No description available.

Post-translational modifications (PTMs), which biochemically modify proteins to generate diversity and control functionality, are important in the field of chemical biology research. However, typical bioconjugation methods involving nucleophilic addition of cysteine and lysine residues are limited by their lack of site-specificity. In this thesis, we have demonstrated the site-selective chemical modification of a variety of proteins through protein halogenation and subsequent pallado-biology strategies. The "Tag-and-Modify" approach, developed previously in our group and involving the creation of a protein tag for further manipulation, is a useful tool for site-selective chemical modification. Here, we envisioned extending its utility to a novel concept of [¹⁸F]-radiolabelling of proteins involving direct electrophilic fluorination, thus forming a C-F bond at a specific modification site and leading to the generation of protein species applicable for in vivo [¹⁸F]-PET imaging and diagnosis. We also explored several alternative methods for the installation of C-Br/C-I bonds onto a protein of interest. Our first attempts involved the biosynthetic incorporation of a synthetic bromotryptophan into the protein, however these processes were unsuccessful. As a workaround, we found IPy₂BF₄, an iodinating agent, to be very effective as a direct method for the site-specific installation of C-I bonds on tyrosine/histidine residues of proteins. We subsequently demonstrated novel and efficient palladium-catalysed cross-couplings of the resultant iodinated tyrosine/histidine moieties, leading to the creation of new C-C bonds. This approach was compatible with a wide range of functionally diverse boronic acids, using a water-soluble and phosphine-ligand-free Pd-complex catalyst under fully aqueous conditions. Our novel, successful method for C-C bond formation onto proteins is potentially applicable to the initial investigation of challenging in vivo Pd cross-coupling of thyroglobulin, based on our achievement of protein labelling under ex vivo conditions. In summary, we are able to create new chemical tools for the site-selective/site-specific chemical modification of proteins, enabling their use as biological probes.

Bioorthogonal reactions, due to its excellent selectivity and time-efficiency, have emerged as a popular tool for protein and cell probing. Among all the bioorthogonal reactions, the inverse electron-demand Diels-Alder reaction (IEDDA) reaction has its advantage of bearing the fastest kinetics. Although the IEDDA reaction drew considerable attention in chemical biology in the last decade, challenges lie in finding the suitable dienophiles. Strained dienophiles, for example, trans-cyclooctene derivatives, can undergo ultrafast IEDDA reactions and therefore have been extensively developed. Unstrained alkenes and alkynes, however, have not been well investigated as IEDDA handles. In general, unstrained dienophiles are more straightforward to synthesise compared with strained dienophiles, therefore they are more accessible to researchers. In addition, the absence of a highly reactive bond makes unstrained dienophiles inert to biological nucleophiles, which allows effectively cellular labelling. In this dissertation, I described three different unstrained dienophiles for different biological purposes. Allyl handle is thiol-stable and non-toxic, which was utilised to label apoptotic cells in a pre-targeting manner. Enol ethers can react with tetrazines to decage protected amino acids and prodrugs. Potassium arylethynyltrifluoroborate, as a novel dienophile, was shown to react fast with pyridyl tetrazines controllably and this new IEDDA was applied to label proteins site-selectively and to fluorescently label two proteins orthogonally. In addition to IEDDA reactions, other bioorthogonal reactions were also developed using these versatile unstrained handles. Allyl-bearing amino acids and proteins can undergo an acetophenone-mediated hetero-[2+2] photocycloaddition with maleimide derivatives, expanding the toolbox of photo-triggered chemistry for protein modification. The potassium arylethynyltrifluoroborate handle was also found reactive in copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) and showcased the huge potential for protein labelling and multicolour cellular labelling.

Doctor of Philosophy / Department of Grain Science and Industry / X. Susan Sun / Oilseed protein is a promising renewable source to be used as the replacement of petroleum-based materials for adhesion purpose, and it has drawn increasing attention since soy-based adhesives were developed for wood glues. However, soy protein comprises a portion of humans’ diets, thereby creating competition between utilization of soy protein for protein-based products or human food. Therefore, alternative bio-resources must be discovered. Proteins from camelina sativa provide such potential. Similar to other protein-based polymers, low mechanical strength and poor water resistance are the major drawbacks limiting camelina protein’s further applications. In this research, camelina protein (CP) was modified by unfolding, crosslinking, and co-polymerization treatment for improved flow-ability, adhesion properties and water resistance, which facilitates the industrialization of camelina as an alternative to soy-based adhesives. The physicochemical properties and microstructures of CP were also investigated. To increase the reactivity of CP adhesive, the first step is to denature the folded structure of native proteins. Camelina protein was extracted from defatted camelina meal through alkali solubilization and acid precipitation and modified with varying amount of NaHSO₃ (0-12% of the protein dry base) and Gdm.Cl (0-250% of the protein dry base). NaHSO₃ treatment broke the disulfide bonds of the CP and thus increased its free sulfhydryl content and surface hydrophobicity. As NaHSO₃ concentration increased, the viscosity, elastic modulus (G') and water resistant of NaHSO₃-modified camelina protein (SMCP) dispersion decreased, and the protein became hydrophobic. Gdm.Cl treatment broke the CPI’s hydrogen bonds but decreased their surface hydrophobicity. Similarly, viscosity, G', and water resistant of Gdm.Cl-modified camelina protein (GMCP) dispersions decreased as Gdm.Cl increased and protein became to aggregate. The reducing effect of NaHSO₃ was more obvious than Gdm.Cl to disrupt CPI’s intermolecular protein interaction but less obvious than Gdm.Cl to reduce the viscosity and water resistant. To further increase the CP’s water resistance, a coupling agent, Ethyl-3-(3-dimethyl-aminopropyl-1-carbodiimide) (EDC), was applied to stabilize the protein structure by crosslinking the free carboxyl groups and amino groups. The cross-linked CP exhibited increased molecular weight and particle size. Microstructures of modified CP also became rigid and condensed. Accordingly, CP’s increased intermolecular protein interaction resulted in its higher elastic modulus, viscosity and water resistance. The ultrasound pretreatment further increased the crosslink degree of CP, which resulted in protein’s increased aggregation behaviors and compact micro-structures. Consequently, the elastic modulus, viscosity, and water resistance of CP increased accordingly. Copolymerization with hydrophobic enhancers was also an effective method to improve CP’s water resistance. In this study, kraft lignin was oxidized by H₂O₂ and then copolymerized with CP as wood adhesives, which exhibited increased wet strength. In the presence of ultrasound irradiation, the H₂O₂-depolymerized kraft lignin exhibited reduced particle size, thermal stability and increased content of hydroxyl groups. Fluorescence spectroscopy analysis revealed that after coupling with pristine or de-polymerized lignin, CP exhibited increased hydrophobicity due to lignin’s increased reactivity with camelina protein. Accordingly, the water resistance of CP-based adhesives improved. In the optimized condition, when CP was copolymerized with ultrasound-induced oxidized lignin, it had increased wet shear adhesion strength from 0.28 MPa to 1.43 MPa, with wood panels passing the three-cycle water soaking test.

No description available.

<p>We have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics. </p><p>We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein-small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced.</p><p>We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells</p> / Dissertation

<p>Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates.</p><p>Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity.</p><p>To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations. </p><p>The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide. </p><p>The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.</p>

Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates. Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity. To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations. The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide. The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.

The need for an alternative to red cells in transfusions has led to the creation of hemoglobin-based oxygen carriers (HBOCs). However, evaluations of all products tested in clinical trials have noted cardiovascular complications, raising questions about their safety that led to the abandonment of all those products. It has been considered that the adverse side effects come from the scavenging of the vasodilator – nitric oxide (NO) by the deoxyheme sites of the hemoglobin derivatives. Another observation is that HBOCs with lower oxygen affinity than red cells release oxygen prematurely in arterioles, triggering an unwanted homeostatic response. Since the need for such a product remains critical, it is important to understand the reactivity patterns that contribute to the observed complications. Various alterations of the protein have been attempted in order to reduce HBOC-induced vasoconstriction. Recent reports suggest that a safe and effective product should be pure, homogenous and have a high molecular weight along with appropriate oxygenation properties. While these properties are clearly important, vasodilatory features of hemoglobin through its nitrite reductase activity may also act as an in situ source of NO. It follows that HBOCs with an enhanced ability to produce NO from endogenous nitrite may serve to counteract vasoactivity associated with NO-scavenging by hemoglobin. Here we characterize the effects of different protein modifications on the nitrite reductase activity of hemoglobin. We produced a variety of HBOCs that include cross-linked tetramers, polyethylene glycol (PEG) conjugates and bis-tetramers of hemoglobin. We report that the rate of NO production strongly depends on the conformational state of the protein, with R-state stabilized proteins (PEG-Hbs), exhibiting the fastest rates. In particular, we found that PEGylated bis-tetramers of hemoglobin (BT-PEG) exhibit increased nitrite reductase activity while retaining cooperativity and stability. Animal studies of BT-PEG demonstrated that this material is benign: it did not cause significant increases in systemic blood pressure in mice, the major side effect associated with existing HBOCs. BT-PEG exhibits an enhanced nitrite reductase activity together with sample purity and homogeneity, molecular size and shape, and appropriate oxygenation properties, characteristics of a clinically useful HBOC.

The need for an alternative to red cells in transfusions has led to the creation of hemoglobin-based oxygen carriers (HBOCs). However, evaluations of all products tested in clinical trials have noted cardiovascular complications, raising questions about their safety that led to the abandonment of all those products. It has been considered that the adverse side effects come from the scavenging of the vasodilator – nitric oxide (NO) by the deoxyheme sites of the hemoglobin derivatives. Another observation is that HBOCs with lower oxygen affinity than red cells release oxygen prematurely in arterioles, triggering an unwanted homeostatic response. Since the need for such a product remains critical, it is important to understand the reactivity patterns that contribute to the observed complications. Various alterations of the protein have been attempted in order to reduce HBOC-induced vasoconstriction. Recent reports suggest that a safe and effective product should be pure, homogenous and have a high molecular weight along with appropriate oxygenation properties. While these properties are clearly important, vasodilatory features of hemoglobin through its nitrite reductase activity may also act as an in situ source of NO. It follows that HBOCs with an enhanced ability to produce NO from endogenous nitrite may serve to counteract vasoactivity associated with NO-scavenging by hemoglobin. Here we characterize the effects of different protein modifications on the nitrite reductase activity of hemoglobin. We produced a variety of HBOCs that include cross-linked tetramers, polyethylene glycol (PEG) conjugates and bis-tetramers of hemoglobin. We report that the rate of NO production strongly depends on the conformational state of the protein, with R-state stabilized proteins (PEG-Hbs), exhibiting the fastest rates. In particular, we found that PEGylated bis-tetramers of hemoglobin (BT-PEG) exhibit increased nitrite reductase activity while retaining cooperativity and stability. Animal studies of BT-PEG demonstrated that this material is benign: it did not cause significant increases in systemic blood pressure in mice, the major side effect associated with existing HBOCs. BT-PEG exhibits an enhanced nitrite reductase activity together with sample purity and homogeneity, molecular size and shape, and appropriate oxygenation properties, characteristics of a clinically useful HBOC.