Title: Engineering Biomolecular Interfaces for Applications in Biotechnology
Description: Protein interactions occurring through biomolecular interfaces play an important role in the circle of life. These interactions are responsible for cellular function, including RNA transcription, protein translation, cell division and cell death among many others. There are different types of interactions based on the strength and the duration of the association. Transient interactions govern most steps of the cellular metabolism, where the associations between two or more molecules are responsive to environmental cues. Among the participants of transient interactions, intrinsically disordered proteins are employed in signaling and other regulatory events within the cell. These proteins exhibit allosteric regulation and gain secondary structure when they bind other proteins or small molecules. In this doctoral thesis work, the biochemical and biophysical principals governing protein associations are investigated and using protein engineering tools, novel biomolecular interfaces are engineered, with potential applications in different areas of biotechnology.

The first part of the thesis (Chapter 2) focuses on the investigation of supramolecular enzyme association among tricarboxylic acid cycle enzymes, specifically between citrate synthase and mitochondrial malate dehydrogenase. In this study, the interactions between these enzymes are examined, both among their natural and synthetically produced recombinant versions. In addition, mutational analysis of the amino acid residues at the complex interface was performed to explore the importance of the positively charged patch connecting the active sites of the enzymes. It was discovered that the channeling of the negatively charged intermediate is severely impaired upon mutation of surface residues contributing to the electrostatic channeling. This work provides an important insight into understanding the coupled reaction-transport systems and metabolon formation in general. In addition, it constitutes a great example for substrate channeling in leaky systems, which are relevant to most biological processes.

The next section of the thesis (Chapter 3) focuses on an intrinsically disordered peptide, the β-roll. This peptide is isolated from the Block V repeats-in-toxin (RTX) domain of adenylate cyclase from Bordetella pertussis. It is disordered in the absence of calcium and it folds into a β-roll secondary structure composed of two parallel β-sheet faces upon binding to calcium ions. This way, the peptide can transition between its unfolded state and the β-roll structure in a reversible way. We have utilized the allosteric regulation of this domain as a tool to engineer new protein interfaces. In its folded state, the peptide has two faces, serving as binding surfaces available for interaction with other proteins. Our work involved the alteration of the residues, which form these faces upon calcium binding, via combinatorial protein design techniques. The potential of this peptide is evaluated as a cross-linking domain for hydrogel formation. By rationally engineering the two faces of the folded β-roll to contain leucine residues, we have created hydrophobic interfaces, serving as environmentally-responsive cross-linking domains.

When there is no calcium, the β-roll domains remain unstructured, delocalizing the leucine rich patches. After calcium binding, the β-rolls fold and the leucine rich faces are exposed creating a hydrophobic driving force for self-assembly. This way, we showed that the β-roll peptide can function as a biomaterials building block capable of proteinaceous hydrogel formation, only in the presence of calcium. The next study (Chapter 4) demonstrates the utilization of this peptide as an alternative scaffold for biomolecular recognition applications. A library of mutant β-rolls was constructed by randomizing the amino acid residues on one of the β-sheet forming faces. Mutant peptides demonstrating an affinity for hen egg white lysozyme were selected, which was chosen as a model target molecule. The thermodynamic parameters of the interactions between the β-roll mutants and the lysozyme were quantified. Upon performing further protein engineering (e.g. concatenation of the single mutants on the DNA level), a mutant with mid-nanomolar affinity was identified.

Affinity chromatography experiments showed that this mutant was capable of capturing the target, in the presence of calcium. The captured target was easily released upon removal of the calcium ions. The reversibility of the calcium binding allowed the engineered molecular interface to be controllable. Throughout this study, the β-roll peptide was explored as an allosterically-regulated protein switch for on/off biomolecular recognition, which can be mediated by simply changing the calcium concentration, allowing control over the binding behavior between molecules. The last part of the thesis (Chapter 5) expands on the calcium dependent network formation study. A hydrogel construct was genetically built by fusing the cross-linking β-roll domain and the lysozyme binding β-roll mutant, resulting in a smart biomaterial with dual-functionality. The network-assembly and target capture functions of this construct were tested by various assays including hydrogel erosion experiments. This allosterically-regulated biomaterial exhibited promising results, where calcium-dependent lysozyme entrapment within the assembled network and lysozyme capture on the hydrogel surface were demonstrated.

The work presented in this thesis demonstrates different approaches to understand and engineer molecular interfaces in both natural and recombinant systems. In the future, these approaches and the knowledge gained from these studies can be further built upon for different biotechnological applications and can also be applied to other synthetic systems.