Model membrane-studies

Antimicrobial peptide interactions with model cell membranes
In the face of the rapidly emerging bacterial resistance to conventional antibiotics as a serious threat to public health, antimicrobial peptides (AMPs) are being increasingly recognized as potential candidate antibacterial, antifungal and in certain cases, antitumor drugs. Antimicrobial peptide action is quick and strong, mediated by a direct interaction and destabilization of the pathogenic cell membrane. However, a precise understanding of their cidal activity in a range of organisms is still lacking owing to the complex nature of the interactions of antimicrobial peptides with the cell membrane, the mechanism of which can vary considerably between different classes of antimicrobial peptides.

Our group focuses on characterizing the molecular basis of antimicrobial peptide/membrane interactions using model cell membranes that mimic the ones in bacterial and mammalian cells. In collaboration with Prof. A. Ouellette from the Department of Pathology, University of California, Irvine and Prof. R. Jelinek from the Ben Gurion University, Israel, we are investigating membrane interactions of cryptdins (mouse AMPs that belong to the α-defensin family). By means of fluorescence spectrophotometry we examine the membrane disruptive ability of the peptides on liposomes (induction of leakage of liposomal contents), as well as their capacity to translocate from the outer to the inner membrane leaflet. We are also applying biosensor technology (surface plasmon resonance (SPR) and acoustic biosensors) which allows the real-time measurement of peptide binding to phospholipid bilayers. This project aims at the development of highly negatively charged membrane bilayers composed of biologically relevant anionic lipids, as biosensor platforms for peptide-membrane interaction studies. Understanding the mechanism of peptide binding and perturbation of biological membranes is pivotal in allowing the exploitation of the potential of antimicrobial peptides as drugs.

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Whole cell-bound receptors/ligand interactions
Despite the widespread use of biosensors with soluble analytes, limited data exist on the detection of whole cells and cell-bound membrane receptor interactions. Acoustic wave biosensors have an important advantage over optical biosensors as it can offer analysis of intercellular interactions at the molecular level. Due to the confinement of the wave close to the interface (~100 nm), the sensor can focus on the protein-protein binding that mediates cell-substrate interactions; the bulk cell mass does not affect the acoustic signal so that non-specific adsorption of cells can be distinguished from specific interactions of interest. The observed sensitivity of acoustic damping to the number of cell/surface specific bonds provides a unique sensing mechanism for investigating membrane interactions. Moreover, the acoustic signal change, together with a modified 3D kinetic analysis, can be used to measure detailed kinetics and derive both two-dimensional association and dissociation rate constants in a fast and simple way.

One current project in the Biosensors laboratory is the characterisation of the interaction of Major Histocompatibility Complex (MHC) glycoproteins with antigenic peptides. The antigenic peptides belong to known and putative tumour-associated antigens; the current research focuses on the development of a fast, label-free and non invasive acoustic technique for the determination of novel tumour peptide epitopes. Several biorecognition surfaces have been developed and studied acoustically, including antibodies and peptides coupled directly to the acoustic sensor or associated with supported lipid bilayers. The modified surfaces have been employed in combination with whole cells and with heterologously expressed MHC molecules.

Cell adhesion on biocompatible surfaces
Adhesion of bone-forming cells is a parameter tested during investigation of the potential of a biomaterial surface for supporting osteogenic differentiation and tissue integration. Specifically, for new bone induction and osteointegration of bone implants, osteoblast adhesion plays an important role. The attachment of murine osteoblast-like cells is, therefore, investigated on titanium-modified acoustic sensor surfaces. These studies can be applied to the screening of various potential bone implant materials and validation of their interactions with different cell types.

Study of the conformation of surface-attached DNA molecules
A major focus of our research is the elucidation of the way by which acoustic waves interact with biological molecules attached to the device surface and specifically, the molecular mechanisms involved in the interaction processes leading to acoustic energy dissipation. A novel theory developed in the group has shown that acoustic measurements can be directly related to DNA intrinsic viscosity, which, in turn, can be used to provide quantitative data on the shape and size of the molecules. The theory has been tested in by resolving via acoustic measurements conformational changes of pre-designed model double stranded (ds) DNA molecules of same shape (rod) but different size and also molecules of the same size (90 bp) but various conformations ("straight", "curved" at various positions along the chain, "triangle"-shaped). Experimentally more challenging DNA molecules with a pre-determined triple helical "straight" and "bent" conformation have also been used to test the validity of the model. The theory is currently being extended to the study of conformational changes of other surface-attached biological molecules.

DNA/protein interaction and DNA hybridization
The significance of the above approach is currently applied in the study of DNA/protein interactions leading to DNA bending. Bending, curving or straightening of DNA or RNA are important structural changes that occur in nature and regulate processes such as transcription, replication, and DNA packaging into nucleosomes. In addition, defects in DNA(RNA)-bending proteins are responsible for certain human diseases while artificial DNA(RNA)-bending proteins can also be made to bend specific genetic sequences to turn genes "on" and "off". Today, such structural changes can be studied in the lab using standard gel-based molecular biology assays. Our approach allows the real time and label-free measurement of structural changes of surface-immobilized DNA(RNA) molecules in a quantitative way regarding the shape of the molecule before and after the interaction. Specific examples include the detection of the binding of histone protein to surface-attached DNA molecules of various sizes. Acoustic data can be used to monitor and quantify the transition from a free-standing, surface protruding DNA molecule to a bent and collapsed DNA/histone complex.

Detection of DNA hybridization is also a challenge currently under study in the group. Changes in the intrinsic viscosity (measured via acoustic wave devices) of surface attached single stranded DNA molecules during their hybridization by a complementary strand are recorded and related to the efficiency of hybridization as well as mismatching effects between the two strands.