Fluorescent-peptide conjugates for biosensing applications

This thesis has studied the different ways that fluorescent peptide conjugates can be used to study biological systems. Phenylene vinylene (PV)-lysine dendrimers were synthesized that assemble in aqueous solution into aggregates, placing the lysine residues on the periphery. The aggregates were not found to be cytotoxic, and were endocytosed by fibroblast cells and visualized via fluorescence. This lack of toxicity correlates with the self-assembly behavior of the amphiphiles, as a pyrene-lysine amphiphile was found to kill cells after uptake. These PV fluorophores were then used as a model for the synthesis of a calcium sensor that can be attached to peptides on solid phase in order to target specific organelles or proteins inside a cell. The calcium sensors have one carboxylic acid on each arm of the PV structure to create a binding site in the space between them. Derivatives of the PV acids showed a selective shift in fluorescence emission upon calcium binding, and were found to bind calcium with micromolar affinity. These fluorescent moieties have other uses as well; they can be incorporated into peptide-amphiphile (PA) self-assembling systems to form fluorescent PA (FPA) aggregates. The PA systems investigated here form nanofibers that can interact with cells through bioactive epitopes placed on their surfaces. These epitopes can be novel binders generated through phase display for proteins such as the growth factor bone morphogenic protein (BMP). A model system was designed that coassembled PAs of opposite peptide polarity in order to expose these bioactive sequences on the periphery and increase the stability of the aggregates. This stability was due to the potential for these systems to form antiparallel arrangements. Annealing these assemblies at physiological temperatures allowed access to these more thermodynamically stable assemblies with enhanced coherence lengths. These opposite polarity coassemblies were also investigated between the FPA and a PA of complementary charge. By varying the ratio of the two components in the assembly, the spacing, organization, and fluorescence of the systems could be controlled. As the FPA was diluted out in these systems, a recovery of fluorescence was observed, followed by a chiral transfer from the peptide portion of the aggregate to the fluorophore. These systems can be used to monitor binding of biomolecules to the PA nanofibers in vitro via fluorescence resonance energy transfer (FRET). To test this principle, fluorescent coassemblies of the FPA with a heparin binding PA (HBPA) were studied in the presence of a fluorescent heparin moiety. A specific FRET response was observed both in dilute solution and in PA gels, and this effect was not seen in the absence of the HBPA component. These results were corroborated by obtaining the binding constants of the PA molecules to their targets (either BMP or heparin) through the use of both fluorescence anisotropy and isothermal calorimetry. In summary, coassembling PA systems and fluorescent conjugates have great potential to help understand and track biological processes in vitro and in vivo.