Protein and Peptide Based Sensors and Switches

This thesis describes the design, development and examination of new materials and reactions from naturally occurring molecules and systems for applications in medicinal and material science. Chapter 1 describes the development of a pyridoxal-5'-phosphate (PLP) based bioconjugation strategy. As an enzyme cofactor, PLP forms an imine with aliphatic amines under physiological conditions. Kinetic parameters of forming hydrazones and oximes with PLP were determined. A convenient phosphate/amine conjugation protocol was developed to covalently link PLP to proteins, affording proteins capable of hydrazone formation with bioorthogonal hydrazinyl functional groups. Different proteins were labeled with PLP and sequential in vitro experiments showed that such protein-PLP conjugates were capable of efficient cell labeling. Chapter 2 describes the development of targeted magnetic resonance imaging (MRI) agents using an antibody (D93) and peptides (LK) that bind selectively to damaged/denatured collagen type-IV. A small MRI contrast agent was conjugated onto D93. The conjugate was characterized and in vivo experiments with mice showed excellent contrast enhancement. The work with LK peptides was focused on characterization and functionalization with specific side chain modification. Chapter 3 describes the rational design and development of peptides that adopt different conformations when bound to copper ions at different oxidation states and switch between those conformations in response to redox stimuli. The first generation of peptides was designed with glutamic acid residues incorporated for Cu(II) binding. The peptide, in the presence of copper ions, was reversibly reconfigured between alpha-helical monomer to beta-sheet aggregates upon oxidation and reduction of Cu(I)/Cu(II). The second generation of peptides was further modified with methionine or histidine residues for Cu(I) binding. These peptides were at a more alpha-helical state when Cu(I) was present, while Cu(II) still made the peptides adopt the beta-sheet state. Chapter 4 describes the development of a carbon nanosheet (CNS) based enzymatic electrode which displayed ultra-high current response through the bioelectrocatalytic oxidation of glucose by glucose oxidase. Both as-manufactured and acid-treated CNS were tested and both showed large enhancement of current. Different immobilization methods were tested, which proved that both physical absorption and covalent linkage resulted in stable enzymatic modification.