Determining the molecular mechanisms at the origin of protein function remains a challenge due to the complex non-covalent interactions that shape their structure. Since the non-covalent interactions arise from charge fluctuations, electric fields can be used as a tool to quantify the interactions between a target and its environment. The contribution of each component of the system is reflected in the direction and strength of the electric field exerted on the target, which can be calculated from molecular dynamics simulations. The interactions experienced by ligands in enzymatic active sites determine the catalytic activity of the enzyme. Ligands in synthetic enzymes lack interactions with the protein scaffold, which limit their efficiency. To substitute for the role of non-effective protein scaffold, we introduced a polar DNA fragment to the enzyme vicinity, inducing electrostatic interactions that will facilitate the reaction. We found that the introduction of a DNA fragment enhanced the original interactions between the residues in the active site and the ligand, without creating new interaction hot spots. Using electric fields, we calculated a reduction in activation energy of 2.0 kcal/mol when introducing the DNA fragment, indicating a promising avenue for catalytic improvement. Inspired by the success in using electric fields to understand enzyme catalysis in the context of electrostatic preorganization theory, we generalized these fundamental concepts to another type of proteins: voltage-gated ion channels. Our results indicate that electric fields also report on channel activity. We find an asymmetry in the number of active residues for channel function between the four domains and between the two gating motifs of the permeation pathway, with domain I being the major contributor in both cases. The importance of residues for channel activity is not a simple linear correlation of their distance with the functional motif, but a relationship dominated by non-covalent interactions. Finally, we investigate the effects of loop dynamics on enzyme product inhibition. We modify the chemical nature of the unstructured loops that obstruct the active site of DszB by glycosylating serine and threonine residues. We monitor the corresponding variations in loop dynamics and their effect on the interaction between the enzyme and the product. Overall, promising results were found using electric fields in the investigation of protein mechanisms that are mainly dominated by non-covalent interactions and provide insight into the role of the individual components in the system. / Doctor of Philosophy / Although weaker than covalent interactions, non-covalent interactions play a crucial role in molecular biological processes, especially in protein mechanisms. In order to modify the properties of proteins to our advantage, we need a metric with which we can map these interactions onto the protein structure. Different types of non-covalent interactions share one similarity: they originate from the change of electron distribution of interacting atoms, therefore can be captured by analyzing the protein- generated electric fields. Synthetic enzymes are designed to better adapt to varying environments and catalyze a broader reaction range. However, they are less effective than natural enzymes because the protein scaffold does not contribute to catalysis. Indeed, protein scaffolds in natural enzymes generate an electric field that lowers the reaction activation energy in the active site. Protein scaffolds in synthetic enzyme do not generate such electric fields. To address this issue, we modified the environment of synthetic enzyme KE15, introducing a polar DNA fragment to induce interactions in the active site. This modification strengthen the interactions between protein and ligand, leading to a decrease in the energy required for the reaction. While enzymes are famous for their generation of electric fields facilitating function, we demonstrated that this phenomenon also exist in voltage-gated ion channels Nav1.7. Residues were found to exert an electric field that can facilitate ion permeation. This is not simply because of their distance to the key regions, but a result of the non-covalent interactions regulating the mechanism, with different regions showing asymmetric importance in the process. Since the governing non-covalent interactions are relatively weak, proteins are flexible, especially protein loops. In enzyme DszB, this loop flexibility enables a conformational change when the ligand binds the active site. The change in loop conformation traps the product inside the active site, limiting enzymatic turnover. To prevent active site obstruction by these flexible loops, we attached glucose to a few loop residues to modify the hydrophobicity profile near the active site. The introduction of hydrophilic glucoses helps to pull the loops towards the solvent, rather than towards the active site, limiting product inhibition while preserving catalytic activity. Overall, our results show that electric field can be applied as a general method for protein studies, relating structure to function.

Intramembrane proteases from the rhomboid-like superfamily are enzymes widely distributed and conserved in all domains of life. They participate in many important processes such as membrane protein quality control or mitochondrial dynamics. Their activity is also linked with diseases like Parkinson's disease or cancer. This makes them potential therapeutic targets. In this work we tried to elucidate in more detail the mechanism of action of the main model intramembrane protease, GlpG from E. coli. We also focused on the mechanism of eukaryotic rhomboid RHBDL2, one of the four mammalian rhomboids, function of which is poorly understood. To acquire more detailed information about substrate-enzyme interaction, we synthesized a series of novel peptidyl-chloromethylketone inhibitors derived from natural rhomboid substrate TatA from P. stuartii. Crystal structure of the complex of GlpG with these inhibitors revealed four substrate binding subsites (S1 to S4) of the enzyme and explained its observed substrate specificity structurally. This study showed that substrate cleavage rate can be dramatically modified by changing the substrate sequence in positions P1 to P5. This helped us develop fluorogenic transmembrane peptide substrates for rhomboid proteases, which are usable in detergent and liposomes, and...