This thesis details the novel theory and characterization of surface-induced peptide folding as a mechanism of biological activation with potential in targeted drug delivery and activity. The design of two hydroxyapatite (HA)-folding peptides was based on the bone-binding protein, osteocalcin. The amino-terminal helix of osteocalcin contains three γ-carboxyglutamic acid (Gla) residues on the HA-binding face of the folded protein in an i, i+4, i+7 motif. This motif was used to design two HA-binding peptides with high helicity and monomeric structure.
Based on the folding propensity of control peptides containing glutamic acid (Glu) in place of the Gla residues, the 36-residue peptide, JAK1, was favored. JAK1 displayed calcium-induced helical folding, which was similar to that observed for osteocalcin. Circular dichroism showed that JAK1 bound to HA as a helix, reversibly unfolded, and desorbed from HA at high temperatures. This is the first de novo designed peptide to fold in a surface-induced manner and the first circular dichroism method capable of examining peptide structure HA-particles.
Surface binding was characterized by X-ray photoelectron spectroscopy (XPS), absolute coverage, and binding affinity. These studies showed that JAK1 coverage was nearly quantitative and Gla-dependent. The affinity of JAK1 was nearly twice that of BSA and the dissociation constant was similar to that of osteocalcin.
The force of the Gla-calcium interaction was directly measured using an atomic force microscope (AFM). Model pentapeptides, α-helices, and proteins all showed greater bond-rupture forces when Gla residues were used in place of Glu residues. This study showed that the bond-rupture force associated with osteocalcin was higher than that of BSA, which may explain its higher coverage, binding affinity, and concentration in bone.
A ligand-bound variant of JAK1 was designed, and evaluated for osteogenic potential. This study showed that surface-bound BMP2 can enhance osteogenesis at the 3-day time point. Lastly, the adsorption of an amphiphilic β-hairpin peptide to hydrophobic surfaces was characterized by showing the role of peptide folding and concentration on adsorbate structure. These studies show the feasibility of designing surface-folding peptides and outline their application towards targeted drug delivery.
|Advisor:||Beebe, Thomas P., Jr.|
|Commitee:||Johnston, Murray, Rabolt, John, Schneider, Joel|
|School:||University of Delaware|
|Department:||Department of Chemistry and Biochemistry|
|School Location:||United States -- Delaware|
|Source:||DAI-B 70/07, Dissertation Abstracts International|
|Subjects:||Analytical chemistry, Biochemistry, Organic chemistry, Biophysics|
|Keywords:||Carboxyglutamic, Circular dichroism, Peptide folding, Surface folding|
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