Creating Lasso Peptide Building Blocks for Higher Order Structures

Abstract

The 2016 Noble prize in Chemistry was awarded for research into molecular machines. These machines are created from mechanically interlocked molecules (MIMs) which can dynamically moved around one another. However, most molecular machinery research to date has been carried out in organic solvents. There is a lack of understanding on how we can effectively build molecular machines in an aqueous solution which is necessary to build something which can be utilised in vitro. Lasso peptides offer a natural starting point into this research. Previous research in the Link lab has delved into creating static MIMs from lasso peptides. In this thesis we leverage off past research and aim to make more dynamic variants of lasso peptides which could be used as building blocks for higher order MIMs and potentially be used as precursors to create natural molecular machines. Two different project pipelines were developed in order to do this. Firstly, we altered the upper steric lock of microcin J25 (MccJ25), a well-characterized lasso peptide known for its stable macrolactam ring structure. Six different variants of MccJ25 were designed, four of which were successfully tolerated and produced. Secondly, we mutated benenodin-1, recognized for its ability to adopt alternative conformations under thermal stimuli, to create a potential molecular shuttle. The mutated variants of MccJ25 and benenodin-1 were analysed on LC-MS and HPLC. Collectively, these studies advance the potential of lasso peptides as scaffolds for developing complex MIMs and contribute to the growing field of bioengineering by demonstrating how molecular dynamics can be harnessed to create novel protein architectures.