Activated neutrophils release a neutrophil serine proteinase (NSP) called Proteinase 3 (PR3). In granulomatosis with polyangiitis (GPA), an autoimmune vasculitis, enhanced PR3 release results in endothelial damage. Serine proteinase inhibitors (serpins) such as α-1 proteinase inhibitor (API) inhibit NSPs through the serpin’s reactive center loop (RCL). However, API is known to bind PR3 with a low specificity, compared to its main inhibitory target Human Neutrophil Elastase (HNE). The current treatment for GPA is immunosuppression, which leaves patients immunocompromised. Thus, the overall aim of this study was to engineer an API variant with a higher specificity to PR3 than HNE, which could serve as a possible novel therapeutic strategy for GPA. We created an API expression library, hypervariable at RCL residues A355-I356-P357-M358-S359, and expressed it in a T7 bacteriophage display system. This phage library was then biopanned for PR3 binding. Two conditions were used for each round of biopanning: experimental, with PR3, and the negative control, without PR3. The library was biopanned for a total of five consecutive rounds, with the product of one screen serving as the starting material for the next. A bacterial mass lysate screen was also employed to further probe the library with PR3. The phage-display and bacterial lysate screens resulted in the selection of two novel variants API-DA (D357/A358) and API-N (N359). Serpin-proteinase gel complexing assays indicated that API-N formed complex with PR3 similar to API-WT (wild-type), while API-DA was mainly cleaved as a substrate. There was no significant difference between the second order rate constants of API-N and API-WT reactions with PR3. Rate constants for API-DA binding to PR3 or for API-HNE reactions were not completed due to novel coronavirus (COVID-19) restrictions. However, this project successfully demonstrated the ability to screen a hypervariable API phage library with PR3, yielding two new novel API variants. / Thesis / Master of Science in Medical Sciences (MSMS) / When harmful substances enter our body such as bacteria or viruses, we have ways of protecting ourselves from them. One of those ways is through a cell called the neutrophil. This is an immune cell that can release “fighting tools” into our blood to combat the harm. Some of these tools are called proteins. One of those proteins is Proteinase 3. However, sometimes our neutrophils can be activated without the presence of viruses or bacteria by products made in our bodies called autoantibodies. When this happens, too many of the “fighting tool” Proteinase 3 is released leading to damage to the tubes or vessels that our blood flows through. This project aimed to find a new possible way to stop these extra fighting tools from doing harm to our body. We did this by creating a library of different proteins that can stop Proteinase 3 once it is released by the neutrophil.

The goals of this research were 1) to produce hydrogen in high yields from cellulosic materials and water by synthetic pathway biotranformation (SyPaB), and 2) to increase the hydrogen production rate to a level comparable to microbe-based methods (~ 5 mmol H2/L/h). Cell-free SyPaB is a new biocatalysis technology that integrates a number of enzymatic reactions from four different metabolic pathways, e.g. glucan phosphorylation, pentose phosphate pathway, gluconeogenesis, and hydrogenase-catalyzed hydrogen production, so as to release 12 mol hydrogen per mol glucose equivalent. To ensure the artificial enzymatic pathway would work for hydrogen production, thermodynamic analysis was firstly conducted, suggesting that the artificial enzymatic pathway would spontaneously release hydrogen from cellulosic materials. A kinetic model was constructed to assess the rate-limited step(s) through metabolic control analysis. Three phosphorylases, i.e. α-glucan phosphorylase, cellobiose phosphorylase, and cellodextrin phosphorylase, were cloned from a thermophile Clostridium thermocellum, and heterologously expressed in Escherichia coli, purified and characterized in detail. Finally, up to 93% of hydrogen was produced from cellulosic materials (11.2 mol H2/mol glucose equivalent). A nearly 20-fold enhancement in hydrogen production rates has been achieved by increasing the rate-limiting hydrogenase concentration, increasing the substrate loading, and elevating the reaction temperature slightly from 30 to 32°C. The hydrogen production rates were higher than those of photobiological systems and comparable to the rates reported in dark fermentations. Now the hydrogen production is limited by the low stabilities and low activities of various phosphorylases. Therefore, non-biologically based methods have been applied to prolong the stability of α-glucan phosphorylases. The catalytic potential of cellodextrin phosphorylase has been improved to degrade insoluble cellulose by fusion of a carbohydrate-binding module (CBM) family 9 from Thermotoga maritima Xyn10A. The inactivation halftime of C. thermocellum cellobiose phosphorylase has been enhanced by three-fold at 70°C via a combination of rational design and directed evolution. The phosphorylases with improved properties would work as building blocks for SyPaB and enabled large-scale enzymatic production of cellulosic hydrogen. / Ph. D.

Yes / The crotonases comprise a widely distributed enzyme superfamily that has multiple roles in both primary and secondary metabolism. Many crotonases employ oxyanion hole-mediated stabilization of intermediates to catalyze the reaction of coenzyme A (CoA) thioester substrates (e.g., malonyl-CoA, α,β-unsaturated CoA esters) both with nucleophiles and, in the case of enolate intermediates, with varied electrophiles. Reactions of crotonases that proceed via a stabilized oxyanion intermediate include the hydrolysis of substrates including proteins, as well as hydration, isomerization, nucleophilic aromatic substitution, Claisen-type, and cofactor-independent oxidation reactions. The crotonases have a conserved fold formed from a central β-sheet core surrounded by α-helices, which typically oligomerizes to form a trimer or dimer of trimers. The presence of a common structural platform and mechanisms involving intermediates with diverse reactivity implies that crotonases have considerable potential for biocatalysis and synthetic biology, as supported by pioneering protein engineering studies on them. In this Perspective, we give an overview of crotonase diversity and structural biology and then illustrate the scope of crotonase catalysis and potential for biocatalysis. / Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the Wellcome Trust

Molecular dynamics simulations and QM/MM calculations can provide insights into the structure and function of enzymes as well as changes due to mutations of the protein sequence.

Elevated reaction temperatures are crucial for the efficient enzymatic degradation of polyethylene terephthalate (PET). A disulfide bridge was introduced to the polyester hydrolase TfCut2 to substitute its calcium binding site. The melting point of the resulting variant increased to 94.7°C (wild-type TfCut2: 69.8 °C) and its half-inactivation temperature to 84.6 °C (TfCut2: 67.3 °C). The variant D204C-E253C-D174R obtained by introducing further mutations at vicinal residues showed a temperature optimum between 75 and 80 °C compared to 65 and 70 °C of the wild-type enzyme. The variant caused a weight loss of PET films of 25.0 +/- 0.8% (TfCut2: 0.3 +/-0.1%) at 70 °C after a reaction time of 48 h. The results demonstrate that a highly efficient and calcium-independent thermostable polyester hydrolase can be obtained by replacing its calcium binding site with a disulfide bridge.

Indiana University-Purdue University Indianapolis (IUPUI) / Computational protein design aims at designing amino acid sequences that can fold into a target structure and perform a desired function. Many computational design methods have been developed and their applications have been successful during past two decades. However, the success rate of protein design remains too low to be of a useful tool by biochemists whom are not an expert of computational biology. In this dissertation, we first developed novel computational assessment techniques to assess several state-of-the-art computational techniques. We found that significant progresses were made in several important measures by two new scoring functions from RosettaDesign and from OSCAR-design, respectively. We also developed the first machine-learning technique called SPIN that predicts a sequence profile compatible to a given structure with a novel nonlocal energy-based feature. The accuracy of predicted sequences is comparable to RosettaDesign in term of sequence identity to wild type sequences. In the last two application chapters, we have designed self-inhibitory peptides of Escherichia coli methionine aminopeptidase (EcMetAP) and de novo designed barstar. Several peptides were confirmed inhibition of EcMetAP at the micromole-range 50% inhibitory concentration. Meanwhile, the assessment of designed barstar sequences indicates the improvement of OSCAR-design over RosettaDesign.

Genome engineering, the deliberate alteration of an organism's genetic material, has revolutionized biotechnology and biomedical research, enabling precise modifications to DNA sequences. Among the tools developed for this purpose, site-specific recombinases (SSRs) stand out for their ability to catalyze targeted DNA rearrangements between defined target sites. The Cre/loxP system, in particular, has been widely used for conditional gene inactivation and recombinase-mediated cassette exchange, facilitating targeted DNA excision, inversion, or integration through the recognition and recombination of loxP target sites. While the inherent specificity of Cre towards the loxP target sequence has been invaluable, it also limits its application to other genomic loci of therapeutic interest. Understanding the factors that govern the enzyme’s DNA specificity opens the possibility to engineer and retarget the complex to non-native sequences, significantly broadening the range of targetable genomic loci. To address this challenge, I describe the development of a high-throughput method to quantify Cre recombination efficiency across a library of loxP-like spacer variants. This method systematically analyzes the impact of spacer sequence alterations to reveal DNA specificity determinants. Through comprehensive screening, the study identified spacer sequences that exhibit inefficient recombination by Cre, despite both full lox sites having matching spacer sequences. Directed evolution was used to enhance Cre activity on these previously 'inert' spacer sequences, generating variants with altered spacer specificity. Detailed molecular analyses, including mutational studies and molecular dynamics simulations, elucidated the structural basis for altered spacer selectivity in evolved Cre variants. The study provides mechanistic insights into the role of specific amino acid residues in determining spacer specificity and highlights the potential for the rational design of recombinases with tailored spacer preferences. Building upon this foundation, I describe the engineering of heterospecific Cre-type SSRs capable of recombining asymmetric DNA target sites. By combining two evolved Cre variants with unique half-site specificities, a functional heterotetrameric complex forms, capable of excising DNA fragments flanked by asymmetric target sequences naturally occurring in the human genome. This approach expands the applicability of SSRs and holds promise for correcting chromosomal inversions underlying genetic disorders, as demonstrated in the correction of the int1h inversion associated with hemophilia A. However, harnessing the full potential of heterospecific SSRs presents challenges, particularly concerning off-target effects resulting from the formation of undesired functional homotetrameric complexes. To mitigate these risks, I investigated strategies to render SSR monomers functionally active in heterotetrameric, but not homotetrameric complexes. Through substrate-linked directed evolution, I identified mutations that confer obligate heterospecificity, leading to safer and more precise genome engineering applications. Together, these studies highlight the transformative potential of engineered SSRs in genome editing and underscore the importance of ongoing research efforts to enhance their specificity, efficacy, and safety for therapeutic interventions and biotechnological applications. By manipulating the highly specific Cre/loxP complex to retarget different lox sequences and analyzing evolved or naturally occurring recombinase recombination specificity, we can better understand how these enzymes can be optimized for therapeutic applications. Furthermore, the ability to confer obligate heterospecificity increases the overall safety of these engineered SSRs, expanding their potential applications in genome engineering, particularly for therapeutic targets that require editing asymmetric (non-palindromic) target sites.

The development of targeted therapy has contributed tremendously to the treatment of patients with cancer. The use of highly specific affinity proteins to target cancer cells has become a standard in treatment strategies for several different cancers. In light of this, many cancer cell markers are investigated for their potential use in diagnostics and therapy. One such marker is the human epidermal growth factor receptor 3, HER3. It has been established as an important contributor to many cancer types. The function of HER3 is to relay cell growth signals from outside of the cell to the inside. Interfering with- and inhibit- ing the function of HER3 has emerged as an interesting strategy for cancer therapeutics. The studies presented in this thesis aim to target HER3 with small, engineered affinity domain proteins for therapeutic purposes. Monomeric affibody molecules have previously been engineered to bind and inhibit HER3 in vitro. Due to the relatively low expression of HER3, an increase in valency appears promising to strengthen the therapeutic potential. Affibody molecules targeting the receptor were thus linked to form bivalent and bispecific constructs and evaluated both in vitro and in vivo. In the first study of this thesis affibody molecules specific for HER3 and HER2 were fused to an albumin binding domain to form bivalent and bispecific construct. The constructs inhibited ligand-induced receptor phos- phorylation of both HER2 and HER3 more efficiently than monomeric affibody molecules. A second approach to enhance the potential of affibody molecules in tumor targeting is described in the second study, where monomeric HER3-binding affibody molecules were engineered to increase their affinity for HER3. The resulting variants showed a 20-fold in- creased affinity and higher capacity to inhibit cancer cell growth. Combining the findings of the first two studies, the third study describes the evaluation of a HER3-targeting bivalent affibody construct for potential application as a therapeutic. Here, the bivalent construct inhibited cancer cell growth in vitro and was found to slow down tumor growth in mice, while being well tolerated and showing no visible toxicity. The fourth study built upon these findings and compares a very similar bivalent construct to the clinically-investigated HER3-specific monoclonal antibody seribantumab. The affibody construct showed very comparable efficacy with the antibody in terms of decreasing tumor growth rate and ex- tending mouse survival. Collectively, these works describe for the first time the use of alternative affinity protein constructs with therapeutic potential targeting HER3. / <p>QC 20170330</p>

Biocatalysis represents an attractive route to the production of various compounds which are difficult or impossible to synthesise and isolate using traditional chemical synthesis. In particular, the production of chiral molecules is a function ideally suited to biocatalysis, due to the natural stereospecificity of enzymes. The synthesis of such chiral molecules is essential in the production of pharmaceuticals, additives for the food and drinks industry and the creation of specialist polymers. CYP105AS1, isolated from Amycolatopsis orientalis, is a cytochrome P450 enzyme which produces the inactive 6-epi-pravastatin of the blockbuster anti-cholesterol drug pravastatin. Previous directed evolution efforts have engineered this enzyme to produce a five-point mutant, known as P450prava, which partially reversed the stereospecificity of the enzyme to produce a majority pravastatin product mixture. This thesis details work to use structure-led engineering approaches to redesign the active site of P450prava to introduce stringent stereospecificity. A combinatorial approach of manual and computational rational design was pursued, leading to the creation of a novel T95F/V180M double mutant of P450prava. This double mutant was found to have successfully eliminated the unwanted 6-epi-pravastatin enantiomer from the product mix, leaving a pure pravastatin product. P450prava was also shown to bind and hydroxylate other statin substrate molecules, demonstrating its versatility in the production of drug metabolites and other high-value oxyfunctionalised molecules. This property, along with its proven tolerance of significant active site engineering efforts, demonstrates the viability of the P450prava as a platform for the creation of novel biocatalysts for the production of various hydroxylated products from diverse substrate molecules.

A specific double-stranded DNA sensing system is of great interest for diagnostic and other biomedical applications. Zinc finger domains, which recognize double-stranded DNA, can be engineered to form custom DNA-binding proteins for recognition of specific DNA sequences. As a proof of concept, a sequence-enabled reassembly of TEM-1 β- lactamase system (SEER-LAC) was previously demonstrated to develop zinc finger protein (ZFP) arrays for the detection of a double-stranded bacterial DNA sequence. Here, we implemented the SEER-LAC system to demonstrate the direct detection of pathogenspecific DNA sequences present in E. coli O157:H7 on the lab-on-a chip. ZFPs customdesigned to detect shiga toxin in E. coli O157:H7 were immobilized on the cyclic olefin copolymer (COC) chip, which can function as a non-PCR based molecular diagnostic. Pathogen-specific double-stranded DNA was directly detected by engineered ZFPs immobilized on the COC chip, providing a detection limit of 10 fmole of target DNA in colorimetric assay. Therefore, in this study, we demonstrated a great potential of ZFP arrays on the COC chip for further development of a simple and novel lab-on-a chip technology for detection of pathogens. Antibiotic resistance is a serious, and rapidly growing global threat. Here, we designed a novel screening method to detect antibiotic resistance genes (ARGs) in bacteria using a graphene oxide-based biosensor utilizing engineered ZFPs. Two-dimensional graphene oxide (GO) sheet possesses unique electronic, thermal, and mechanical properties. The quenching ability of GO can create novel methods for detection of biomolecules. Our approach utilizes quenching of fluorescence signal by GO in the absence of target ARGs, but restoring the signal in the presence of target ARGs. Quantum dot (QD)- labeled ZFP can bind to GO via stacking interactions of aromatic and hydrophobic residues in conjunction with hydrogen bonding interaction between hydroxyl or carboxyl groups of GO and hydroxyl or amine groups of the protein. Due to fluorescence resonance energy transfer (FRET) between QD and GO when they are in close proximity, fluorescence signal of QD-labeled ZFP is expected to be quenched. In the presence of target DNA, the bound DNA-protein complex is released from GO, restoring the fluorescence signal.