Structural study of unconventional proteins, the membrane Hairpin Proteins, using DISCO light.

Membrane proteins are proteins that interact with biological membranes. They are targets of over 50% of all modern medicinal drugs. Membrane proteins perform a variety of functions vital to the survival of organisms: signal reception and transduction, transport of molecules and ions across the membrane, enzymatic activities, etc. Various topologies are described such as, transmembrane proteins spanning the phospholipid bilayer or proteins attached to only one side of the membrane via hydrophobic loop, amphipathic helices or lipid anchor. However, one class of membrane protein is poorly described in the literature: membrane hairpin proteins. These proteins are present in all organisms and some of them are found associated with lipid droplets (LD). These observations suggest that membrane hairpin proteins share biological and biophysical properties. LD are lipid storage intracellular nanoparticles consisting mainly of a core of neutral lipids (triacylglycerols and/or steryl esters) enclosed in a monolayer of phospholipids. LD contain a number of proteins which vary considerably with the species. Some structural hydrophobic proteins stabilize the interface between lipid core and cellular aqueous environment. Seed LD are completely covered by oleosins, which belong to the membrane hairpin protein family. We developed a genetic approach using Saccharomyces cerevisiae to obtain heterologous expression of plant oleosins, but also mammalian stomatin and caveolin, and hepatitis C virus core protein. These transformed yeasts and purified LD were used for functional and structural studies. Yeast expressing oleosins overaccumulated LDs leading to a specific increase of storage lipids and these LDs harbor a high level of oleosins. Purified LDs were exposed to synchrotron light on DISCO beamline. We obtained structural data on whole cellular organelle and on oleosins inserted in LD using Sychrotron Radiation Circular Dichroism (SRCD)

Synchrotron X-ray footprinting – Mass-spectrometry to probe structure of proteins in interactions

National audienceThe meeting provides an invaluable forum for the synchrotron radiation user community, presenting an important opportunity to obtain the latest information on beamline performance at SOLEIL, to hear about the latest and most exciting results obtained at SOLEIL and to share scientific, technical and practical issues about the synchrotron radiation use

Dynamic and structural studies of lipid droplets using synchrotron light

In yeast, animals and other organisms, storage lipids are maintained in the cytoplasm in specialized organelles called lipid droplets (LDs) or oil bodies. These structures consist mainly of a core of neutral lipids (triacylglycerols and/or steryl esters) enclosed in a monolayer of phospholipids, and contain a number of proteins which vary considerably with the species. In particular, some structural hydrophobic proteins stabilize the interface between lipid core and cellular aqueous environment (perilipins, apolipoproteins, oleosins). Oleosins, with their mass between 15 and 25 kDa, are the major proteins of plant LDs, and completely cover the LD surface. In the last decade, proteomic and genetic studies of this compartment have shown that LDs appear as a complex dynamic organelle with a role in metabolism control and cell signaling. LDs have received a lot of attention as their abnormal dynamics is associated with several metabolic diseases (obesity, diabetes, atherosclerosis and myopathies). In the context of green chemistry, LDs are also promising sources of neutral lipids for the development of derived products, such as biofuels and alternative molecules for food, medicine and cosmetics. For these reasons, understanding dynamics of LD and structure of LD integral proteins is of major importance. We developed a genetic approach using Saccharomyces cerevisiae to obtain heterologous expression of Arabidopsis thaliana LD proteins [1]: oleosin AtOle1 or caleosin AtClo1. These transformed yeasts overaccumulated LDs leading to a specific increase of storage lipids and these LDs harbor a high level of oleosins. These cells or their purified LDs were exposed to the Soleil synchrotron light to understand the dynamics of lipid storage and carbon fluxes by measuring the biochemical changes on single cells using the Synchrotron FTIR microspectroscopy on SMIS beamline [2]. to obtain structural data on whole cellular organelle and on oleosins inserted in LD using Sychrotron Radiation Circular Dichroism (SRCD) [3], Deep UV imaging on DISCO beamline and Synchrotron water radiolysis footprinting of accessible oleosin amino-acids on METROLOGY beamline

Synchrotron X-ray footprinting – Mass-spectrometry to probe structure of proteins in interactions

National audienceThe meeting provides an invaluable forum for the synchrotron radiation user community, presenting an important opportunity to obtain the latest information on beamline performance at SOLEIL, to hear about the latest and most exciting results obtained at SOLEIL and to share scientific, technical and practical issues about the synchrotron radiation use