Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Darrin J. Pochan, Dept. of Materials Science & Engineering. Includes bibliographical references.

A series of polypeptides with well-defined sequences, (Asp3Phe1)n, (Asp2Phe1)n, (Asp1Phe1)n, (Asp1Phe2)n, and (Asp1Phe3)n, containing the hydrophilic amino acid, aspartic acid (Asp) and the hydrophobic amino acid, phenylalanine (Phe), were synthesized. Their behaviour in aqueous solution was investigated by performing fluorescence quenching and non-radiative energy transfer (NRET) experiments which were complemented by dynamic (DLS) and static (SLS) light scattering. The photophysical properties of the polypeptides were dependent on their Phe content. An increase in the Phe content led to an increase in the extinction coefficient, fluorescence quantum yield, and fluorescence average lifetime of the polypeptides. Circular dichroism experiments revealed that except for the (Asp1Phe3)n polypeptide, which adopts an alpha-helical conformation in aqueous solution, the other polypeptides did not adopt any known conformation in solution. The fluorescence quenching studies performed using molecular pyrene physically bound to the polypeptide via hydrophobic interactions resulted in protective quenching for the (Asp1Phe1)n, (Asp1Phe2)n, and (Asp1Phe3)n polypeptides, with some pyrenes having a lifetime of ~300 ns. Evidence for protective quenching was also observed in the polypeptides richer in Phe, when pyrene was covalently attached onto the polypeptides. However pyrene was found to be fully exposed to the quencher solution for the more hydrophilic polypeptides. The presence of NRET between a naphthalene labeled polypeptide and a pyrene labeled polypeptide for the (Asp1Phe2)n and (Asp1Phe3)n polypeptides and its absence for the (Asp3Phe1)n, (Asp2Phe1)n, and (Asp1Phe1)n polypeptides led to the conclusion that those polypeptides richer in Phe generate interpolymeric aggregates which provide protection to a hydrophobic cargo like molecular pyrene whereas the hydrophilic polypeptides exist predominantly as unimolecular micelles. These results were confirmed by DLS and SLS experiments.

A series of polypeptides with well-defined sequences, (Asp3Phe1)n, (Asp2Phe1)n, (Asp1Phe1)n, (Asp1Phe2)n, and (Asp1Phe3)n, containing the hydrophilic amino acid, aspartic acid (Asp) and the hydrophobic amino acid, phenylalanine (Phe), were synthesized. Their behaviour in aqueous solution was investigated by performing fluorescence quenching and non-radiative energy transfer (NRET) experiments which were complemented by dynamic (DLS) and static (SLS) light scattering. The photophysical properties of the polypeptides were dependent on their Phe content. An increase in the Phe content led to an increase in the extinction coefficient, fluorescence quantum yield, and fluorescence average lifetime of the polypeptides. Circular dichroism experiments revealed that except for the (Asp1Phe3)n polypeptide, which adopts an alpha-helical conformation in aqueous solution, the other polypeptides did not adopt any known conformation in solution. The fluorescence quenching studies performed using molecular pyrene physically bound to the polypeptide via hydrophobic interactions resulted in protective quenching for the (Asp1Phe1)n, (Asp1Phe2)n, and (Asp1Phe3)n polypeptides, with some pyrenes having a lifetime of ~300 ns. Evidence for protective quenching was also observed in the polypeptides richer in Phe, when pyrene was covalently attached onto the polypeptides. However pyrene was found to be fully exposed to the quencher solution for the more hydrophilic polypeptides. The presence of NRET between a naphthalene labeled polypeptide and a pyrene labeled polypeptide for the (Asp1Phe2)n and (Asp1Phe3)n polypeptides and its absence for the (Asp3Phe1)n, (Asp2Phe1)n, and (Asp1Phe1)n polypeptides led to the conclusion that those polypeptides richer in Phe generate interpolymeric aggregates which provide protection to a hydrophobic cargo like molecular pyrene whereas the hydrophilic polypeptides exist predominantly as unimolecular micelles. These results were confirmed by DLS and SLS experiments.

Understanding protein stability and folding is of central importance in chemistry, biology, and medicine. Despite its importance, a molecular level understanding of protein stability still remains illusive due to the complexity of the system. In this study, we employed protein-like polypeptides to study several aspects of protein stability in different aqueous environments. The model system employed here is elastin-like polypeptides (ELPs). First, the modulation of the lower critical solution temperature (LCST) of neutral ELPs was investigated in the presence of 11 sodium salts that span the Hofmeister series for anions. It was found that the hydrophobic collapse/aggregation of these ELPs generally followed the series. Specifically, kosmotropic anions decreased the LCST by polarizing interfacial water molecules involved in hydrating amide groups on the ELPs. By contrast, chaotropic anions lowered the LCST through a surface tension effect. Additionally, chaotropic anions showed salting-in properties at low salt concentrations that were related to the saturation binding of anions with the biopolymers. These overall mechanistic effects were also compared to the results previously found for the hydrophobic collapse and aggregation of poly(N-isoproplyacrylamide). A positively charged ELP, ELP KV6-112, was used as a next model system. We observed both inverse and direct Hofmeister effects on LCST with five chaotropic salts. Next, the solvent isotope effects on the LCST of ELPs were investigated as a function of ELP chain length and guest residue chemistry using D2O and H2O. Differences in the LCST values with heavy and light water were correlated with secondary structure formation of the polypeptide chains which was quantified by circular dichroism, FTIR, and differential scanning calorimetry measurements. It was found that there is a great change in the LCST values between H2O and D2O for those polypeptides which form the greatest amount of b-spiral structure. This study suggests that hydrogen bonding rather than hydrophobicity is the key factor in the stabilization of ELPs in D2O over H2O. The phase transition property of ELPs can also be applied to development of stimuli responsive biosensor system. In this study, we employed ELP-conjugate solid supported lipid bilayer as a size selective binding sensor.

No description available.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 147-169) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

to evaluate the GIP response to feeding and oral glucose tolerance tests. Initial evidence for the insulinotropic action of GIP was presented. Indicated also was an implication of GIP involvement in various pathological conditions. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Currently used small diameter synthetic vascular grafts are prone to high rates of failure related to thrombosis and neointimal hyperplasia. Biomimetic materials, based on the extracellular matrix (ECM) composition of native tissues, represent an attractive solution to address these complications. The inherent low thrombogenicity and cell signalling properties of elastin-like polypeptides (ELPs) make them a suitable option for these applications. In this thesis, ELP surface modification has been achieved through the use of elastin cross-linking peptide bioactive fluorinated surface modifiers (ECP-BFSMs). The synthesis of these low molecular weight fluorinated additives was described and their subsequent blending with a base polycarbonate-urethane (PCNU) was shown to successfully enrich the surface to allow for ELP surface cross-linking. The kinetic surface migration of fluorescent ECP-BFSMs was studied over a 2 week casting period by two-photon confocal microscopy. Contact angle and x-ray photoelectron spectroscopy (XPS) confirmed the surface localization of the ECP-BFSMs. Changes in contact angle and XPS spectrums following ELP surface cross-linking confirmed the success of the surface modification approach. The novel ELP surface modified materials were demonstrated to inhibit fibrinogen surface adsorption and platelet adhesion under physiological flow conditions and inhibit bulk platelet activation following blood-material contact. Moreover, these ELP modified surfaces were shown to promote increased endothelial and smooth muscle cell adhesion, spreading and retention over a 7 day culture period relative to their non-ELP analogs. Endothelial and smooth muscle cells seeded on the elastin-like materials were shown to express endothelial nitric oxide synthase (eNOS) and smooth muscle myosin heavy chain (SM-MHC) cell specific phenotypic markers, respectively. Furthermore, competitive inhibition experiments revealed that initial smooth muscle cell adhesion to ELP surface modified materials was mediated through elastin-laminin cell surface receptors binding to VGVAPG peptide sequences on the ELP molecules. Hence, these materials may have broad applicability in cardiovascular applications, from blood contacting materials to scaffold structures for vascular graft tissue engineering. Furthermore, this surface modifying additive approach represents a versatile technique that can be custom tailored for various biomimetic applications to generate stable bioactive ECM-like surfaces retained onto a relatively inert fluorinated background.