Interfaces of biophysical interest

Orateur : Soutenance HDR de Marco MACCARINI (Laboratoire TIMC-IMAG)

My HDR thesis describes the biophysical studies I conducted on different type of interfaces with a significant relevance in biology. In the first part of the thesis I describe my work on the interfaces between hydrophobic surfaces and water. These are fundamental to address the hydrophobic interaction that is responsible for key biological phenomena such as protein folding, self‐assembling, colloids stability and membrane fusion. The thin layer of water in contact with the planar surface has properties different compared to the corresponding bulk phase. I conducted neutron reflectometry experiments on planar surfaces molecularly modified with self‐assembled monolayers to reveal evidences for a density reduction and to study how this density reduction depends on parameters like the interfacial energy, the temperature, and the presence of electrolytes. I rationalised the behaviour of the density depletion as governed by two effects : the surface energy difference between water and the substrate and the chemical potential of the aqueous phase. The results, supported by computer simulations, were compatible with th e presence of a thin layer of water vapour at the hydrophobic interface. In the second part I describe my study on the interaction between nanoparticles and model membranes in the context of nanotoxcitity. The development of novel nano‐engineered materials poses important questions regarding the impact of these new materials on living systems. Possible adverse effects must be assessed in order to prevent risks for the health and the environment. On the other hand, a thorough understanding of their interaction with biological systems might also result in the creation of novel biomedical applications. A key issue in making use of nanoparticles (NP) for biomedical purposes is an understanding of their interaction with cells beyond the desired and planned functions. Since the first contact that all nanomaterials will always have with any living organism is through the cell membrane, a ∼5 nm thick lipid bilayer, I focused my study on the interaction of a particular class of NP with model cell membranes. I conducted neutron reflectometry experiment on mode l membranes composed by a solid supported double lipid bilayer, in which the second bilayer floats 20‐30 Å above the first one. This choice gives access to a highly hydrated, fluctuating bilayer that represents a membrane system with dynamic properties comparable to biological membranes. The experiments provided a characterisation of the effect of cationic and anionic nanoparticle on the nanostructure of the lipid bilayers. This knowledge is crucial to effectively design safe nanoparticles for biomedical application and also to define practices and procedures for the secure handling of nanoparticles in general. In the last part of my thesis I discuss my work biomimetic membranes obtained by incorporating membrane proteins into tethered lipid bilayers, model systems that serve as a tool to better understand biological systems. Recent developments however, have seen the use of model biomimetic membranes as more than an aid to better understand biological function and have now advanced to become a core engineered element in a new generation of biomimetic molecular devices. Biological membranes in living organisms are made up by a variety of lipid molecules, and a plethora of different proteins dedicated to different functions. The proteins incorporated into those membranes provide the nanostructured pathways for the transmembrane transport of charged ions, essential for many cellular functions. In this context, I will present my study about the incorporation and characterisation of functional recombinant protein OprF into tethered bilayer systems. OprF is the main outer membrane protein of the Pseudomona Aeruginosa, an antibiotic resistant bacterium responsible for 10% of all hospital‐ acquired infections. The crystallographic high‐resolution structure of OprF is not available due to the difficulties involved in crystallising membrane proteins. By the use of neutron reflectometry (NR), we provided an alternative route to obtain fundamental low ‐resolution information on the nanoscale structure OprF, in an environment that mimics its native condition. NR allowed us to characterize the nanostructural details of the lipid membrane, the amount of the protein embedded in the bilayer, the thickness of the extramembrane domain of the proteins, which extends from the membrane. This study showed that OprF purified using the cell free protocol and then incorporated into tethered lipid bilayer yelds a controllable (and measurable at the nanoscale) biomimetic system constructed in vitro, and sets off a proof of concept method that is being extended to various other membrane transport proteins used for biotechnological devices.

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