Refolding G protein-coupled receptors: a novel approach using polymeric surfactants – Refolding GPCRs

In spite of their key physiological roles, an understanding of the molecular basis for the function and dysfunction of membrane proteins (MPs) is rarely available. A main obstacle to establishing the atomic structure of MPs is their low natural abundance and the absence of established methods to overexpress them under their native form. Large amounts of MPs can be obtained in denatured form from inclusion bodies. Refolding them, however, has proven extremely difficult. One of the snags in renaturation attempts is that the detergents used to handle MPs in aqueous solutions tend to be inactivating. This makes it very difficult to identify conditions under which they keep refolding proteins from aggregating while allowing them to refold. We have found that it is possible to get around this difficulty thanks to amphipathic polymers ('amphipols'), which can keep MPs soluble under very mild conditions. Recent data obtained on six different MPs, including three model proteins and three G protein-coupled receptors (GPCRs), show that amphipols constitute extremely favorable media for their renaturation, probably because they keep them from aggregating while being unable to block their refolding. We propose to build upon these observations by extending the number of target GPCRs, broadening the range of renaturation conditions, and studying the underlying mechanisms, so as to develop and optimize a general method for renaturing MPs and to produce, in particular, refolded GPCRs for functional and structural studies. The project associates four laboratories with demonstrated expertise in synthesizing amphipols, in developing their applications to membrane biology, in overexpressing MPs, most specifically GPCRs, in refolding them, and in studying their structure by X ray crystallography and NMR spectroscopy. It is built in three overlapping stages of increasing difficulty: i) identifying optimal amphipols and conditions for refolding model MPs and studying the underlying mechanisms; ii) transferring this know-how to the refolding of GPCRs that have already been either totally or partially refolded; iii) applying this experience to refolding GPCRs that can be mass-produced but whose renaturation has until now proven intractable. Refolding will be initiated by supplementing solutions of denatured proteins with the amphipol to be tested and removing the denaturing agent(s) by any of various methods. Renaturation will be monitored by spectroscopic methods and/or ligand binding assays. Whenever practical, the rate of renaturation will be followed in real time. Parameters to be varied will include the nature and concentration of the constituents, temperature, pH, ionic strength, the degree of unfolding of the denatured protein, the nature and rate of removal of the denaturant(s), the addition of cofactors such as lipids etc. New amphipols will be designed and synthesized in view of this particular application. Series of homologous GPCRs will be overexpressed and tested in order to examine the generality of any emerging procedure and to increase the number of successfully refolded targets. The functionality, structure, dispersity and stability of the refolded receptors will be examined by biochemical, biophysical, spectroscopic and binding assays and, whenever the preparations will be found of sufficient quality, they will be subjected to crystallization attempts and NMR investigations.