Proteins that have been modified by attaching them to a surface or to a polyethylene glycol (PEG) molecule can see many uses in therapeutics and diagnostics -- these unique proteins are called protein devices. Current techniques can perform these functionalizations at a specific residue on the protein, but what remains is identifying what happens to protein structure when mutated, and where to perform the attachment. Both of these issues can be examined using molecular dynamic (MD) simulations. Currently, simulations of the unnatural amino acid (uAA) mutations necessary for protein device functionalization cannot be executed, and full-protein screens of all possible protein device models have never been attempted. Results from this dissertation first employs a new model for simulating PEGylated protein devices building off of previous studies that explore where to attach functional groups. Next, many current assumptions in the community regarding ideal attachment sites are examined. Some of these factors include primary chain location, amino acid type, solvent accessibility, and secondary structure. The focus then turns to novel tertiary structure factors that could influence how well attachment locations affect overall protein device stability. The usefulness of each factor is analyzed to show what factors provided the best predictive power for a site's performance in the screen. A general heuristic is given that could aid in future screens of other protein devices to reduce compute time and quickly identify sites for experimental examination. To explore uAA mutation effects on protein structure, parameters are developed for linear moiety R-groups present in these novel amino acids. The CHARMM and CGenFF force fields currently lack parameters for most linear-angle molecular moieties. This work proposes a method that (1) develops CHARMM parameters for four small molecules that contain terminal azido and alkynyl groups using ffTK, (2) addresses linearity issues, and (3) validates ffTK results via in silico MD simulation. Dihedral analysis examines the linear-angle-containing dihedrals and compares methods for the moiety parameterization. Next, the small molecule parameters are combined with CGenFF to generate parameters for unnatural amino acid MD simulation in a protein. Finally, validation confirms the parameters derived in this work to appropriately simulate unnatural amino acids and small molecules with azido and alkynyl groups.

Proteins that have been modified by attaching them to a surface or to a polyethylene glycol (PEG) molecule can see many uses in therapeutics and diagnostics -- these unique proteins are called protein devices. Current techniques can perform these functionalizations at a specific residue on the protein, but what remains is identifying what happens to protein structure when mutated, and where to perform the attachment. Both of these issues can be examined using molecular dynamic (MD) simulations. Currently, simulations of the unnatural amino acid (uAA) mutations necessary for protein device functionalization cannot be executed, and full-protein screens of all possible protein device models have never been attempted. Results from this dissertation first employs a new model for simulating PEGylated protein devices building off of previous studies that explore where to attach functional groups. Next, many current assumptions in the community regarding ideal attachment sites are examined. Some of these factors include primary chain location, amino acid type, solvent accessibility, and secondary structure. The focus then turns to novel tertiary structure factors that could influence how well attachment locations affect overall protein device stability. The usefulness of each factor is analyzed to show what factors provided the best predictive power for a site's performance in the screen. A general heuristic is given that could aid in future screens of other protein devices to reduce compute time and quickly identify sites for experimental examination. To explore uAA mutation effects on protein structure, parameters are developed for linear moiety R-groups present in these novel amino acids. The CHARMM and CGenFF force fields currently lack parameters for most linear-angle molecular moieties. This work proposes a method that (1) develops CHARMM parameters for four small molecules that contain terminal azido and alkynyl groups using ffTK, (2) addresses linearity issues, and (3) validates ffTK results via in silico MD simulation. Dihedral analysis examines the linear-angle-containing dihedrals and compares methods for the moiety parameterization. Next, the small molecule parameters are combined with CGenFF to generate parameters for unnatural amino acid MD simulation in a protein. Finally, validation confirms the parameters derived in this work to appropriately simulate unnatural amino acids and small molecules with azido and alkynyl groups.

abstract: Continuing and increasing reliance on fossil fuels to satisfy our population’s energy demands has encouraged the search for renewable carbon-free and carbon-neutral sources, such as hydrogen gas or CO2 reduction products. Inspired by nature, one of the objectives of this dissertation was to develop protein-based strategies that can be applied in the production of green fuels. The first project of this dissertation aimed at developing a controllable strategy to incorporate domains with different functions (e. g. catalytic sites, electron transfer modules, light absorbing subunits) into a single multicomponent system. This was accomplished through the rational design of 2,2’-bipyridine modified dimeric peptides that allowed their metal-directed oligomerization by forming tris(bipyridine) complexes, thus resulting in the formation of a hexameric assembly. Additionally, two different approaches to incorporate non-natural organometallic catalysts into protein matrix are discussed. First, cobalt protoporphyrin IX was incorporated into cytochrome b562 to produce a water-soluble proton and CO2 reduction catalyst that is active upon irradiation in the presence of a photosensitizer. The effect of the porphyrin axial ligands provided by the protein environment has been investigated by introducing mutations into the native scaffold, indicating that catalytic activity of proton reduction is dependent on axial coordination to the porphyrin. It is also shown that effects of the protein environment are not directly transferred when applied to other reactions, such as CO2 reduction. Inspired by the active site of [FeFe]-hydrogenases, the second approach is based on the stereoselective preparation of a novel amino acid bearing a 1,2-benzenedithiol side chain. This moiety can serve as an anchoring point for the introduction of metal complexes into protein matrices. By doing so, this strategy enables the study of protein interactions with non-natural cofactors and the effects that it may have on catalysis. The work developed herein lays a foundation for furthering the study of the use of proteins as suitable environments for tuning the activity of organometallic catalysts in aqueous conditions, and interfacing these systems with other supporting units into supramolecular assemblies. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019

Thesis advisor: Abhisheck Chatterjee / The genetic codon expansion (GCE) is a technique that uses an orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair to incorporate noncanonical amino acids (ncAA) into proteins, to enable more protein-based chemistry. In the past two decades, more than 200 ncAAs have been site-specifically introduced into proteins in E. coli, and facilitated studies of protein structures, functions and interaction with other molecules. Although a large variety of ncAAs are available for incorporation in the bacterial systems, significantly fewer ncAAs are accessible for incorporation in eukaryotic cells. An expanded GCE toolbox will be beneficial for numerous applications in eukaryotic systems. Currently, introducing ncAAs in eukaryotes predominantly relies on the archaeal pyrrolysyl tRNA/aaRS pair. Such a strong dependence on a single platform has precluded genetic encoding of many desirable ncAAs, including structural mimics of many important post-translational modifications. The work presented in this thesis first developed an engineered E. coli leucyl tRNA/aaRS pair to enable site-specific incorporation of citrulline, an important PTM, into proteins expressed in mammalian cells. This technology was used to reveal the role of citrullination on site R372 and R374 of PAD4. Additionally, aiming at genetically encoding more diverse ncAAs, all 20 E. coli derived tRNA/aaRS pairs were screened for their ability to suppress TAG and TGA in mammalian cells. This study revealed several tRNA/aaRS pairs that are suitable for ncAA incorporation in mammalian cells, including those selective for phenylalanine, lysine, arginine, serine and glutamine. Efforts are currently under way to engineer these pairs to genetically encode new structural classes of ncAAs. / Thesis (PhD) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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While protein therapeutics are indispensable in the treatment of a variety of diseases, including cancer, rheumatoid arthritis, and diabetes, key limitations including short half-lives, high immunogenicity, protein instability, and centralized production complicate long-term use and on-demand production. Site-specific polymer conjugation provides a method for mitigating these challenges while minimizing negative impacts on protein activity. However, the location-dependent effects of polymer conjugation are not well understood. Cell-free protein synthesis provides direct access to the synthesis environment and rapid synthesis times, enabling rapid evaluation of multiple conjugation sites on a target protein. Here, work is presented towards developing cell-free protein synthesis as a platform for both design and on-demand production of next generation polymer-protein therapeutics, including (1) eliminating endotoxin contamination in cell-free reagents for simplified therapeutic preparation, (2) improving shelf-stability of cell-free reagents via lyophilization for on-demand production, (3) coupling coarse-grain simulation with high-throughput cell-free protein synthesis to enable rapid identification of optimal polymer conjugation sites, and (4) optimizing cell-free protein synthesis for production of therapeutic proteins

RNAs have become increasingly recognized as possible drug targets due to their involvement in important biochemical functions, as well as their unique but well-defined structures. Recently published crystal structures depict the binding of a series of aminoglycosides- or more specifically- 2-deoxystretamine (2-DOS), the most preserved central scaffold of aminoglycosides, to a conserved 5'-GU-3'region on their target RNAs. A novel unnatural γ-amino acid, 1, has been synthesized using 2-deoxystreptamine as a template through structure-based rational design. The unnatural amino acid has been designed to replace a glycosidic linkage with an amide bond, which may limit the promiscuous binding characteristics of aminoglycosides through increased rigidity of the ligands and additional hydrogen bonding. The binding selectivity and affinity will be studied in the future through a fluorescence assay.

<p>The glutathione transferases (GSTs) represent a superfamily of dimeric proteins involved in cellular detoxication by catalyzing the nucleophilic addition of the reduced glutathione (GSH) to the hydrophobic electrophiles. The present work focuses on the functional role of the conserved structures of GSTP1-1. The lock-and-key motif is a highly conserved hydrophobic interaction in the subunit interface of Pi, Mu, and Alpha class GSTs. The key residue (Tyr⁵⁰ in hGSTP1-1) of one subunit is wedged into a hydrophobic pocket of the neighboring subunit. The heterodimer GSTP1/Y50A was constructed from the fully active wild-type GSTP1-1 and the nearly inactive Y50A in order to study how an essentially inactive subunit influences the activity of the neighboring subunit. The results illuminate the vital role of the lock-and-key motif in modulating the GSH binding and the rate of catalysis. Additionally, the two active sites of the dimeric enzyme work synergistically. An observed water network, in hGSTP1-1 structures, connects the two active sites, thereby offering a mechanism for communication between the two active sites.</p><p>Cys⁴⁸ and Tyr⁵⁰ were targeted by mutations and chemical modifications for understanding how the α2 loop residues modulate GSH binding and catalysis. The replacement of Tyr⁵⁰ with different unnatural amino acids showed that the nature of the key residue side-chain influences the interaction with the lock structure and, consequently, the catalytic activity. The K_M^GSH, GSH affinity and protein stability can be modulated by fitting key residue into the lock cavity of the neighbor subunit and, consequently, restriction of the flexibility of the α2 loop. Optimization of the interaction between the key residue and the lock-cavity increases k_cat. Also, the crystal structure of the Cys-free variant was determined. The result indicated that Cys⁴⁸ restricts the flexibility of the α2 loop by interacting with surrounding residues and, consequently, contributes to GSH binding and protein stability.</p>

The glutathione transferases (GSTs) represent a superfamily of dimeric proteins involved in cellular detoxication by catalyzing the nucleophilic addition of the reduced glutathione (GSH) to the hydrophobic electrophiles. The present work focuses on the functional role of the conserved structures of GSTP1-1. The lock-and-key motif is a highly conserved hydrophobic interaction in the subunit interface of Pi, Mu, and Alpha class GSTs. The key residue (Tyr50 in hGSTP1-1) of one subunit is wedged into a hydrophobic pocket of the neighboring subunit. The heterodimer GSTP1/Y50A was constructed from the fully active wild-type GSTP1-1 and the nearly inactive Y50A in order to study how an essentially inactive subunit influences the activity of the neighboring subunit. The results illuminate the vital role of the lock-and-key motif in modulating the GSH binding and the rate of catalysis. Additionally, the two active sites of the dimeric enzyme work synergistically. An observed water network, in hGSTP1-1 structures, connects the two active sites, thereby offering a mechanism for communication between the two active sites. Cys48 and Tyr50 were targeted by mutations and chemical modifications for understanding how the α2 loop residues modulate GSH binding and catalysis. The replacement of Tyr50 with different unnatural amino acids showed that the nature of the key residue side-chain influences the interaction with the lock structure and, consequently, the catalytic activity. The KMGSH, GSH affinity and protein stability can be modulated by fitting key residue into the lock cavity of the neighbor subunit and, consequently, restriction of the flexibility of the α2 loop. Optimization of the interaction between the key residue and the lock-cavity increases kcat. Also, the crystal structure of the Cys-free variant was determined. The result indicated that Cys48 restricts the flexibility of the α2 loop by interacting with surrounding residues and, consequently, contributes to GSH binding and protein stability.

Chemical biology encompasses the study and manipulation of biological system using chemistry, often by virtue of small molecules or unnatural amino acids. Much insight has been gained into the mechanisms of biological processes with regards to protein structure and function, metabolic processes and changes between healthy and diseased states. As an ever expanding field, developing new tools to interact with and impact biological systems is an extremely valuable goal. Herein, work is described towards the synthesis of a small library of heterocyclic-containing small molecules and the mechanistic details regarding the interesting and unexpected chemical compounds that arose; an alternative set of non-toxic copper catalyzed azide-alkyne click conditions for in vivo metabolic labelling; and the synthesis of an unnatural amino acid for further chemical modification via [3+2] cycloadditions with nitrones upon incorporation into a peptide of interest. Altogether, these projects strive to supplement pre-existing methodology for the synthesis of small molecule libraries and tools for metabolic labelling, and thus provide further small molecules for understanding biological systems.