de novo protein-protein interfaces
AJ Smith et al. bioRxiv 486902; doi: https://doi.org/10.1101/486902
8. Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm.
MJ Lee et al., Nature Chem Biol 14, 142-147, 2018