Graduation Year

2026

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Physics

Major Professor

Jianjun Pan, Ph.D.

Committee Member

Ghanim Ullah, Ph.D.

Committee Member

Theresa Evans-Nguyen, Ph.D.

Committee Member

Robert Hoy, Ph.D.

Keywords

AFM, AMPs, CPPs, SLBs, Magainin 2, Penetratin, Pep-1

Abstract

Membrane-active peptides (MAPs) are a diverse group of biologically relevant molecules that induce nanoscale modulations during their interactions with cell membranes. Understanding the precise mechanism by which these MAPs perturb or modulate the cell membranes is a cornerstone of membrane biophysics, as it facilitates rational design of useful therapeutics. That is, if we have the knowledge to determine what the peptide can do, how selective it is, or how toxic it might be, we can optimize them and design new peptide therapeutics such as for antimicrobial, anticancer, or drug-delivery applications. In general, MAPs can be categorized into two classes based on how they interact with the lipid bilayer. Membrane disruptors or antimicrobial peptides (AMPs) are known for their ability to combat wide range of microorganisms, such as bacteria, fungi, and viruses. On other hand, membrane translocators or cell-penetrating peptides (CPPs) are capable of traversing cell membranes, facilitating intracellular delivery of various cargo molecules. However, despite extensive research on their interactions with lipid membranes, the detailed mechanisms underlying the peptide-induced membrane perturbations remain poorly understood.

In my research, I look at the nanoscale lipid bilayer morphology, where the peptide’s mechanism becomes more clearly visible. I employ fluid-based atomic force microscopy (AFM) imaging to investigate structural changes induced by MAPs in lipid bilayers. This method is specifically useful as it visualizes label-free morphological alterations under physiological conditions. Moreover, high-resolution AFM imaging helps distinguish even minute structural changes that are difficult to infer from bulk assays alone. Furthermore, AFM-based force spectroscopy is utilized to probe peptide-induced nanomechanical modulations in the lipid bilayer, that often provide insights on early physical signs of how a peptide works.

In this dissertation, I investigate morphological perturbations of three therapeutically relevant MAPs at the nanoscale. First, the impact of the AMP Magainin 2 (Mag2) on various lipid bilayers was explored. AFM imaging uncovered interesting formation of “surface pores” in POPC bilayers, characterized by a limited depth that differs from transmembrane pores. In contrast, Mag2-treated E.coli bilayer demonstrated a fluctuation-like morphology, with an increased surface roughness. Addition of POPG into POPC bilayers exposed its modulatory effects on Mag2 activity, and force spectroscopy measurements on Mag2-induced POPC membrane revealed a weakening of bilayer stability and stiffness. Collectively, these findings of nanoscale perturbations elucidated useful insights into the mechanism leading Mag2-bilayer interactions. Next, I describe nanoscopic bilayer remodeling events caused by Penetratin. This CPP initially induced elevated protrusions at POPC bilayer edges and progressed to form shallow pits across the patch interior at higher concentrations. Anionic POPC/POPS bilayers showcased enhanced susceptibility to Penetratin, proceeding from peripheral disruption to full fragmentation into lipid-peptide assemblies. However, cholesterol-containing bilayers remained highly resistant, forming only a few isolated deep defects. Similar but synthetic CPP named Pep-1 was studied thereafter to reveal its curvature-induced remodeling in bilayers with and without free edges. Pep-1 perturbed POPC patch boundaries preferentially, leading to detergent-like disintegration. Inclusion of POPS in POPC patches enhanced peptide binding and localized disruption, resulting elevated annular rims, holes, and peptide–lipid aggregates. But in continuous POPC/POPS bilayers, Pep-1 formed flower-like protrusions that combined into an interconnected network of peptide-rich domains. Inclusion of cholesterol revealed protruded, ridge-like features, consistent with lipid redistribution and curvature generation. These findings of Penetratin and Pep-1 elucidated composition-dependent remodeling pathways, providing new insights relevant to CPP translocation.

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