The main research objective of this project is to explore effects of nano-scale confinement on the structure and dynamics of self-assembled phospholipid membranes by means of advanced High Field (HF) EPR spectroscopy in combination with novel spin labeling methods.

Our research objective is motivated by the needs of merging two main strategies of making novel hybrid devices with unique capabilities provided by nanotechnology and addressable machine architectures. These are so-called "bottom-up" and "top-down" strategies. The "bottom-up" strategy for hybrid devices involves molecular engineering and self-assembly of functional biomolecules (proteins) and their complexes for highly specific nano-scale tasks while the "top-down" is based on various forms of lithography and replication. The lithographic techniques are now reaching ca. 100 nm spatial scale thus creating an unprecedented opportunity for a seamless transition to a still smaller protein scale of just 3-10 nm. However, this transition from rigid lithographic structures to "soft" biomolecules is not without difficulties. The two major problems are yet to be solved are:

1. compatibility of biomolecules and the solid support including development of new attachment method(s), and
2. a scale mismatch between state-of-the-art lithographic technologies and protein sizes.

One of the most promising direction to solve these problems is to mimic the nature which utilizes phospholipid bilayers to support, protect, and organize membrane proteins. Phospholipids represent a natural environment for the membrane proteins and this solves the compatibility issue. Under normal conditions, complex electrostatic interactions between proteins and phospholipids ensure a proper protein attachment to the membrane as well as its optimum transmembrane location and a correct folding conformation. In addition, the phospholipids have a property of being self-assembled into bilayers, micelles, and other structures. This self-assembly is an ideal feature for filling the existing scale gap between the sizes of wells that could be created lithographically (or by other means) and dimensions of biomolecules. Thus, phospholipids could be viewed as an ideal self-organizing "packing" material for fitting "soft" biomolecules into "hard" man-made "boxes".

Because the geometry of solid-state support materials, their complex surface and electrostatic properties are very different from natural cellular environments, these artificial solid-state substrates are expected to have a profound effect on phospholipid bilayers. What is needed is a detailed fundamental understanding of the confinement effects on self-assembly and properties of phospholipid membranes. This puts forward the following specific goals for our research project:

• To determine how the order parameter, viscosity, phase transition, and other properties of self-organized phospholipid structures (monolayers, bilayers, and vesicles) are affected by the nano-scale confinements and increased surface area and how these confinement effects scale with the size of the structure;
• To investigate what is the minimum confinement size at which the self-assembly is still possible;
• To study how the properties of the surface and various surface treatments affect the supported bilayer;
• To examine our hypothesis that the hydrophobic/hydrophilic mismatch at the borders of substrate-supported lipid corrals can be minimized by a mixture of short- and long-chain phospholipids. Figure 1 shows one possible arrangement of a lipid corral on a shallow substrate well. Clearly, such kind of arrangement could create a hydrophobic/hydrophilic mismatch region (shown by arrows). If so, this mismatch could affect stability of the bilayer. We propose to eliminate/minimize this mismatch by using a mixture of long- and short-chain phospholipids such as DMPC and DHPC (and also other short detergents). These mixtures are known to form bicelle structures and were previously developed for high field NMR studies of membrane peptides and proteins (in solutions the bicelles spontaneously align in high magnetic fields). We hypothesize that the bicelle structure could also be useful for preparing substrate-supported membranes for the needs of nanotechnology (Figure 2).
• To investigate the effects of incorporation of model membrane peptides (melittin, gramicidin) and proteins (bacteriorhodopsin) on the structure and dynamic of nano-confined lipids.

Figure 1. A lipid bilayer on a solid substrate support	Figure 2. A bicelle on a solid substrate: filled circles - long chain phospholipids, open circles - short-chain phospholipids.

This work represents a developing multidisciplinary collaboration between North Carolina State University (NC State) and the Argonne National Laboratory (Argonne). The project combines cross-cutting technologies in materials science with recent advances in high resolution high field EPR complemented by an array of modern spectroscopic methods. This work explores a new area of science of discovering nano-scale phenomena on the interface of biophysics, physical chemistry, and materials science.