Projet ANR-06-PCVI-0019 – Stabicalix : Design of calix[n]arenic detergents for structural and functional studies of ABC transporters
Programme : Physique et Chimie du Vivant (PCV) (PCV) 2006
Année de financement : 2006
Coordinateur du projet : CNRS DELEGATION REGIONALE RHONE AUVERGNE (CNRS DELEGATION REGIONALE RHONE AUVERGNE)
Partenaires : Centre national de la recherche scientifique; Centre national de la recherche scientifique; Centre national de la recherche scientifique; Centre national de la recherche scientifique
Montant : 500000.0 euros
Date de début : 2006
Durée : 36 mois
Résumé : 1-Context and objectives of the project  Our main objective is to obtain the first 3D-structure at atomic resolution of a MultiDrug-Resistance (MDR) ATP-Binding Cassette (ABC) transporter, a class of proteins for which biomedical interest is considerable and for which functional and structural data are essential to develop adapted treatments. Our strategy is based on two original concepts. The first one consists in developing detergents stabilizing the solubilised transporters in a unique conformation, favourable for crystallogenesis experiments. Two distinct approaches will either use the polyspecific character of these proteins by developing detergents with high affinity for their transport site or, on the contrary aimed at developing bulky and polycharged detergents, surrounding the membrane areas. The second original concept consists of synthesizing all these detergents by using the calix[n]arene chemical scaffold on which hydrophilic/polar heads and hydrophobic tails can be dissymmetrically grafted, generating a series of detergents designated "stabicalix". This study, with a marked interdisciplinary character, gathers 4 complementary teams, of international level, of biologists, chemists and physicists. Dysfunction of ABC transporters is responsible to genetic diseases while other ABC transporters are at the origin of chemo-resistances which are developed during treatments against cancer or pathogenic micro-organisms. These proteins are inserted into membranes and their purification requires extraction using detergents. This step is critical because the replacement of the lipids by detergents can promote unfolding. In addition, the polyspecific ABC transporters are able to interact with a large variety of chemicals and bind also at their transport site the detergents precisely used to solubilise them. This functional duality may generate a conformational heterogeneity, which is harmful for the stabilization of protein and consequently prejudicial for crystallization.  2-Description of the project, methodology  Our idea is thus to create a type of detergent optimized for the structural study of these transporters by exploiting their capacity to interact with the polar head of the detergents. They will be conceived with a head having a strong affinity for the site of transport of a given transporter, in order to block it in a given conformation and thus to stabilize it sufficiently for favouring crystallization. The length of the hydrophobic tail will be also adapted to modulate the critical micellar concentration, an important parameter during the concentration step. In parallel, our idea is also trying to stabilize these membrane proteins by developing bulky and polycharged detergents, therefore interacting preferentially outside the protein and maintaining it solubilised without denaturation. The design of these detergents requires using a chemical motif onto which one can easily graft each part at a given position. Calix[n]arenes fit well with this profile, consisting of 4 to 8 condensed phenol, which form a two-faced crown on which it is possible to graft various groups in an dissymmetrical way. The detergents will be 1°/designed in collaboration by the 4 teams; 2°/ synthesized by the Chemistry team which has a well established capacity in calix[n]arene chemistry; 3°/ tested by the Biochemistry teams which study human, fungal and bacterial MDR ABC transporters and have set up expression systems compatible with the crystallogenesis studies, some of which are currently in progress. The synthesized detergents will be evaluated for their capacity to solubilize the proteins overexpressed from their native membranes, and to maintain them stable in solution. Their effects on the ATPase activity, and drug binding and transport will be also evaluated. The detergents will be also tested on conformation-stabilized mutants generated in the frame of that project 4°/ Each transporter will then be purified and checked for homogeneit