2024 Theses Doctoral

Engineering Enzymatic Modulation Platforms for Biomolecular Applications

Gokulu, Ipek Simay

DNA serves as a mode of storage for genetic information and carries essential biophysical functions which continue to gain importance for new biomolecular applications. The functionalization of proteins with nucleic acids has found several different uses in a variety of fields such as DNA origami due to the highly programmable nature of DNA. The modulation of key biomolecular properties of proteins by the conjugation of DNA nanotools, mechanical forces and potential catalytic modulators is an under discovered area of protein engineering.

In this doctoral thesis work, functionalization strategies of proteins with DNA oligonucleotides, the catalytic mechanism of AdhD from Pyrococcus furiosus and effects of mechanical forces on enzymatic catalysis have been investigated through the utilization of DNA tools.

In the first part of the thesis (Chapter 2), several bioconjugation strategies which can be used to attach DNA oligonucleotides to proteins have been reviewed and assessed for emerging biomolecular applications. This work provides insights about commonly used bioconjugation reactions and investigates the performance of each conjugation reaction in terms of yield, site- specificity, flexibility in conjugation position, steric hinderance, cost, reagent availability and risk of altering native protein properties.

In the next section of the thesis (Chapter 3), DNA spring attachment sites are strategically modeled and created on the model enzyme for assigned bioconjugation strategies and the DNA springs are assembled on this model enzyme. The effect of the forces generated by the DNA springs during bulk catalysis is investigated and the changes in binding pockets and substrate specificity properties are demonstrated. A novel magnetic bead-based purification strategy for the separation of DNA spring conjugated enzyme is established in this work to ensure homogenous catalysis conditions. This construct allows the tuning of catalytic properties by the usage of different lengths of DNA and is shown to be reversible. As enzymatic catalysis is investigated under bulk conditions and the amino acid sequence within the active site is not altered, this strategy provides a new platform for the modulation of enzymatic trajectories without isolation and altered microenvironment limitations as well as the irreversible effects of conventional techniques such as mutagenesis and directed evolution.

In the last part of the thesis (Chapter 4), the effects of different DNA constructs on AdhD are investigated. Convex DNA springs are constructed on AdhD molecules with the goal of applying compressive forces over the active site. In addition, DNA tweezers are designed to apply comparable forces to the DNA spring studied in Chapter 3, assembled on AdhD and purified using the same magnetic bead-based strategy. This construct, as discussed in Chapter 1, allows the modulation of substrate specificity profiles consistent with its mechanical design.

As a continuation of the last part of the thesis, the rare earth element (REE) binding capacity of AdhD and the effect of metal binding on its enzymatic trajectory are investigated in Chapter 5. The rare earth element (REE) binding affinity of AdhD and its effect on enzymatic catalysis on both the forward and reverse reactions are demonstrated with and without the presence of a metal chelator. This discovery sheds light on the potential allosteric regulation mechanisms of AdhD and the catalytic regulator effect of REEs.

The work presented in this doctoral thesis demonstrates different biomolecular approaches towards the modulation of enzymatic properties through DNA oligonucleotide conjugation, application of mechanical forces and potential catalytic regulators. In the future, the results presented in this work can be utilized to initiate in depth studies about protein-DNA conjugation and modulation of enzymatic catalysis, and discover extended applications which can be used universally across different biomolecular platforms.