Enzyme networks by design

This research project was completed in the last funding period.

Project history

Molecular networks are the basis of life. Enzymes play a central role in the regulation of this network. This network is based on coupled enzymatic reactions. Both the limitations in size as well as in numbers impose incisive boundary conditions, which give rise to characteristic properties of cells and govern their fundamental biochemical processes. Our goal right from the beginning on to elucidate the stochastic nature of these networks and to understand their impact on live systems. Our approach was to mimic such processes and to reconstitute basic elements of these regulatory loops in a synthetic biology approach, to this end we proposed to design and assemble networks of enzymes, molecule by molecule, and to control and follow the response function of each of these networks by tracking the products at the single molecule level. Understanding by building has been key to this project from the beginning on. With this improved understanding of spatio-temporal control we hoped to finally contribute to an optimization of functional synergies in the design of complex enzyme composites. Our strategy was to employ Single Molecule Cut & Paste technology to assemble patterns of individual enzymes, specifically RNA polymerases and restriction endonucleases. Zero mode waveguides were envisioned to provide 3d confinement of substrates and products with an AFM-controlled lid to allow the coupling to the bulk reservoir. In the first funding period, we established the experimental platform to assemble enzymatic networks by single molecule cut & paste (SMC&P). We investigated several model enzyme systems and identified the most suitable handle and anchoring strategies. In the second funding period, we built on this know- how and we intend to design and assemble networks of enzymes, molecule by molecule. The goal was to control and follow the response function of each of these networks by tracking the products at the single molecule level. With this improved understanding of spatio-temporal control we hoped to finally contribute to an optimization of functional synergies in complex enzyme composites such as the cellulosome. One essential ingredient for success was the control of stoichiometry, composition, and interaction based on individual molecules with the option to adjust their relative couplings by the geometry of their arrangement. During the past funding period we had to learn that the hierarchy of binding forces, which is key to a successful SMC&P assembly required novel molecular binding partners for a thermally stable assembly, which reliably operates for hours, as some of the envisioned enzymatic reactions required such long observation time spans. The DNA duplexes that we had used in the past proved to interfere with the enzymatic activity of the RNA polymerases that we had used in the first funding period. We therefore explored the broad repertoire of cohesion:dockerin complexes of the large family of cellulytic bacteria. We expressed and characterized them and we identified suitable complexes.
We also revisited the well-known Biotin:Streptavidin complex. We were able to analyze the unbinding paths of the complex and identified the highest unbinding barrier. In combination with the retained longevity of the complex this system proved to be well suited for SMC&P. A second key feature is the covalent and site-specific anchoring of these proteins to the surfaces. We developed several strategies to achieve this goal and successfully implemented the couplers for SMC&P.