The processes localized to biological membranes are of great interest, both from a scientific and pharmaceutical point of view. Understanding aspects such as the detailed mechanism and regulation of these processes requires investigation of the structure and function of the membrane-bound proteins in which they take place. The study of these processes is often complicated by the need to create in vitro systems that mimic the environment in which these proteins are normally found in vivo. This thesis describes some of the methods available for membrane-protein studies in membrane-mimetic systems, as well as our work aimed at developing such systems. Furthermore, results from studies using these systems are described. In the first two studies, described in Papers I &amp; II, we investigated the use of silica particle-supported lipid bilayers, both for membrane-protein studies and as possible drug-delivery vehicles. Successful reconstitution of a multisubunit proton-pump, cytochrome c oxidase is described and characterized. Initial attempts to develop drug-delivery systems with two different targeting peptides are also described in the thesis. The second part of this thesis revolves around our work with membraneprotein dependent pathways. Results from studies of systems where the proton- pump bo3 oxidase and ATP synthase work in concert are described. The results show a surprising lipid-composition dependence for the coupled bo3- ATP-synthase activity (Paper III). Finally, a new system utilizing synaptic vesicle-fusion proteins for coreconstitution of membrane proteins is described, showing successful coreconstitution of a small respiratory chain, delivery of soluble proteins to preformed liposomes and reconstitution of ATP synthase in native membranes (Paper IV). / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

The lipid bilayer, the fundamental structure of cell membranes, exemplifies a highly adaptable molecular assembly with characteristics that have been fine-tuned through evolution to meet the diverse functional needs of cells. These bilayers must strike a delicate balance: they need to be sufficiently rigid to act as protective barriers, yet fluid enough to facilitate the diffusion of proteins and molecular clusters crucial for various biological processes. Owing to their multifunctional nature, lipid membranes are not only vital in biological contexts but also in numerous practical applications, such as artificial cells, drug-delivery nanocarriers, and biosensors. Both biological and synthetic lipid membranes frequently incorporate molecular or nanoscale additives that modify their properties through a range of mechanisms. Gaining a comprehensive understanding of how lipid membranes interact with these additives is an area of active research, particularly with the advent of advanced high-resolution characterization techniques that reveal both the static and dynamic behaviors of these systems. This dissertation investigates the impact of small molecular additives – specifically natural and synthetic sterols – on the structure, elasticity, and organization of biomimetic lipid membranes. Utilizing advanced scattering techniques and other methods, the research elucidates the intricate interplay between the membrane composition, structure, and elasticity. Key findings demonstrate that, unlike previous observations, cholesterol significantly affects the bending rigidity of lipid membranes regardless of chain unsaturation, when measured on mesoscopic length and time scales. Interestingly, the replacement of cholesterol with engineered molecules, comprised of a sterol unit that is chemically conjugated to one or both of the lipid chains, results in further enhancement in the membrane bending rigidity and mechanical stability, making them a promising additive for advanced liposomal drug delivery systems. Further studies on phase-separating membranes illustrate the effective use of sterol-modified lipids in regulating the formation and size of distinct lipid domains implicated in protein recruitment and biological function. This work advances the current understanding of membrane biophysics and paves the way for novel therapeutic strategies and biomaterial designs. / Doctor of Philosophy / Cells are the central unit of life found in all living organisms. The outermost layer of the cell, the plasma membrane, is quite complex. Yet it is primarily formed by the self-assembly of lipids and sterols that form a bilayer structure that mediates important biological functions. To understand the properties of plasma cell membranes and their implication in function, biophysicists use model cell membranes to reduce biological complexity. This dissertation explores changes in the structure and dynamics of model lipid membranes in response to small molecular additives, including cholesterol and hybrid cholesterol analogues. Using sophisticated scientific techniques, experiments reveal that cholesterol stiffens lipid membranes, regardless of the architecture of their molecular building blocks. These findings challenge previous beliefs and suggest a universal rule for membrane stiffness with cholesterol. What is more, synthetic additives formed by conjugating lipid and cholesterol structures are even better than cholesterol at stabilizing membranes. This discovery has practical implications for improving drug delivery systems. Additional studies provide new insights into how additives can control how lipids organize into distinct domains that are important for many biological processes. Overall, this work enhances our understanding of cell membranes and opens up new possibilities for developing advanced medical treatments and tunable biomaterials.

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