NADPH:cytochrome P450 reductase (CPR) is a 78 kDa flavoprotein, which is together with cytochrome P450 component of monooxygenase system bound in the membrane of the endoplasmic reticulum. Monooxygenase system is involved in the metabolism of a wide range of organic substances, including drugs or various pollutants present in the environment (polycyclic aromatic hydrocarbons, aromatic amines, etc.). CPR works as a transporter of reducing equivalents from NADPH to the cytochromes P450. For proper interaction with cytochromes P450, intact N-terminal hydrophobic domain anchoring protein in the membrane is needed. Removing this domain, e.g. during trypsin proteolysis, gives rise a soluble CPR (72 kDa) and cause loss of catalytic activity towards cytochrome P450. During heterologous expression in E. coli proteolytically sensitive site of CPR (Lys56 - Ile57) is cleaved by intracellular trypsin-like proteases, that may negatively affect the yields of native 78 kDa protein. This thesis describes the heterologous expression, purification and characterization of two forms of rat CPR. WtCPR is a protein naturally occurring in rats (Wistar strain), while mCPR contains one amino acid substitution (K56Q) in the site of proteolytic degradation. The result of that substitution is proteolytically stable CPR,...

PEGylation can improve the pharmacokinetic properties of protein therapeutics via decreasing renal clearance and shielding the protein surface from proteases, antibody neutrailization, and aggregation. Conformational stability enhancement can provide criteria for the identification of optimal sites for PEGylation, but how PEG influence the noncovalent interactions from the surface of proteins has not been well illustrated. Macrocyclization can effectively enhance the conformational stability of small peptides and large proteins. Combination of PEG-based conformational stability enhancement and macrocyclization-based conformational constraint has not been explored. Macrocycliziation has been employed to stabilize protein tertiary structures, but there are no general guidelines for interhelical staple to stabilize coiled-coil motifs of proteins. Chapter 1 is an introduction to peptide stapling and macrocyclization of proteins. Chapter 2 describes our test of the hypothesis that PEG increases the conformational stability of proteins by desolvating nearby salt bridges. In chapter 3, we explore the combination of PEG-based conformational stability enhancement with macrocyclization on WW domain, and find that the most important criteria for PEG stapling is ensuring the side chains cross-linked by PEG are distant in primary sequence but close in tertiary structure. In chapter 4, we further apply this macrocyclization criteria to another -sheet-based protein, SH3 domain of the chicken Src protein, and to a disulfide-bonded parallel coiled-coil heterodimer derived from the yeast transcription factor GCN4. In chapter 5, we explore the determinants of PEG-staple-based stabilization by changing the distance of the staple to the terminal interhelical disulfide bond, varying the length of staple, exploring different solvent exposed positions for stapling and employing heterochiral residues for stapling. We further apply the interhelical PEG staple to a HER-2 affibody, and find that PEG-stapling increases the conformational stability and proteolytic resistance of the stapled affibody relative to its non-stapled counterpart and to the native unmodified affibody.

This dissertation primarily covers the impact of bulky ,-dehydroamino acids on the proteolytic stability and enhanced folding of -hairpins. It partly describes the progress towards the total synthesis of yakuamide A, a potent anticancer peptide with an IC50 value of 14 ng/mL against leukemia cells. Proteins and peptides are a very attractive source of potential medicinal agents as they can target various protein“protein interactions that are implicated in several diseases and disorders. The global sales of peptide drugs in 2013 were estimated to be about $28 billion and are constantly rising at an appreciable rate. However, peptide drugs have a short plasma half-life because of their susceptibility to proteolysis. Multiple approaches have been discovered to overcome this shortcoming, but there is still an urgent need for better peptidomimetics to increase the stream of peptides entering the pharmaceutical market. Here, it has been demonstrated that the incorporation of a bulky ,-dehydroamino acid in the turn regions of -hairpins can substantially increase their proteolytic stability and folding. Insertion of a dehydrovaline (ΔVal) residue at the i+1 position imparted ca. 7-fold increase in proteolytic resistance and ca. 15% increase in folding when compared to the parent peptide. Since the insertion of a bulky ,-dehydroamino acid into the turn regions of -hairpins can promote proteolytic stability without perturbing the secondary structures, it is believed that this novel approach is very promising in stabilizing bioactive turn-containing peptides for therapeutic use.Yakuamide A is a medium-sized peptide that contains several bulky dehydroamino acids, -hydroxyamino acids and unique N- and C-termini. It has an unprecedented anticancer profile, and potent bioactivity, hence it was imperative to accomplish its total synthesis to elicit its unique mode of action and biological target. More efficient methods were developed to synthesize bulky dehydroamino acids and -hydroxyamino acids. A regioselective base-free aminohydroxylation was developed for the synthesis of -hydroxyamino acids. The major focus was the three-step synthesis of the N-terminal acyl group from a known compound by a one-pot indium-catalyzed cross-Claisen condensation/reduction and the synthesis of (2S,3R)--hydroxyisoleucine, and racemic -hydroxyisoleucine, which are the precursors of E- and Z-dehydroisoleucine.