Integral membranes proteins have a crucial role in biological processes and are the principal targets of pharmacological agents. Despite their importance, there remains a lack of biophysical and structural data regarding membrane proteins. This lack of information is mainly due to the membranous properties and naturally low abundance of these proteins. Two significant bottlenecks in the study of membrane proteins are their expression and purification.
In this context, we are developing original strategies for the overexpression and purification of membrane proteins to obtain large quantities of functional protein for biophysical and structural studies, mostly by NMR spectroscopy.
Proteins of interest:
G-protein coupled receptors (GPCRs)
Our target GPCRs are the opioid receptors (MOP, KOP, DOP) and the proteins involved in the nociceptive response (NOP, NPFF1, NPFF2). Several strategies are used for their expression, including overexpression in the yeast Pichia pastoris to obtain functional GPCRs and expression, as inclusion bodies, in the bacteria E. coli. Different strategies are being developed to optimize purification and refolding processes. The receptors can then be solubilized in micelles for structural characterization and for ligand binding studies, or can be reconstituted in proteoliposomes to investigate the importance of phospholipid composition on protein structure and function.
Prokaryotic outer membranes form natural barriers that need to be selectively permeable to various products, such as ions or nutrients. Selective permeability is ensured by membrane proteins, such as porins, which are attractive pharmacological targets in host pathogen interactions.
Porins of Corynebacteria
PorA and PorH are the most important porins in Corynebacterium glutamicum. Their expression in various systems (C. glutamicum, E. coli, cell free) has revealed the presence of post-translational modifications that are crucial for channel activity. PorA and PorH form a hetero-oligomer, the structure of which is being investigated by NMR spectroscopy. Highly efficient cell free expression of PorA and PorH has been developed in collaboration with Dr F. Bernhard, Frankfurt. The homologous expression in C. Glutamicum provides fully functional proteins possessing their required post translational mycoloylation.
Outer membrane protein A (OmpA) of Klebsiella pneumonia
KpOmpA is expressed as inclusion bodies in E. coli. Following purification and refolding, the structure of KpOmpA was resolved by NMR spectroscopy, in a DHPC solubilized state (by solution state NMR) as well as in lipid bilayers (by solid state NMR). AFM-SMFS studies have revealed a reversible unfolding process of the membrane domain at a single molecule level ( in coll. with D. Müller, ETH Basel).
Sample preparation : from the heterologous expression to NMR spectrum
In order to obtain large quantities of functional proteins of interest, we are using various systems suitable for the expression of membrane proteins. Membrane proteins are purified by chromatography and are folded in detergent micelles for structure determination by liquid state NMR spectroscopy or are reconstituted in liposomes for solid state NMR spectroscopy.
- The first part of our work consists in the comparison and optimization of different heterologous gene expression systems; for example, cell free, bacterial and yeast-based.
- The second part of our work involves establishing biochemical and biophysical conditions for pharmacological and structural studies of membrane proteins in detergent micelles or reconstituted in proteoliposomes.
- J.L. Banères, IBMM, Montpellier, FRANCE
- M. Daffé, IPBS-CNRS, Toulouse, France
- M. Tropis, IPBS-CNRS, Toulouse, France
Method of molecular evolution with synthetic DNA Inventors: Didier Fournier, Pascal Demange Number EP1818415, 2007
- Saurel, O., Iordanov, I., Nars, G., Demange, P., Le Marchand, T., Andreas, L. B., Pintacuda, G., and Milon, A., Local and Global Dynamics in Klebsiella pneumoniae Outer Membrane Protein a in Lipid Bilayers Probed at Atomic Resolution, J.A.C.S. 139, 1590-1597 (2017).
- Planchard, N., E. Point, T. Dahmane, F. Giusti, M. Renault, C. Le Bon, G. Durand, A. Milon, E. Guittet, M. Zoonens, J. L. Popot, and L. J. Catoire. The use of amphipols for solution NMR studies of membrane proteins: advantages and constraints as compared to other solubilizing media. J. Membr. Biol. 247, 827-842 (2014).
- Nars, G., O. Saurel, F. Bordes, I. Saves, M. Remaud-Simeon, I. Andre, A. Milon, and A. Marty. Production of stable isotope labelled lipase Lip2 from Yarrowia lipolytica for NMR: investigation of several expression systems. Protein Expr. Purif. 101, 14-20 (2014).
- Iordanov I, et al. (2012) Dynamics of Klebsiella pneumoniae OmpA transmembrane domain: The four extracellular loops display restricted motion behavior in micelles and in lipid bilayers. Biochim. Biophys. acta 1818:2344-2353.
- Bosshart PD, et al. (2012) The transmembrane protein KpOmpA anchoring the outer membrane of Klebsiella pneumoniae unfolds and refolds in response to tensile load. Structure 20:121-127.
- Rath P, et al. (2011) Functional expression of the PorAH channel from Corynebacterium glutamicum in cell-free expression systems: implications for the role of the naturally occurring mycolic acid modification. J Biol Chem 286:32525-32532.
- Renault M, Saurel O, Demange P, Reat V, Milon A. (2010) Solution−state NMR spectroscopy of membrane proteins in detergent micelles: structure of the Klebsiella pneumoniae outer membrane protein A, KpOmpA. Methods Mol Biol. 654:321−39
- Muller, I., Sarramegna, V., Milon, A., Talmont, F. J. (2010). The N-terminal end truncated mu-opioid receptor: from expression to circular dichroism analysis. Appl Biochem Biotechnol 160: 2175-86
- Renault, M., Saurel, O., Czaplicki, J., Demange, P., Gervais, V., Lohr, F., Reat, V., Piotto, M., and Milon, A. (2009) Solution State NMR Structure and Dynamics of KpOmpA, a 210 Residue Transmembrane Domain Possessing a High Potential for Immunological Applications, J. Mol. Biol., 385:117−30
- Denis-Quanquin, S., Lamouroux, L., Lougarre, A., Maheo, S., Saves, I., Paquereau, L., Demange, P. and Fournier, D. (2007). Protein expression from synthetic genes: selection of clones using GFP. J Biotechnol 131, 223-30
- Sarramegna V., Muller I., Milon A., and Talmont F. (2006). Recombinant G-Protein Coupled Receptors : from expression to renaturation: a challenge towards structure. Cell. Mol. Life Sci. 63, 1149-1164
- Sarramegna V., Talmont F., Demange P., and Milon A. (2003) GPCRs heterologous expression : comparison of the main expression systems from the standpoint of large scale production Cell. Mol. Life Sci., 60, 1-18.
- Massou S., Puech V., Talmont F., Demange P., Lindley N., Tropis M., and Milon A. (1999) Heterologuous expression of a deuteurated membrane receptor and partial deuteration in methylotrophic yeast. J. Biomol.NMR , 14, 231-239.