PhD projects


Structural Biology of Membrane Proteins

PhD project:

Solution state NMR structure determination of PorA, the major porin of Corynebacterium glutamicum

Principal Investigator:

Prof A. Milon, IPBS, 205 rte de Narbonne, 31077 Toulouse, France; alain.milon|

SBMPs partner:

Dr F. Bernhard, CBMR, Inst. Biophys. Chem., Frankfurt, Germany; fbern|

In collaboration with:

Dr M. Daffé, Dr M. Tropis, Dr V. Réat, IPBS, Toulouse, France.

Corynebacterineae is a suprageneric actinomycete group that includes corynebacteria and mycobacteria.

Figure 1

The cell wall of these bacteria, which share the property of having an unusual composition and architecture, is a target for several anti-tubercular agents and Corynebacterium glutamicum has proven to be useful in the study of orthologues of Mycobacterium tuberculosis genes that are essential for its viability. Moreover, C. glutamicum is widely used in the industrial production of amino acids such as L-glutamate and L-lysine. For all these reasons, the comprehension of the cell envelope biogenesis remains a challenge. The cell envelope of Corynebacterineaea consists of a typical plasma membrane surrounded by a cell wall skeleton formed by a peptidoglycan covalently linked to an arabinogalactan, which in turn is esterified by mycolic acids (long chain α-alkyl, β-hydroxyl fatty acids)1. Mycolic acids play a crucial role in determining the fluidity and permeability of the cell wall. Consistently, many cell envelope proteins with pore forming activity which certainly facilitate the passage of small hydrophilic molecules through their outer membrane have been identified in all Corynebacterineae examined so far2, 3.

C. glutamicum contains a low molecular protein, PorA (5kDa), recently characterized and localized in the mycolic acid bilayer of the cell wall4. PorA is a small and very hydrophobic polypeptide containing 45 amino acids with an excess of four negative charges. It has been show that PorA is active as a selective cation porine5, 6. Because of its small size, one PorA can not form a channel through mycolic acid bilayer (10 nm thickness). One can therefore assume that the pore should be composed of a homo-oligomer of PorA like the protein MspA (octamer), found in the M. smegmatis mycobacteria3.

The serine 15 presents a post-translational modification whereas neither N-terminal modification nor a signal sequence were found.

Figure 2

A preliminary work showed that the activity of this porin in black lipid membrane seems to be dependent on the post-translational modification.

Structure determination of integral membrane proteins (MPs) is one of the most important challenges of structural biology. However, the number of high resolution structures of integral MPs in biological databases lags far behind that of soluble proteins. Over the last decade, only six three-dimensional folds of integral membrane proteins have been determined by application TROSY-based NMR pulses sequences 7-12. One of them, the outer membrane protein A from klebsiella pneumoniae, has been recently determined by Milon’s group, by using the state of the art of the liquid NMR spectroscopy. This is one of the largest integral membrane proteins (210 residues) for which nearly complete resonance assignment including side-chains could be performed, using TROSY-based 3D and 4D experiments at 800-900 MHz (made available via the EU-NMR platform and a collaboration with Dr F. Lohr, CBMR frankfurt), on a [15N,13C,2H] fully labeled sample and a methyl protonated, otherwise perdeuterated sample. The structure was refined from 1147 experimental constraints giving an ensemble of 20 best structures with a r.m.s. deviation of 0.56 Å for the main chain atoms in the core eight stranded β-barrel.

Figure 3 Figure 4

Secondary structure of the transmembrane domain of KpOmpA. (a) Representative 15N (ω3) / 1HN (ω>4) planes extracted from the 4D 15N, 15N-edited [1H, 1H]-HMQC-NOESY-TROSY (mixing time 250 ms) spectrum recorded with 1 mM u-[2H, 13C, 15N]-labeled KpOmpA/DHPC sample at 800 MHz showing sequential and long range HN-HN NOEs correlations (colored in blue) to T159 and L161 (colored in red). The most intense correlations that identify cross-stranded HN-HN NOEs (underlined residues) establish unambiguously the anti-parallel orientation of sequential β strands. This experiment allows then to determine 75 distances characteristic of anti parallel β sheets (i, j); (i+2, j-2) etc... and the β barrel organization

Key to the success of any project on structural biology of membrane proteins is the capacity to produce routinely and efficiently large amount of pure, stable isotope labelled protein. One strategy concerning PorA is the direct purification from corynebacteria, which was proven to be feasible at a level of 10 mg/L. This has the advantage to produce a functional protein containing the post-traductional modification in the right position.  Nevertheless, a complementary approach based on recombinant protein is required in order to increase the production level and to produce the unmodified PorA.

In vivoexpression approaches can be critical when MPs become toxic to the host cells upon overproduction as a result of system overloads or pore and channel forming activities. Recent advancements in cell-free expression technologies have provided new and highly promising alternatives for the production of problematic MPs13-16. These technologies allow a direct translation into detergent micelles combined with the elimination of toxic effects and other intrinsic barriers of MP overproduction. Furthermore, fast and efficient labelling protocols by cell-free expression have been developed and they open completely new avenues for the structural analysis of MPs by NMR14.

This PhD project is focused on understanding of oligomerization state and the structure of PorA forming form the pore, with or without the post-translational modification. This implies various activities:

1) Expression – purification in corynebacterium glutamicum (group of M. Daffe, IPBS, Toulouse); currently available, 2 month

2) Expression – purification in cell free expression system (group of F. Bernhard, CBMR, Frankfurt); to be established in Frankfurt, 3-6 months

3) Functional reconstitution in detergent micelles and in lipid bilayers; biophysical and biochemical studies; Oligomerisation states and structure – function relationship; Toulouse, 3-6 months.

4) 3D structure resolution of PorA by solution state NMR; group of A. Milon, IPBS with possible access to the EU-NMR infrastructures in Frankfurt; Toulouse and Frankfurt, 12 months

5) 3D structure in bilayers by solid state NMR; group of A. Milon, Toulouse, 12 months.

  1. McNeil M, Daffe M, Brennan PJ. 1990.
    Evidence for the nature of the link between the arabinogalactan and peptidoglycan of mycobacterial cell walls. J. Biol. Chem. 265, 18200-18206.
  2. Costa-Riu N, Burkovski A, Kramer R, Benz R. 2003.
    PorA represents the major cell wall channel of the Gram-positive bacterium Corynebacterium glutamicum. J. Bacteriol. 185, 4779-4786.
  3. Faller M, Niederweis M Schltz GE 2004.
    The structure of a mycobacterial outer-membrane channel. Science 303, 1189-1192
  4. Puech, V., Chami, M., Lemassu, A., Lanéelle, M.A., Schiffler, B., Gounon, P., Bayan, N., Benz, R. and Daffé, M. 2001.
    Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall fracture plane, Microbiology 147, 1365-1382.
  5. Lichtinger T, Burkovski A, Niederweis M, Kramer R, Benz R. 1998.
    Biochemical and biophysical characterization of the cell wall porin of Corynebacterium glutamicum: the channel is formed by a low molecular mass polypeptide. Biochemistry 37, 15024-32.
  6. Lichtinger T, Riess FG, Burkovski A., Engelbrecht F, Hesse D, Kratzin HD, Kramer R, Benz R. 2001.
    The low-molecular-mass subunit of the cell wall channel of the Gram-positive Corynebacterium glutamicum. Immunological localization, cloning and sequencing of its gene porA Eu.r J. Biochem. 268, 462-469.
  7. Arora, A., Abildgaard, F., Bushweller, J.H. & Tamm, L.K. 2001.
    Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nature Structural Biology 8, 334-338.
  8. Hwang, P.M. et al. 2002.
    Solution structure and dynamics of the outer membrane enzyme PagP by NMR. PNAS USA 99, 13560-13565.
  9. Fernandez, C., Hilty, C., Wider, G., Guntert, P. & Wuthrich, K. 2004.
    NMR structure of the integral membrane protein OmpX. Journal of Molecular Biology 336, 1211-1221.
  10. Roosild, T.P. et al. 2005.
    NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science 307, 1317-1321.
  11. Liang, B.Y. & Tamm, L.K. 2007.
    Structure of outer membrane protein G by solution NMR spectroscopy. PNAS USA 104, 16140-16145.
  12. Renault M., Saurel O., Czaplicki C., Demange P., Gervais V., Lohr F., Réat V., Piotto M. and Milon A., 2008.
    Solution state NMR structure and dynamics of KpOmpA, a 210 residues transmembrane domain possessing a high potential for immunological applications. Submitted to Nature struct Biol
  13. Klammt C, Schwarz D, Lohr F, Schneider B, Dotsch V, Bernhard F, 2006.
    Cell-free expression as an emerging technique for the large scale production of integral membrane protein. FEBS J 273, 4141-53.
  14. Koglin A, Klarnmt C, Trbovic N, Schwarz D, Schneider B, Schafer B, Lohr F, Bernhard F, Dotsch V, 2006.
    Combination of cell-free expression and NMR spectroscopy as a new approach for structural investigation of membrane proteins J. Magn Reson in Chemistry 44, 17-23.
  15. Schwarz D, Junge F, Durst F, Frölich N, Schneider B, Reckel S, Sobhanifar S, Dötsch V, Bernhard F.
    Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. Nat Protoc. 2007; 2(11), 2945-57.
  16. Klammt C, Schwarz D, Dötsch V, Bernhard F.
    Cell-free production of integral membrane proteins on a preparative scale. Methods Mol Biol. 2007; 375, 57-78. Review.