Giovanna Fragneto

The focus of my research activities is the structural characterisation and  the study of fluctuations of model biological membranes with the aim of understanding phenomena at cell surfaces. Examples include the interaction of membranes with peptides, proteins, drugs or drug delivery systems. Systems are mainly planar and studied by neutron or x-ray reflectometry. 

• Structure of natural and model membranes by reflectometry and diffraction: interaction of lipids with biomolecules
• Deuterated Natural Lipid Extraction for structural characterisation of model membranes (coll.: H. Wacklin-Knecht, K. Batchu, A. Luchini, G. Corucci)
• Determination of bending modulus and in-plane features of floating bilayers by grazing-incidence synchrotron radiation (coll.:  T. Charitat, J. Daillant & al.)
• Lipid rafts mimicks structural characterisation (coll.: L. Cantù, V. Rondelli)
• Lipid Flip-Flop (coll.: Yuri Gerelli, Lionel Porcar, Ursula Perez-Salas)
• Interaction of nano particles with membranes (coll. Maccarini, Baldelli)
• Deuterated protein/PEG interactions (coll.: E. Schneck, M. Sferrazza, A. Halperin) 
• Liquid/liquid interfaces (coll.: E. Scoppola, O. Diat, O. Konovalov, E. Watkins, R. Campbell, P. Gutfreund)
• Flash-cooling phospholipid bilayers (coll.: M. Weik, J. Zaccai)

Real biological membranes have different components, including lipids, sterols and proteins, which are distributed very heterogeneously, forming domains that are involved in the transmission of signals between the inside and the outside of the cell. Current efforts are devoted to exploring systems, such as natural lipids extracted from cells, to get closer to this complex reality.

Optical, electron, and atomic force microscopies have come a long way in recent decades. However, neutron scattering gives complementary and unique information and is increasingly widely used. One of its more and more frequent applications is the study of the nanoscale structure of model membranes and their interactions with proteins or peptides participating in important cellular functions.

Protein deuteration is becoming a common technique coupled to neutrons to refine details of protein-lipid interactions and protein structure at interfaces: deuteration of other membrane components like lipids and sterols have also made steady progress in recent years. Neutron scattering at small angles has nanometer resolution and, unlike crystallographic techniques, can be used to probe membranes in physiological conditions or in-vivo and follow processes in-situ.

Recently we have shown that it is possible to prepare extracts of true natural yeast membranes and prepare with them reproducible and stable bilayers. This allowed the study of the mechanism of interaction of an antibiotic molecule like amphotericin with natural membranes containing sterol.
Work is in progress to optimize methods to produce bilayers from natural lipid extracts on soft cushions and to separate the different lipid species from the extracts.

A very topical issue in this field is also the development of reliable protocols for reconstitution of membrane proteins into model membranes. Smart measurement setups and data analysis procedures have already allowed the acquisition of precious information on membrane protein function. The coupling of scattering and MD simulations has been recently reviewed.
Interesting developments for the study of liquid-liquid interfaces with neutron reflectometry have been possible recently thanks to the possibility to strike the interface with the neutron beam from both sides of a liquid-liquid interface. For example, planar lipid bilayers were prepared and characterized both in the gel and fluid phases at a fluorocarbon-aqueous solution interface.
 
Neutron work at the liquid-liquid interface has been limited in the past by the poor transmission of neutrons through liquids, even if deuterated. The universal law of attenuation of radiation passing through matter (Beer-Lambert law) is also valid for neutron transmission. Fluorocarbons transmit neutrons well (larger penetration depth than for hydrocarbons) and are denser than water. This allows the use of bulk liquids with large enough surfaces to illuminate the sample away from the meniscus (for reflectometry measurements samples must be flat to avoid cumbersome procedures of data normalization). Partially negatively charged bilayers from POPC containing 30 mol % POPS were formed from vesicle fusion in the presence of salt from the aqueous side of the interfaces on a positively charged monolayer from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and di-stearoylpropyl trimonium chloride (DSTAP) (7:3 mol:mol) created at the fluorocarbon-aqueous solution interface. A bilayer with good coverage was produced from which the distance of the interface varied with salt content. The interest of producing bilayers at a liquid-liquid interface lies in the possibility to have a model fluctuating between fluid media adapted for characterization at the fraction of nanometer level with ease of manipulation and possibility to determine the effect of thermodynamic parameters or interacting biomolecules. The transport across the interface should be easier than when bilayers are adjacent to a solid surface.

This and other interesting developments confirm that in the biology of the future, physics should play a major role. If we fully exploit their potential, neutrons can help unravel the secrets of life.