Research

A wide variety of diseases are results of deficient or abnormal protein-lipid interactions. The elucidation of the interactions between specific proteins and lipids, and the ability to examine and manipulate biomembranes that mimic real life systems hold the key to a better understanding of these diseases. Our research interests lie in the interdisciplinary area which can be termed as “interfacial medicine”. Using two-dimensional monolayers, either at the air-water interface or transferred onto solid substrates, and supported bilayers as model systems, along with various microscopy and scattering techniques, we plan to carry out fundamental studies on the interactions between lipids and proteins to gain insights into the biophysical aspects of these diseases. Two diseases of particular interest are listed below.

A complex mixture of lipids and proteins, known as lung surfactant, forms monolayers at the alveolar air-water interface. The surfactant lowers the surface tension to near zero, and is responsible for reducing the work of breathing. A lack of surfactant, either due to immaturity in premature infants or disease or trauma in adults, can lead to RDS. In spite of the serious morbidity and mortality of the disease, a firm understanding of the role of surfactant in both normal and diseased lungs is still lacking. My group is interested in developing a detailed structure-function relationship for the various components of lung surfactant. In particular, we will examine the phase behavior of various mixtures of lung surfactant components, as well as the interactions between lung surfactant specific proteins and the surrounding lipid matrix. We will explore the effect of lung surfactant proteins on monolayer surfactant. The knowledge gained from this should lead to an understanding of the morphological consequences of monolayer phase separation and collapse, which is necessary for the continued development of positive interventions for patients suffering from RDS.

An artistic movie rendition of a model lung surfactant monolayer can be seen HERE.

Aβ, a self-assembling 39-43 residue peptide generated by the proteolytic processing of the amyloid precursor protein, comprises the major proteinaceous component of neuritic plaques and vascular deposits that appear in Alzheimer’s disease, and is implicated as one of the causal factors in the pathology of the disease. Since the Aβ peptide fragment includes 28 residues just outside the membrane plus the first 11-15 residues of the transmembrane domain, it has been shown to display properties commonly associated with surfactants. My group is interested in understanding the aggregation of the Aβ peptides, and in using two-dimensional thin films (either free-standing monolayers or supported bilayers) as “templates” to explore the possibility of surface-induced aggregation. We plan to study various isoforms of Aβ and examine their surface activities and their association with model membrane systems in both their monomeric and aggregated states. This can elucidate the residue length dependence of the aggregation process, and help explain why the longer Aβ isoforms may be more intimately associated with Alzheimer’s disease pathology than their shorter counterparts. Aβ is also known to aggregate and form fibrils, though the mechanism involved is still not well understood. Since the rate of this formation process can be adjusted by various experimental parameters, we plan to monitor the formation process, and characterize the structure of the fibrils formed. Our goal is to provide a model for Aβ aggregation. Other research interests of the group includes protein and lipid diffusion in model membrane systems, structures and dynamics of monolayer domains, and the manipulation of supported bilayers via electric field.

The principles of selectivity and differing levels of activity of antimicrobial peptides with cell membranes are not well understood, nor is the exact mechanism of action. Bacterial membranes contain negatively charged lipids on the outer leaflet, whereas the outer leaflets of eukaryotic cell membranes are predominantly zwitterionic. Protegrin-1 (PG-1) is a β-sheet 18-amino acid peptide that was originally isolated from pig leukocytes. NMR studies of the peptide in solution determined the structure to be a one-turn β-hairpin. Oriented circular dichroism studies revealed two different states of PG-1 in aligned multilayer phospholipid samples depending on peptide concentration. Using fluorescence microscopy, x-ray reflectivity and grazing incidence x-ray diffraction techniques, we have studied the structure and behavior of PG-1 at the air-water interface in the presence of various phospholipids. PG-1 was injected underneath DPPG, DPPE, DPPC, and Lipid A monolayers at surface pressure of 20, 25, and 30 mN/m. We found that PG-1 readily penetrates into anionic DPPG monolayers, but have little to no insertion into zwitterionic DPPC and DPPE monolayers, respectively. In the case of Lipid A, the insertion of PG-1 into the film leads to complete disruption to the stability of the film (see pubs. 33 & 40). We are currently exploring the effects of lipid unsaturation and cholesterol on PG-1/lipid interaction, and are trying to understanding the mechanism of interaction involved.

P188, an amphipathic triblock copolymer of the form poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) is known to help seal electroporated cell membranes, arresting leakage of intracellular materials of the damaged cell. The hydrophobic PPO and the hydrophilic PEO portions are responsible for the surface activity of P188, and probably aid in its adsorption into the bilayer of the damaged cell. To mimic the outer leaflet of the cell bilayer, monolayers of DPPC and DPPG were used. Experiments were conducted on a Langmuir trough equipped with a Wilhelmy plate coupled to a fluorescence microscope. Monolayers of the desired lipid were spread and P188 was injected into the subphase at various surface pressures. At 30°C, P188 inserts into both DPPC and DPPG monolayers at surface pressures equal to and lower than 23 mN/m; this pressure is also equivalent to the maximum surface pressure attainable by P188 on a pure water subphase. Poor adsorption of P188 at higher pressures implies that it only inserts into damaged portions of electroporated membranes, thereby localizing its effect. Similar results observed with different lipid headgroups suggest that P188 insertion is not influenced by electrostatics. The question we would like to address is how the structure of the polymer affects its ability to interact with the membrane. In this light, we are examining triblock copolymers of different architecture, and testing polymers with different motifs. High resolution imaging are being carried out to pinpoint the location of the polymer in the lipid matrix using AFM.

Antimicrobial peptides can be found in our innate immune system. They fight against bacteria or other pathogens by causing leakage in the membrane. We directly visualized the process by atomic force microscopy, recording a formation of pores and wormlike micelles in model lipid membranes. The formation of these structures can be understood by considering the peptide as a line-active agent. Understanding the effect of lipid compositions would help to elucidate the fundamental biophysics of membrane disruption and the mechanism by which antimicrobial peptides use to distinguish between bacterial and mammalian cells. A video of PG-1 mediated membrane disruption can be viewed at lipid.uchicago.edu/~kinlok/a.gif

(A) Schematic sketch of the monolayer in the boundary region. A boundary lying parallel to the y axis separates two large domains denoted by 1 and 2. (B) Cross-section parallel to the xz plane. The monolayer conformation is parametrized by the angle q(s). It makes with the xy reference plane at curvilinear distance from the boundary.

Fluorescence microscope imaging a lipid monolayer at the air-water interface.

Effects of PG-1 on E. coli morphology. (Upper) Bacteria were exposed to 50 mg/mL PG-1 for 15 min. (Lower) Bacteria are untreated controls. The outer membrane of the treated organisms is greatly expanded and displays innumerable microvilli.

7:3 DMPC:DMPG lipid system with 5 μg/mL of PG-1 in water.

2008 The University of Chicago Chemistry Department