Designing Peptides to Target Membrane Lipids and to Evaluate Fluorination of Proteins

Abstract

My graduate research has used engineered peptides to perturb the non-covalent interactions in protein folding, protein-protein association and protein-membrane association. We have focused on understanding the fundamental principles of molecular recognition behind protein-protein and protein-membrane interactions, and further using these principles in protein engineering. This thesis includes three projects. I) Towards Small Molecule Receptors for Membrane Lipids: A Case Study on Phosphatidylserine The lipid composition and distribution of cell membranes play important roles in regulating the physiology of the cell. The lipid composition of plasma membranes is one characteristic feature that can be used to identify cell types and functions. Molecules that specifically recognize a particular lipid are useful as imaging probes for targeting cells or tissues of interest. Protein based lipid binding probes have intrinsic limitations due to their large size and poor pharmacokinetic properties such as slow clearance rate and poor in vivo stability. A plausible strategy to achieve a probe with small size and high binding affinity and selectivity is to use a peptide to mimic the protein lipid-binding domains. As a case study, a cyclic peptide that specifically targets phosphatidylserine containing membranes has been developed. This cyclic peptide is potentially capable of imaging apoptosis in vivo, and the strategy of developing this cyclic peptide can be generalized to the design of peptide-based probes for other lipid species. My research has pointed out a challenging but feasible way to design a peptide that achieves specificity and affinity similar to lipid-binding proteins. (II) Study of Apoptotic Cell Membrane (ACM) Permeant Molecules Noninvasive imaging of apoptosis is highly desirable for the diagnosis of a variety of diseases, as well as for the early prognosis of anticancer treatments. One characteristic feature of apoptotic cells that has been targeted for developing specific biomarkers is enhanced membrane permeability compared to that of healthy cells. Several unrelated molecules that are capable of selectively penetrating the apoptotic cell membrane (ACM) have recently been reported. However, the origin of the altered ACM permeability is poorly understood, as is the scope of molecular structures that can permeate through the ACM. Herein, we report a systematic investigation on the altered ACM permeability. Our results show that simple modifications of commonly used dyes (e.g. fluorescein) afford specific entry into cells at the early stages of apoptosis. The ACM appears to be permeable to molecules of various functional groups and charge, but does discriminate against molecules of large size. The new findings reported here greatly expand the pool of small molecules for imaging cell death, thus facilitating the development of noninvasive imaging agents for apoptosis. (III) Study of Aromatic-Fluorinated Aromatic Interactions in Peptide Systems Therapeutic proteins have been through a remarkable expansion in the last two decades. A general problem that they are facing is poor stability. Protein engineering focuses on solving this problem by incorporating unnatural amino acids into protein sequences to purposefully modify protein structures. Fluorinated aliphatic amino acids have been demonstrated to be effective in stabilizing protein structures and functioning as recognition motifs. In contrast, fluorinated aromatic amino acids are less studied. We investigated the effect of perturbation of fluorination on aromatic residues on the stability of protein model systems, as well as the influence on protein-protein association behavior. The results of this study provided a fundamental understanding of aromatic interactions in protein systems, and guidelines for protein engineering with fluorinated aromatics for stabilizing protein structures or directing specific protein-protein interactions.